Targeted Resequencing of the Pericentromere of Chromosome 2 Linked to Constitutional Delay of Growth and Puberty

Constitutional delay of growth and puberty (CDGP) is the most common cause of pubertal delay. CDGP is defined as the proportion of the normal population who experience pubertal onset at least 2 SD later than the population mean, representing 2.3% of all adolescents. While adolescents with CDGP spontaneously enter puberty, they are at risk for short stature, decreased bone mineral density, and psychosocial problems. Genetic factors contribute heavily to the timing of puberty, but the vast majority of CDGP cases remain biologically unexplained, and there is no definitive test to distinguish CDGP from pathological absence of puberty during adolescence. Recently, we published a study identifying significant linkage between a locus at the pericentromeric region of chromosome 2 (chr 2) and CDGP in Finnish families. To investigate this region for causal variation, we sequenced chr 2 between the genomic coordinates of 79–124 Mb (genome build GRCh37) in the proband and affected parent of the 13 families contributing most to this linkage signal. One gene, DNAH6, harbored 6 protein-altering low-frequency variants (< 6% in the Finnish population) in 10 of the CDGP probands. We sequenced an additional 135 unrelated Finnish CDGP subjects and utilized the unique Sequencing Initiative Suomi (SISu) population reference exome set to show that while 5 of these variants were present in the CDGP set, they were also present in the Finnish population at similar frequencies. Additional variants in the targeted region could not be prioritized for follow-up, possibly due to gaps in sequencing coverage or lack of functional knowledge of non-genic genomic regions. Thus, despite having a well-characterized sample collection from a genetically homogeneous population with a large population-based reference sequence dataset, we were unable to pinpoint variation in the linked region predisposing delayed puberty. This study highlights the difficulties of detecting genetic variants under linkage regions for complex traits and suggests that advancements in annotation of gene function and regulatory regions of the genome will be critical for solving the genetic background of complex phenotypes like CDGP.     

Bone‐forming capacity of adult human nasal chondrocytes

Nasal chondrocytes (NC) derive from the same multipotent embryological segment that gives rise to the majority of the maxillofacial bone and have been reported to differentiate into osteoblast‐like cells in vitro. In this study, we assessed the capacity of adult human NC, appropriately primed towards hypertrophic or osteoblastic differentiation, to form bone tissue in vivo. Hypertrophic induction of NC‐based micromass pellets formed mineralized cartilaginous tissues rich in type X collagen, but upon implantation into subcutaneous pockets of nude mice remained avascular and reverted to stable hyaline‐cartilage. In the same ectopic environment, NC embedded into ceramic scaffolds and primed with osteogenic medium only sporadically formed intramembranous bone tissue. A clonal study could not demonstrate that the low bone formation efficiency was related to a possibly small proportion of cells competent to become fully functional osteoblasts. We next tested whether the cues present in an orthotopic environment could induce a more efficient direct osteoblastic transformation of NC. Using a nude rat calvarial defect model, we demonstrated that (i) NC directly participated in frank bone formation and (ii) the efficiency of survival and bone formation by NC was significantly higher than that of reference osteogenic cells, namely bone marrow‐derived mesenchymal stromal cells. This study provides a proof‐of‐principle that NC have the plasticity to convert into bone cells and thereby represent an easily available cell source to be further investigated for craniofacial bone regeneration.     

Short-term impacts of energy wood harvesting on ectomycorrhizal fungal communities of Norway spruce saplings

The increased demand for harvesting energy wood raises questions about its effects on the functioning of the forest ecosystems, soil processes and biodiversity. Impacts of tree stump removal on ectomycorrhizal fungal (EMF) communities of Norway spruce saplings were studied with 454-pyrosequencing in a 3-year field experiment replicated in 3 geographical areas. This is possibly the most thorough investigation of EMF communities associated with saplings grown on sites subjected to energy wood harvesting. To separate impacts of tree stump and logging residue removal on EMF and plant variables, we used three harvesting treatments with increasing complexity from patch mounding alone (P) to patch mounding combined with logging residue removal (RP), and patch mounding combined with both logging residue and stump removal (SRP). Saplings grown in uncut forests (F) served as references for harvesting treatments. A majority of sequences (>92%) and operational taxonomic units (OTUs, 55%) were assigned as EMF. EMF OTU richness, fungal community composition or sapling growth did not differ between harvesting treatments (P, RP and SRP), while EMF OTU richness, diversity and evenness were highest and sapling growth lowest in the undisturbed reference forests (F). The short study period may partially explain the similarities in fungal and sapling variables in different harvesting treatments. In conclusion, our results indicate that neither stump removal nor logging residue removal have significant additional negative impacts on EMF communities or growth of Norway spruce saplings in the short-term compared with the impacts of more conventional harvesting methods, including clear cutting and patch mounding.     

