RNA Interference Can Rebalance the Nitrogen Sink of Maize Seeds without Losing Hard Endosperm

Background: One of the goals of plant breeding is to create crops to provide better nutrition for humans and livestock. Insufficient intake of protein is one of the most severe factors affecting the growth and development of children in developing countries. More than a century ago, in 1896, Hopkins initiated the well-known Illinois long-term selection for maize seed protein concentration, yielding four protein strains. By continuously accumulating QTLs, Illinois High Protein (IHP) reached a protein level 2.5-fold higher than normal maize, with the most increased fraction being the zein protein, which was shown to contain no lysine soon after the long-term selection program initiated. Therefore, IHP is of little value for feeding humans and monogastric animals. Although high-lysine lines of non-vitreous mutants were based on reduced zeins, the kernel soft texture precluded their practical use. Kernel hardness in opaque 2 (o2) could be restored in quality protein maize (QPM) with quantitative trait loci called o2 modifiers (Mo2s), but those did not increase total protein levels. Methods: The most predominant zeins are the 22- and 19-kDa a-zeins. To achieve a combination of desired traits, we used RNA interference (RNAi) against both a-zeins in IHP and evaluated the silencing effect by SDS-PAGE. Total protein, amino acid composition and kernel texture were analyzed. Conclusions: The a-zeins were dramatically reduced, but the high total seed protein level remained unchanged by complementary increase of non-zein proteins. Moreover, the residual zein levels still allowed for a vitreous hard seed. Suc

Immunotherapy of lung cancer: An update

In Germany lung cancer is the leading cause of cancer-associated death in men. Surgery, chemotherapy and radiation may enhance survival of patients suffering from lung cancer but the enhancement is typically transient and mostly absent with advanced disease; eventually more than 90% of lung cancer patients will die of disease. New approaches to the treatment of lung cancer are urgently needed. Immunotherapy may represent one new approach with low toxicity and high specificity but implementation has been a challenge because of the poor antigenic characterization of these tumors and their ability to escape immune responses. Several different immunotherapeutic treatment strategies have been developed. This review examines the current state of development and recent advances with respect to non-specific immune stimulation, cellular immunotherapy ( specific and non-specific), therapeutic cancer vaccines and gene therapy for lung cancer. The focus is primarily placed on immunotherapeutic cancer treatments that are already in clinical trial or well progressed in preclinical studies. Although there seems to be a promising future for immunotherapy in lung cancer, presently there is not standard immunotherapy available for clinical routine

The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

Safety, tumor trafficking and immunogenicity of chimeric antigen receptor (CAR)-T cells specific for TAG-72 in colorectal cancer.

BackgroundT cells engineered to express chimeric antigen receptors (CARs) have established efficacy in the treatment of B-cell malignancies, but their relevance in solid tumors remains undefined. Here we report results of the first human trials of CAR-T cells in the treatment of solid tumors performed in the 1990s.MethodsPatients with metastatic colorectal cancer (CRC) were treated in two phase 1 trials with first-generation retroviral transduced CAR-T cells targeting tumor-associated glycoprotein (TAG)-72 and including a CD3-zeta intracellular signaling domain (CART72 cells). In trial C-9701 and C-9702, CART72 cells were administered in escalating doses up to 1010 total cells; in trial C-9701 CART72 cells were administered by intravenous infusion. In trial C-9702, CART72 cells were administered via direct hepatic artery infusion in patients with colorectal liver metastases. In both trials, a brief course of interferon-alpha (IFN-α) was given with each CART72 infusion to upregulate expression of TAG-72.ResultsFourteen patients were enrolled in C-9701 and nine in C-9702. CART72 manufacturing success rate was 100% with an average transduction efficiency of 38%. Ten patients were treated in CC-9701 and 6 in CC-9702. Symptoms consistent with low-grade, cytokine release syndrome were observed in both trials without clear evidence of on target/off tumor toxicity. Detectable, but mostly short-term (≤14 weeks), persistence of CART72 cells was observed in blood; one patient had CART72 cells detectable at 48 weeks. Trafficking to tumor tissues was confirmed in a tumor biopsy from one of three patients. A subset of patients had 111Indium-labeled CART72 cells injected, and trafficking could be detected to liver, but T cells appeared largely excluded from large metastatic deposits. Tumor biomarkers carcinoembryonic antigen (CEA) and TAG-72 were measured in serum; there was a precipitous decline of TAG-72, but not CEA, in some patients due to induction of an interfering antibody to the TAG-72 binding domain of humanized CC49, reflecting an anti-CAR immune response. No radiologic tumor responses were observed.ConclusionThese findings demonstrate the relative safety of CART72 cells. The limited persistence supports the incorporation of co-stimulatory domains in the CAR design and the use of fully human CAR constructs to mitigate immunogenicity

Performance deficits of NK1 receptor knockout mice in the 5 choice serial reaction time task: effects of d Amphetamine, stress and time of day.

