Tissue culture independent transformation for Corchorus olitorius

An efficient microprogation protocol has been developed for Dendrobium densiflorum Lindl. ex Wall., a traditional medicinal plant, through protocorm-like bodies (PLBs) from nodal stem segments using 6-benzylamino-purine (BAP) and the lanthanoid neodymium. The highest percentage of explants producing PLBs (72%), with an average of 15 PLBs per explant, was induced by culturing stem segments on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ BAP. The newly formed PLBs proliferated well on the basal MS medium and completely converted into shoots on MS medium containing 2.0 mg l⁻¹ BAP. Shoots produced an average of 22 roots per plantlet when cultured on MS medium supplemented with 2.0 mg l⁻¹ neodymium nitrate. Healthy plantlets with well-developed roots were successfully acclimatized. The obtained result suggests that the lanthanoids can be used to effectively initiate rooting in the micropropagation and conservation of D. densiflorum.     

Activation of CFTR by UCCF-029 and genistein

The mechanism of action of a novel CFTR activator UC_CF-029 on NIH3T3 cells stably expressing ΔF508-CFTR was investigated and its effects compared to those of genistein, a known CFTR activator. This study shows that UC_CF-029 and genistein have differing efficacies. The efficacy of UC_CF-029 in the presence of forskolin (10 μM) is 50% that of genistein; however, the EC₅₀’s for both drugs are comparable; 3.5 μM for UC_CF-029 and 4.4 μM for genistein. Using NIH3T3 cells stably transfected with K1250A-CFTR we find that CFTR channel open time is unaffected by UC_CF-029 or genistein, supporting the hypothesis that these compounds stabilize the open state by inhibiting ATP hydrolysis at NBD2. Our data suggest that the ability of UC_CF-029 to augment ΔF508-CFTR channel activity necessitates further interest.     

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method. Joaquin Sanchis, Layla Fernández, and J. Daniel Carballeira contributed equally.     

THetA: inferring intra-tumor heterogeneity from high-throughput DNA sequencing data

Tumor samples are typically heterogeneous, containing admixture by normal, non-cancerous cells and one or more subpopulations of cancerous cells. Whole-genome sequencing of a tumor sample yields reads from this mixture, but does not directly reveal the cell of origin for each read. We introduce THetA (Tumor Heterogeneity Analysis), an algorithm that infers the most likely collection of genomes and their proportions in a sample, for the case where copy number aberrations distinguish subpopulations. THetA successfully estimates normal admixture and recovers clonal and subclonal copy number aberrations in real and simulated sequencing data. THetA is available at http://compbio.cs.brown.edu/software/.     

Crystal structure and allosteric activation of protein kinase C βII

Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C βII was determined at 4.0 ?, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCβII was derived from small-angle X-ray scattering. Together, these results show how PKCβII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism.     

How territoriality affects the density of an egg predator： Habitat renovation and reintroduction as a method of conserving two endangered desert spring fish

Conservation of endangered sympatric species must be carefully executed to ensure that the protection of one species does not negatively impact the survival of another. Critically endangered Pecos gambusia Gambusia nobilis and Leon Springs pupfish Cyprinodon bovinus, which are endemic to a threatened desert spring habitat, exemplify one such scenario. Recently, this habitat has been expanded, and captively bred C. bovinus were released to repopulate an area historically known to contain this species. A previous study suggested that G. nobilis are detrimental to C. bovinus populations, due to their tendency to congregate near spawning pairs and feed on their eggs. Whether G. nobilis are attracted to territorial C. bovinus, regardless of spawning behavior, remains unclear. To determine this, the number of G. nobilis in occupied and unoccupied breeding territories of both wild and captively bred C. bovinus was measured. Gambusia nobilis densities were similar between unoccupied and occupied territorial sites, suggesting that they do not show a preference for C. bovinus territories. Regardless of habitat location or prior captivity, territorial C. bovinus significantly excluded G. nobilis within their direct vicinity （5 cm）, but not from their entire territory. Decreased G. nobilis density within the habitat may allow C. bovinus to defend their breeding territories more readily during the summer spawning season. This study provides empirical evidence of captively raised individuals behaving similarly to wild indi- viduals upon reintroduction to their natural habitat and support for conservation methods that focus on common limiting factors between endangered species [Current Zoology 60 （4）： 527-533, 2014].     

Cardiovascular Risks Associated with Low Dose Ionizing Particle Radiation

Previous epidemiologic data demonstrate that cardiovascular (CV) morbidity and mortality may occur decades after ionizing radiation exposure. With increased use of proton and carbon ion radiotherapy and concerns about space radiation exposures to astronauts on future long-duration exploration-type missions, the long-term effects and risks of low-dose charged particle irradiation on the CV system must be better appreciated. Here we report on the long-term effects of whole-body proton (¹H; 0.5 Gy, 1 GeV) and iron ion (⁵⁶Fe; 0.15 Gy, 1GeV/nucleon) irradiation with and without an acute myocardial ischemia (AMI) event in mice. We show that cardiac function of proton-irradiated mice initially improves at 1 month but declines by 10 months post-irradiation. In AMI-induced mice, prior proton irradiation improved cardiac function restoration and enhanced cardiac remodeling. This was associated with increased pro-survival gene expression in cardiac tissues. In contrast, cardiac function was significantly declined in ⁵⁶Fe ion-irradiated mice at 1 and 3 months but recovered at 10 months. In addition, ⁵⁶Fe ion-irradiation led to poorer cardiac function and more adverse remodeling in AMI-induced mice, and was associated with decreased angiogenesis and pro-survival factors in cardiac tissues at any time point examined up to 10 months. This is the first study reporting CV effects following low dose proton and iron ion irradiation during normal aging and post-AMI. Understanding the biological effects of charged particle radiation qualities on the CV system is necessary both for the mitigation of space exploration CV risks and for understanding of long-term CV effects following charged particle radiotherapy.