Metabolic remodeling in human colorectal cancer and surrounding tissues: alterations in regulation of mitochondrial respiration and metabolic fluxes

The aim of the work was to evaluate whether or not there is glycolytic reprogramming in the neighboring cells of colorectal cancer (CRC). Using postoperative material we have compared the functional capacity of oxidative phosphorylation (OXPHOS) in CRC cells, their glycolytic activity and their inclination to aerobic glycolysis, with those of the surrounding and healthy colon tissue cells. Experiments showed that human CRC cannot be considered a hypoxic tumor, since the malignancy itself and cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The absence of acute hypoxia in colorectal carcinomas was also confirmed by their practically equal glucose-phosphorylating capacity as compared with surrounding non-tumorous tissue and by upregulation of VEGF family and their ligands. Studies indicated that human CRC cells in vivo exert a strong distant effect on the energy metabolism of neighboring cells, so that they acquire the bioenergetic parameters specific to the tumor itself. The growth of colorectal carcinomas was associated with potent downregulation of the creatine kinase system. As compared with healthy colon tissue, the tumor surrounding cells display upregulation of OXPHOS and have high values of basal and ADP activated respiration rates. Strong differences between the normal and CRC cells in the affinity of their mitochondria for ADP were revealed; the corresponding K_m values were measured as 93.6±7.7 µM for CRC cells and 84.9±9.9 µM for nearby tissue; both these apparent K_m (ADP) values were considerably (by almost 3 times) lower in comparison with healthy colon tissue cells (256±34 µM).     

Negative regulation of EGFR signalling through integrin-alpha1beta1-mediated activation of protein tyrosine phosphatase TCPTP

Integrin-mediated cell adhesion regulates a multitude of cellular responses, including proliferation, survival and cross-talk between different cellular signalling pathways. So far, integrins have been mainly shown to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signalling. Here we show that a collagen-binding integrin alpha(1)beta(1) functions as a negative regulator of epidermal growth factor receptor (EGFR) signalling through the activation of a protein tyrosine phosphatase. The cytoplasmic tail of alpha(1) integrin selectively interacts with a ubiquitously expressed protein tyrosine phosphatase TCPTP (T-cell protein tyrosine phosphatase) and activates it after cell adhesion to collagen. The activation results in reduced EGFR phosphorylation after EGF stimulation. Introduction of the alpha(1) cytoplasmic domain peptide into cells induces phosphatase activation and inhibits EGF-induced cell proliferation and anchorage-independent growth of malignant cells. These data are the first demonstration of the regulation of TCPTP activity in vivo and represent a new molecular paradigm of integrin-mediated negative regulation of receptor tyrosine kinase signalling.     

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.     

Antimicrobial activity of the marine alkaloids haminol and pulo'upone and related compounds

The marine alkaloids haminol A, haminol B and pulo'upone as well as 17 related compounds (twelve 2-substituted pyridine derivatives, four 3-substituted ones and one analogue of the bicyclic terminus of pulo'upone) were tested for antimicrobial activity against a panel of six microbes (Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida albicans and Saccharomyces cerevisiae) using the paper disc agar diffusion method. Six compounds were tested also against the mold Aspergillus niger. Some of the compounds displayed noteworthy antimicrobial activity, only one congener being completely devoid of activity. Nearly all compounds had activity against B. cereus and S. epidermidis. The growth of E. coli, C albicans and S. cerevisiae was also distinctly inhibited by many compounds. In contrast, most compounds were inactive or had minimal activity against P. aeruginosa. Interestingly, most of the compounds tested against the opportunistic pathogen A. niger were active, one of them having noteworthy inhibitory potency.     

Ethiopian agriculture has greater potential for carbon sequestration than previously estimated

More than half of the cultivation‐induced carbon loss from agricultural soils could be restored through improved management. To incentivise carbon sequestration, the potential of improved practices needs to be verified. To date, there is sparse empirical evidence of carbon sequestration through improved practices in East‐Africa. Here, we show that agroforestry and restrained grazing had a greater stock of soil carbon than their bordering pair‐matched controls, but the difference was less obvious with terracing. The controls were treeless cultivated fields for agroforestry, on slopes not terraced for terracing, and permanent pasture for restrained grazing, representing traditionally managed agricultural practices dominant in the case regions. The gain by the improved management depended on the carbon stocks in the control plots. Agroforestry for 6–20 years led to 11.4 Mg ha^?1 and restrained grazing for 6–17 years to 9.6 Mg ha^?1 greater median soil carbon stock compared with the traditional management. The empirical estimates are higher than previous process‐model‐based estimates and indicate that Ethiopian agriculture has greater potential to sequester carbon in soil than previously estimated.