Background The neurochemical status and hyperactivity of mice lacking functional substance P-preferring NK1 receptors (NK1R-/-) resemble abnormalities in Attention Deficit Hyperactivity Disorder (ADHD). Here we tested whether NK1R-/- mice express other core features of ADHD (impulsivity and inattentiveness) and, if so, whether they are diminished by d-amphetamine, as in ADHD. Prompted by evidence that circadian rhythms are disrupted in ADHD, we also compared the performance of mice that were trained and tested in the morning or afternoon. Methods and Results The 5-Choice Serial Reaction-Time Task (5-CSRTT) was used to evaluate the cognitive performance of NK1R-/- mice and their wildtypes. After training, animals were tested using a long (LITI) and a variable (VITI) inter-trial interval: these tests were carried out with, and without, d-amphetamine pretreatment (0.3 or 1 mg/kg i.p.). NK1R-/- mice expressed greater omissions (inattentiveness), perseveration and premature responses (impulsivity) in the 5-CSRTT. In NK1R-/- mice, perseveration in the LITI was increased by injection-stress but reduced by d-amphetamine. Omissions by NK1R-/- mice in the VITI were unaffected by d-amphetamine, but premature responses were exacerbated by this psychostimulant. Omissions in the VITI were higher, overall, in the morning than the afternoon but, in the LITI, premature responses of NK1R-/- mice were higher in the afternoon than the morning. Conclusion In addition to locomotor hyperactivity, NK1R-/- mice express inattentiveness, perseveration and impulsivity in the 5-CSRTT, thereby matching core criteria for a model of ADHD. Because d-amphetamine reduced perseveration in NK1R-/- mice, this action does not require functional NK1R. However, the lack of any improvement of omissions and premature responses in NK1R-/- mice given d-amphetamine suggests that beneficial effects of this psychostimulant in other rodent models, and ADHD patients, need functional NK1R. Finally, our results reveal experimental variables (stimulus parameters, stress and time of day) that could influence translational studies

Expansion and Characterization of Human Melanoma Tumor-Infiltrating Lymphocytes (TILs)

Various immunotherapeutic strategies for cancer are aimed at augmenting the T cell response against tumor cells. Adoptive cell therapy (ACT), where T cells are manipulated ex vivo and subsequently re-infused in an autologous manner, has been performed using T cells from various sources. Some of the highest clinical response rates for metastatic melanoma have been reported in trials using tumor-infiltrating lymphocytes (TILs). These protocols still have room for improvement and furthermore are currently only performed at a limited number of institutions. The goal of this work was to develop TILs as a therapeutic product at our institution.TILs from 40 melanoma tissue specimens were expanded and characterized. Under optimized culture conditions, 72% of specimens yielded rapidly proliferating TILs as defined as at least one culture reaching ≥3×10(7) TILs within 4 weeks. Flow cytometric analyses showed that cultures were predominantly CD3+ T cells, with highly variable CD4+:CD8+ T cell ratios. In total, 148 independent bulk TIL cultures were assayed for tumor reactivity. Thirty-four percent (50/148) exhibited tumor reactivity based on IFN-γ production and/or cytotoxic activity. Thirteen percent (19/148) showed specific cytotoxic activity but not IFN-γ production and only 1% (2/148) showed specific IFN-γ production but not cytotoxic activity. Further expansion of TILs using a 14-day "rapid expansion protocol" (REP) is required to induce a 500- to 2000-fold expansion of TILs in order to generate sufficient numbers of cells for current ACT protocols. Thirty-eight consecutive test REPs were performed with an average 1865-fold expansion (+/- 1034-fold) after 14 days.TILs generally expanded efficiently and tumor reactivity could be detected in vitro. These preclinical data from melanoma TILs lay the groundwork for clinical trials of ACT

Nitrogen uptake and internal recycling in Zostera marina exposed to oyster farming: eelgrass potential as a natural biofilter

