Modeling sarcomagenesis using multipotent mesenchymal stem cells

Because of their unique properties, multipotent mesenchymal stem cells (MSCs) represent one of the most promising adult stem cells being used worldwide in a wide array of clinical applications. Overall, compelling evidence supports the long-term safety of ex vivo expanded human MSCs, which do not seem to transform spontaneously. However, experimental data reveal a link between MSCs and cancer, and MSCs have been reported to inhibit or promote tumor growth depending on yet undefined conditions. Interestingly, solid evidence based on transgenic mice and genetic intervention of MSCs has placed these cells as the most likely cell of origin for certain sarcomas. This research area is being increasingly explored to develop accurate MSC-based models of sarcomagenesis, which will be undoubtedly valuable in providing a better understanding about the etiology and pathogenesis of mesenchymal cancer, eventually leading to the development of more specific therapies directed against the sarcoma-initiating cell. Unfortunately, still little is known about the mechanisms underlying MSC transformation and further studies are required to develop bona fide sarcoma models based on human MSCs. Here, we comprehensively review the existing MSC-based models of sarcoma and discuss the most common mechanisms leading to tumoral transformation of MSCs and sarcomagenesis.     

Harmonine, a defence compound from the harlequin ladybird, inhibits mycobacterial growth and demonstrates multi-stage antimalarial activity

The harlequin ladybird beetle Harmonia axyridis has been introduced in many countries as a biological control agent, but has become an invasive species threatening the biodiversity of native ladybirds. Its invasive success has been attributed to its vigorous resistance against diverse pathogens. This study demonstrates that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), which is present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited. Harmonine displays gametocytocidal activity, and inhibits the exflagellation of microgametocytes and zygote formation. In an Anopheles stephensi mosquito feeding model, harmonine displays transmission-blocking activity.     

Role of gamma-subunit N- and C-termini in assembly of the mitochondrial ATP synthase in yeast

The γ-subunit is required for the assembly of ATP synthases and plays a crucial role in their catalytic activity. We stepwise shortened the N-terminus and the C-terminus of the γ-subunit in the mitochondrial ATP synthase of yeast and investigated the relevance of these segments in the assembly of the enzyme and in the growth of the cells. We found that a deletion of 9 residues at the N-terminus or 20 residues at the C-terminus still allowed efficient import of the subunit into mitochondria; however, the assembly of both monomeric and dimeric holoenzymes was partially impaired. γ-Subunits lacking 13 N-terminal residues or 30 C-terminal residues were not assembled. Yeast strains expressing either of the truncated γ-subunits did not grow on non-fermentable carbon sources, indicating that non-assembled parts of the ATP synthase accumulated and impaired essential mitochondrial functions.     

The effect of irradiance on long-term skeletal growth and net photosynthesis in Galaxea fascicularis under four light conditions

The relation between irradiance, skeletal growth and net photosynthesis was studied for the scleractinian coral Galaxea fascicularis to provide experimental evidence for mediation of light-enhanced calcification through photosynthesis. The hypothesis was tested that skeletal growth and photosynthesis are linearly correlated.A long-term experiment was performed in a closed-circuit aquarium system, in which four series of nine nubbins (single polyp clones of a coral colony) of Galaxea fascicularis were exposed to four light treatments (10L:14D): 144 W T8 fluorescent lighting providing an irradiance of 68 µE/m²/s and 70, 250 and 400 W Metal Halide lighting providing an irradiance of 38 µE/m²/s, 166 µE/m²/s and 410 µE/m²/s, respectively. Growth of these nubbins was measured as buoyant weight at different time intervals in a 294 day experiment. A light-saturation curve for photosynthesis was measured in a respirometric flow cell using a 54 week Galaxea fascicularis colony grown at 60 µE/m²/s.No saturation of net photosynthesis of Galaxea fascicularis was found at the irradiances tested. The specific growth rate (µ, in day^- 1) of the coral nubbins increased with irradiance. Whereas irradiance varied 11-fold (38 to 410 µE/m²/s), buoyant weight (increase after 294 days) increased 5.7 times (2243 to 12374 mg), specific growth rate (1-294 days) increased 1.6 times (0.0103 to 0.0161 day^- 1), while net photosynthetic rate increased 8.9 times (0.009 µmol O₂/min/cm² to 0.077 µmol O₂/min/cm²). The increase of specific growth rate with irradiance was less than expected based on the increase in net photosynthetic rate with irradiance. This discrepancy between potential energy produced in photosynthesis and energy used for skeletal growth indicates that skeletal growth is not limited by photosynthetic potential at high irradiance levels.     

