Dynamic observation and quantification of type I/II collagen in chondrogenesis of mesenchymal stem cells by second‐order susceptibility microscopy

Second‐order susceptibility (SOS) microscopy is used to image and characterize chondrogenesis in cultured human mesenchymal stem cells. SOS analysis shows that the SOS tensor ratios can be used to characterize type I and II collagens in living tissues and that both collagen types are produced at the onset of chondrogenesis. Time‐lapse analysis shows a modulation of extracellular matrix results in a higher rate in increase of type II collagen, as compared to type I collagen. With time, type II collagen content stabilizes at the composition of 70% of total collagen content. SOS microscopy can be used to continuously and noninvasively monitor the production of collagens I and II. With additional development, this technique can be developed into an effective quality control tool for monitoring extracellular matrix production in engineered tissues.      

Apoptosis is required during early stages of tail regeneration in Xenopus laevis

The Xenopus tadpole is able to regenerate its tail, including skin, muscle, notochord, spinal cord and neurons and blood vessels. This process requires rapid tissue growth and morphogenesis. Here we show that a focus of apoptotic cells appears in the regeneration bud within 12 h of amputation. Surprisingly, when caspase-3 activity is specifically inhibited, regeneration is abolished. This is true of tails both before and after the refractory period. Programmed cell death is only required during the first 24 h after amputation, as later inhibition has no effect on regeneration. Inhibition of caspase-dependent apoptosis results in a failure to induce proliferation in the growth zone, a mispatterning of axons in the regenerate, and the appearance of ectopic otoliths in the neural tube, in the context of otherwise normal continued development of the larva. Larvae amputated during the refractory stage exhibit a much broader domain of caspase-3-positive cells, suggesting a window for the amount of apoptosis that is compatible with normal regeneration. These data reveal novel roles for apoptosis in development and indicate that a degree of apoptosis is an early and obligate component of normal tail regeneration, suggesting the possibility of the existence of endogenous inhibitory cells that must be destroyed by programmed cell death for regeneration to occur.     

CD36 signals to the actin cytoskeleton and regulates microglial migration via a p130Cas complex

The pattern recognition receptor CD36 initiates a signaling cascade that promotes microglial activation and recruitment to beta-amyloid deposits in the brain. In the present study we identify the focal adhesion-associated proteins p130Cas, Pyk2, and paxillin as novel members of the tyrosine kinase signaling pathway downstream of CD36 and show that assembly of this complex is essential for microglial migration. In primary microglia and macrophages exposed to beta-amyloid, the scaffolding protein p130Cas is rapidly tyrosine-phosphorylated and co-localizes with CD36 to membrane ruffles contemporaneous with F-actin polymerization. These beta-amyloid-stimulated events are not detected in CD36 null cells and are dependent on CD36 activation of Src family tyrosine kinases. Fyn, a Src kinase known to interact with CD36, co-precipitates with p130Cas and is an essential upstream intermediate in the signaling pathways leading to phosphorylation of the p130Cas substrate domain. Furthermore, the p130Cas-interacting kinase Pyk2 and the cytoskeletal adapter protein paxillin also demonstrate CD36-dependent phosphorylation, identifying these focal adhesion molecules as additional members of this beta-amyloid signaling cascade. Disruption of this p130Cas complex by small interfering RNA silencing inhibits p44/42 mitogen-activated protein kinase phosphorylation and microglial migration, illustrating the importance of this pathway in microglial activation and recruitment. Together, these data are the first to identify the signaling cascade that directly links CD36 to the actin cytoskeleton and, thus, implicates it in diverse processes such as cellular migration, adhesion, and phagocytosis.     

Phototropins and Their LOV Domains：Versatile Plant Blue-Light Receptors

The phototropins phot1 and phot2 are plant blue-light receptors that mediate phototropism, chloroplast movements, stomatal opening, leaf expansion, the rapid Inhibition of hypocotyl growth in etiolated seedlings, and possibly solar tracking by leaves in those species in which It occurs. The phototroplns are plasma membrane-associated hydrophilic proteins with two chromophore domains （designated LOV1 and LOV2 for their resemblance to domains In other signaling proteins that detect light, oxygen, or voltage） in their Nterminal half and a classic serine/threonlne kinase domain in their C-terminal half. Both chromophore domains bind flavin mononucleotide （FMN） and both undergo light-activated formation of a covalent bond between a nearby cystelne and the C（4a） carbon of the FMN to form the signaling state. LOV2-cystelnyl adduct formation leads to the release downstream of a tightly bound amphlpathlc α-helix, a step required for activation of the klnase function. This cysteinyl adduct then slowly decays over a matter of seconds or minutes to return the photoreceptor chromophore modules to their ground state. Functional LOV2 is required for light-activated phosphorylation and for various blue-light responses mediated by the phototroplns. The function of LOV1 is still unknown, although It may serve to modulate the signal generated by LOV2. The LOV domain Is an ancient chromophore module found In a wide range of otherwise unrelated proteins In fungi and prokaryotes, the latter Including cyanobacterla, eubacterla, and archaea. Further general reviews on the phototropins are those by Celaya and Liscum （2005） and Christie and Briggs （2005）.     