Oyster farming in estuaries and coastal lagoons frequently overlaps with the distribution of seagrass meadows, yet there are few studies on how this aquaculture practice affects seagrass physiology. We compared in situ nitrogen uptake and the productivity of Zostera marina shoots growing near off-bottom longlines and at a site not affected by oyster farming in San Quintin Bay, a coastal lagoon in Baja California, Mexico. We used benthic chambers to measure leaf NH4 (+) uptake capacities by pulse labeling with (NH4)-N-15 (+) and plant photosynthesis and respiration. The internal N-15 resorption/recycling was measured in shoots 2 weeks after incubations. The natural isotopic composition of eelgrass tissues and vegetative descriptors were also examined. Plants growing at the oyster farming site showed a higher leaf NH4 (+) uptake rate (33.1 mmol NH4 (+) m(-2) day(-1)) relative to those not exposed to oyster cultures (25.6 mmol NH4 (+) m(-2) day(-1)). We calculated that an eelgrass meadow of 15-16 ha (which represents only about 3-4 % of the subtidal eelgrass meadow cover in the western arm of the lagoon) can potentially incorporate the total amount of NH4 (+) excreted by oysters (similar to 5.2 x 10(6) mmol NH4 (+) day(-1)). This highlights the potential of eelgrass to act as a natural biofilter for the NH4 (+) produced by oyster farming. Shoots exposed to oysters were more efficient in re-utilizing the internal N-15 into the growth of new leaf tissues or to translocate it to belowground tissues. Photosynthetic rates were greater in shoots exposed to oysters, which is consistent with higher NH4 (+) uptake and less negative delta C-13 values. Vegetative production (shoot size, leaf growth) was also higher in these shoots. Aboveground/belowground biomass ratio was lower in eelgrass beds not directly influenced by oyster farms, likely related to the higher investment in belowground biomass to incorporate sedimentary nutrients

A Bayesian Model for Detection of Highorder Interactions Among Genetic Variants in Genome-Wide Association Studies

Background: A central question for disease studies and crop improvements is how genetics variants drive phenotypes. Genome Wide Association Study (GWAS) provides a powerful tool for characterizing the genotypephenotype relationships in complex traits and diseases. Epistasis (gene-gene interaction), including high-order interaction among more than two genes, often plays important roles in complex traits and diseases, but current GWAS analysis usually just focuses on additive effects of single nucleotide polymorphisms (SNPs). The lack of effective computational modelling of high-order functional interactions often leads to significant under-utilization of GWAS data. Results: We have developed a novel Bayesian computational method with a Markov Chain Monte Carlo (MCMC) search, and implemented the method as a Bayesian High-order Interaction Toolkit (BHIT) for detecting epistatic interactions among SNPs. BHIT first builds a Bayesian model on both continuous data and discrete data, which is capable of detecting high-order interactions in SNPs related to case—control or quantitative phenotypes. We also developed a pipeline that enables users to apply BHIT on different species in different use cases. Conclusions: Using both simulation data and soybean nutritional seed composition studies on oil content and protein content, BHIT effectively detected some high-order interactions associated with phenotypes, and it outperformed a number of other available tools. BHIT is freely available for academic users at http://digbio.missouri.edu/BHIT/

Hox10 Genes Function in Kidney Development in the Differentiation and Integration of the Cortical Stroma

Organogenesis requires the differentiation and integration of distinct populations of cells to form a functional organ. In the kidney, reciprocal interactions between the ureter and the nephrogenic mesenchyme are required for organ formation. Additionally, the differentiation and integration of stromal cells are also necessary for the proper development of this organ. Much remains to be understood regarding the origin of cortical stromal cells and the pathways involved in their formation and function. By generating triple mutants in the Hox10 paralogous group genes, we demonstrate that Hox10 genes play a critical role in the developing kidney. Careful examination of control kidneys show that Foxd1-expressing stromal precursor cells are first observed in a cap-like pattern anterior to the metanephric mesenchyme and these cells subsequently integrate posteriorly into the kidney periphery as development proceeds. While the initial cap-like pattern of Foxd1-expressing cortical stromal cells is unaffected in Hox10 mutants, these cells fail to become properly integrated into the kidney, and do not differentiate to form the kidney capsule. Consistent with loss of cortical stromal cell function, Hox10 mutant kidneys display reduced and aberrant ureter branching, decreased nephrogenesis. These data therefore provide critical novel insights into the cellular and genetic mechanisms governing cortical cell development during kidney organogenesis. These results, combined with previous evidence demonstrating that Hox11 genes are necessary for patterning the metanephric mesenchyme, support a model whereby distinct populations in the nephrogenic cord are regulated by unique Hox codes, and that differential Hox function along the AP axis of the nephrogenic cord is critical for the differentiation and integration of these cell types during kidney organogenesis