IL-21-induced isotype switching to IgG and IgA by human naive B cells is differentially regulated by IL-4

Naive B cells can alter the effector function of their Ig molecule by isotype switching, thereby allowing them to secrete not only IgM, but also the switched isotypes IgG, IgA, and IgE. Different isotypes are elicited in response to specific pathogens. Similarly, dysregulated production of switched isotypes underlies the development of various diseases, such as autoimmunity and immunodeficiency. Thus, it is important to characterize mediators controlling isotype switching, as well as their contribution to the overall B cell response. Isotype switching in human naive B cells can be induced by CD40L together with IL-4, IL-10, IL-13, and/or TGF-beta. Recently, IL-21 was identified as a switch factor for IgG1 and IgG3. However, the effect of IL-21 on switching to IgA, as well as the interplay between IL-21 and other switch factors, remains unknown. We found that IL-4 and IL-21 individually induced CD40L-stimulated human naive B cells to undergo switching to IgG, with IL-4 predominantly inducing IgG1(+) cells and IL-21 inducing IgG3. Culture of naive B cells with CD40L and IL-21, but not IL-4, also yielded IgA(+) cells. Combining IL-4 and IL-21 had divergent effects on isotype switching. Specifically, while IL-4 and IL-21 synergistically increased the generation of IgG1(+) cells from CD40L-stimulated B cells, IL-4 concomitantly abolished IL-21-induced switching to IgA. Our findings demonstrate the dynamic interplay between IL-4 and IL-21 in regulating the production of IgG subclasses and IgA, and suggest temporal roles for these cytokines in humoral immune responses to specific pathogens.     

Lung epithelial injury markers are not influenced by use of lower tidal volumes during elective surgery in patients without preexisting lung injury

Clara cell protein levels are elevated in plasma of individuals with mild or subclinical lung injury. We studied the influence of two mechanical ventilation strategies on local and systemic levels of Clara cell protein (CC16) and compared them with levels of soluble receptor for advanced glycation end products (sRAGE) and surfactant proteins (SP)-A and -D in patients undergoing elective surgery. Saved samples from a previously reported investigation were used for the study. Forty patients planned for elective surgery were randomized to mechanical ventilation with either a conventional tidal volume (V(T)) of 12 ml/kg without positive end-expiratory pressure (PEEP) or low V(T) of 6 ml/kg and 10 cmH(2)O PEEP. Plasma and bronchoalveolar lavage fluid (BALF) was collected directly after intubation and after 5 h of mechanical ventilation. While systemic levels of SP-A and SP-D remained unchanged, systemic levels of CC16 and sRAGE increased significantly in both groups after 5 h (P < 0.001 for both). BALF levels of SP-A, SP-D, CC16, and sRAGE remained unaffected. No differences were found between the two mechanical ventilation strategies regarding any of the measured biological markers. In conclusion, systemic levels of CC16 and sRAGE rise after 5 h in patients receiving mechanical ventilation for elective surgery. Mechanical ventilation with lower tidal volumes and PEEP did not have a different effect on levels of biomarkers of lung epithelial injury compared with conventional mechanical ventilation.     

Phosphorylation-dependent binding of cyclin B1 to a Cdc6-like domain of human separase

Sister chromatids are held together by the ring-shaped cohesin complex, which likely entraps both DNA-double strands in its middle. This tie is resolved in anaphase when separase, a giant protease, becomes active and cleaves the kleisin subunit of cohesin. Premature activation of separase and, hence, chromosome missegregation are prevented by at least two inhibitory mechanisms. Although securin has long been appreciated as a direct inhibitor of separase, surprisingly its loss has basically no phenotype in mammals. Phosphorylation-dependent binding of Cdk1 constitutes an alternative way to inhibit vertebrate separase. Its importance is illustrated by the premature loss of cohesion when Cdk1-resistant separase is expressed in mammalian cells without or with limiting amounts of securin. Here, we demonstrate that crucial inhibitory phosphorylations occur within a region of human separase that is also shown to make direct contact with the cyclin B1 subunit of Cdk1. This region exhibits a weak homology to Saccharomyces cerevisiae Cdc6 of similar Cdk1 binding behavior, thereby establishing phosphoserine/threonine-mediated binding of partners as a conserved characteristic of B-type cyclins. In contrast to the Cdc6-like domain, the previously identified serine 1126 phosphorylation is fully dispensable for Cdk1 binding to separase fragments. This suggests that despite its in vivo relevance, it promotes complex formation indirectly, possibly by inducing a conformational change in full-length separase.     

Optical measurement of mechanical forces inside short DNA loops

Knowledge of the mechanical properties of double-stranded DNA (dsDNA) is essential to understand the role of dsDNA looping in gene regulation and the mechanochemistry of molecular machines that operate on dsDNA. Here, we use a newly developed tool, force sensors with optical readout, to measure the forces inside short, strained loops composed of both dsDNA and single-stranded DNA. By varying the length of the loops and their proportion of dsDNA, it was possible to vary their internal forces from 1 pN to >20 pN. Surprisingly, internal loop forces changed erratically as the amount of dsDNA was increased for a given loop length, with the effect most notable in the smallest loop (57 nucleotides). Monte Carlo simulations based on the helical wormlike chain model accurately predict internal forces when more than half of the loop is dsDNA but fail otherwise. Mismatches engineered into the double-stranded regions increased flexibility, suggesting that Watson-Crick basepaired dsDNA can withstand high compressive forces without recourse to multibase melts. Fluorescence correlation spectroscopy further excluded transient melting (microsecond to millisecond duration) as a mechanism for relief of compressive forces in the tested dsDNAs. DNA loops with integrated force sensors may allow the comprehensive mapping of the elasticity of short dsDNAs as a function of both sequence and salt.