Metabolomics standards initiative: ontology working group work in progress

In this article we present the activities of the Ontology Working Group (OWG) under the Metabolomics Standards Initiative (MSI) umbrella. Our endeavour aims to synergise the work of several communities, where independent activities are underway to develop terminologies and databases for metabolomics investigations. We have joined forces to rise to the challenges associated with interpreting and integrating experimental process and data across disparate sources (software and databases, private and public). Our focus is to support the activities of the other MSI working groups by developing a common semantic framework to enable metabolomics-user communities to consistently annotate the experimental process and to enable meaningful exchange of datasets. Our work is accessible via a public webpage and a draft ontology has been posted under the Open Biological Ontology umbrella. At the very outset, we have agreed to minimize duplications across omics domains through extensive liaisons with other communities under the OBO Foundry. This is work in progress and we welcome new participants willing to volunteer their time and expertise to this open effort. See the MSI Ontology Working Group website for a complete list of members and contributors. Web URL:     

Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination

Nonhomologous end joining (NHEJ) eliminates DNA double-strand breaks (DSBs) in bacteria and eukaryotes. In Saccharomyces cerevisiae, there are pairwise physical interactions among the core complexes of the NHEJ pathway, namely Yku70-Yku80 (Ku), Dnl4-Lif1 and Mre11-Rad50-Xrs2 (MRX). However, MRX also has a key role in the repair of DSBs by homologous recombination (HR). Here we have examined the assembly of NHEJ complexes at DSBs biochemically and by chromatin immunoprecipitation. Ku first binds to the DNA end and then recruits Dnl4-Lif1. Notably, Dnl4-Lif1 stabilizes the binding of Ku to in vivo DSBs. Ku and Dnl4-Lif1 not only initiate formation of the nucleoprotein NHEJ complex but also attenuate HR by inhibiting DNA end resection. Therefore, Dnl4-Lif1 plays an important part in determining repair pathway choice by participating at an early stage of DSB engagement in addition to providing the DNA ligase activity that completes NHEJ.     

Stable isotope analysis reveals ontogenetic feeding shifts in Pacific blue marlin (Makaira nigricans) off eastern Taiwan

To gain a better understanding of the trophic ecology of Pacific blue marlin Makaira nigricans off eastern Taiwan, nitrogen and carbon stable isotopes (δ¹⁵N and δ¹³C) and Bayesian mixing models were used to explore trophic dynamics and potential ontogenetic feeding shifts across M. nigricans of different size classes. Makaira nigricans samples from east of Taiwan (n = 213) and Palau (n = 37), as well as their prey (n = 70), were collected during 2012 and 2013. Results indicated increases in δ¹⁵N with size, with values of larger size classes (> 200 cm eye-to-fork length; L_EF) significantly higher than those < 200 cm L_EF. Values of δ¹³C were negatively correlated with size. Makaira nigricans > 200 cm L_EF had the highest estimated trophic position (4.44) and also exhibited ontogenetic changes in trophic position. Large M. nigricans fed more on dolphinfish Coryphaena hippurus and hairtail Trichiurus lepturus, while smaller M. nigricans consumed smaller forage fish (e.g., moonfish Mene maculata) and cephalopods. These changes may relate to greater swimming speeds and vertical habitat use in larger M. nigricans, allowing capture and consumption of larger prey items at higher trophic positions. The high trophic level of M. nigricans east of Taiwan confirms its important role as an apex predator in marine food webs and how ecological role changes with size.     

Crosslinking CD81 results in activation of TCRgammadelta T cells

CD81 is expressed on most cells and is associated with other glycoproteins, including CD4 and CD8, to form multimolecular membrane complexes. Crosslinking of CD81 on TCRalphabeta(+) T cells results in costimulatory signals that have been proposed to be mediated via CD4 or CD8. In this study, we show that CD81 is also expressed on TCRgammadelta(+)CD4(-)CD8(-) T cells. CD81 crosslinking greatly enhanced anti-CD3 activation of both TCRalphabeta(+) (CD4+ and CD8+) and TCRgammadelta(+) T cells with regard to IFN-gamma production. However, crosslinking of CD81 molecules on TCRgammadelta(+) T cells, in the absence of anti-CD3 stimulation, resulted in cytokine production and enhanced IL-2-induced proliferation, demonstrating that physical association with CD4 or CD8 is not necessary for CD81 signaling. In contrast, crosslinking of CD81 on TCRalphabeta(+) T cells, in the absence of anti-CD3 stimulation, failed to activate these T cells. These results suggest that CD81 signaling may be mediated via a different mechanism(s) in TCRgammadelta(+) versus TCRalphabeta(+) T cells.     

DNA cloning without restriction enzyme and ligase

One common problem in using the traditional DNA cloning procedure is that suitable natural restriction sites are often unavailable for a given task. Creating new restriction sites is often time consuming. Here, I describe a simple technique of producing "customized cohesive ends" by a combination of PCR primer design and lambda exonuclease digestion. These complementary cohesive ends can form hybrids to link two sequences. Because the overhangs created by lambda exonuclease are slightly longer than the complementary sequence, after hybrid formation, a stretch of single-strand gap remains, which then is repaired by Klenow (3'-->5' exo-) enzyme. The repair process also stabilizes the linkage. Because of the independence from natural or artificial restriction sites, this method allows rapid and precise insertion of one DNA fragment into another at virtually any position. It also simplifies the planning of a cloning strategy, increases recombinant frequency and is suitable for automation.