Lutein can act as a switchable charge transfer quencher in the CP26 light-harvesting complex

Energy-dependent quenching of excitons in photosystem II of plants, or qE, has been positively correlated with the transient production of carotenoid radical cation species. Zeaxanthin was shown to be the donor species in the CP29 antenna complex. We report transient absorbance analyses of CP24 and CP26 complexes that bind lutein and zeaxanthin in the L1 and L2 domains, respectively. For CP24 complexes, the transient absorbance difference profiles give a reconstructed transient absorbance spectrum with a single peak centered at approximately 980 nm, consistent with zeaxanthin radical cation formation. In contrast, CP26 gives constants for the decay components probed at 940 and 980 nm of 144 and 194 ps, a transient absorbance spectrum that has a main peak at 980 nm, and a substantial shoulder at 940 nm. This suggests the presence of two charge transfer quenching sites in CP26 involving zeaxanthin radical cation and lutein radical cation species. We also show that lutein radical cation formation in CP26 is dependent on binding of zeaxanthin to the L2 domain, implying that zeaxanthin acts as an allosteric effector of charge transfer quenching involving lutein in the L1 domain.     

Semliki Forest Virus Expressing Interleukin-12 Induces Antiviral and Antitumoral Responses in Woodchucks with Chronic Viral Hepatitis and Hepatocellular Carcinoma

A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with ≥1.5-log₁₀ reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.Hepatocellular carcinoma (HCC) is a major public health problem worldwide, representing the fifth most common type of cancer. HCC is also the third leading cause of cancer-related death, mainly because only surgical and local ablative therapeutic options have shown efficacy in patients with this type of cancer (21). Approximately 80% of all HCC cases are attributed to chronic infection with hepatitis C virus and/or hepatitis B virus (HBV). Chronic carriers of HBV have a greater than 100-fold-increased relative risk of developing HCC compared to HBV-uninfected humans, with an annual incidence rate of 2 to 6% in cirrhotic patients. The high incidence of HCC, together with its poor prognosis and limited therapeutic options, warrants the development of new treatment strategies for this disease.There is increasing evidence that stimulation of the immune system for subsequent recognition and killing of tumor cells may be a valuable treatment option for liver cancer. In general, HCC appears to be an attractive target for immunotherapy because cases of spontaneous tumor regression have been reported, HCC is often infiltrated with lymphocytes, and HCC-associated proteins such as alpha-fetoprotein may be used as targets for immune-mediated killing of tumors (5, 49).A promising strategy to stimulate the deficient antitumoral immune response is based on the transfer and subsequent expression of immunostimulatory genes in tumor cells using viral or nonviral delivery vectors. One of the most effective immunostimulatory cytokines is interleukin-12 (IL-12), a protein usually expressed by macrophages and dendritic cells. IL-12 has been demonstrated to induce strong antitumoral effects that are mediated by the stimulation of T-helper cell type 1 (Th1) responses, including the activation of cytolytic T lymphocytes (CTL) and natural killer cells, and by the inhibition of angiognesis (48, 50). All of these effects are dependent on the production of gamma interferon (IFN-γ). Viral vectors that are based on adenovirus have been used to deliver IL-12 into several animal models with transplantable HCC, resulting in a localized expression of this cytokine and usually leading to antitumoral effects (3, 14, 37). However, and despite successful treatment of HCC in preclinical studies, a phase I clinical trial with a first-generation adenoviral vector for delivery and expression of IL-12 in patients with primary and metastatic liver cancer produced only a modest antitumoral effect (41). This poor response was probably due to the low and transient IL-12 expression in tumors. These results in humans indicated a need for vectors with higher potency and for preclinical testing in relevant models of HCC (i.e., large animals with spontaneous tumors).Vectors based on Semliki Forest virus (SFV), a member of the alphavirus group, are highly efficient in inducing antitumoral responses in a variety of animal models (2, 9, 10, 39, 44, 53). The SFV vector used in the present study is based on a viral RNA genome in which the region coding for the structural proteins has been replaced by a heterologous gene (24). Recombinant SFV RNA can be transcribed in vitro and transfected into cells, resulting in viral replication and subsequent production of a subgenomic RNA from which the heterologous protein is expressed at very high levels. Recombinant SFV RNA can be packaged into viral particles (vp) by cotransfecting it into cells together with two helper RNAs coding for the capsid and the envelope proteins (43). Compared to adenoviral vectors expressing IL-12, tumor treatment with SFV vectors expressing the same cytokine resulted in greater antitumoral effects in a murine colon adenocarcinoma model and also in a rat orthotopic HCC model (16, 39). The greater antitumoral effect mediated by SFV vectors has been attributed to the higher expression of IL-12 and to the induction of apoptosis caused by SFV replication within tumor cells. Apoptosis leads to the release of tumor antigens that can be taken up by antigen-presenting cells, thereby potentiating the antitumoral response induced by IL-12 (54). Furthermore, SFV vectors have low immunogenicity when delivered intratumorally, allowing repetitive administrations into the same tumor, which is not possible with adenoviral vectors (38).In the present study, the antitumoral efficacy of an SFV vector expressing IL-12 (SFV-enhIL-12) was investigated in woodchucks with HCC. The Eastern woodchuck (Marmota monax) is frequently infected with the woodchuck hepatitis virus (WHV), which is closely related to the human HBV in its structure, genomic organization, mechanism of replication, and course of infection (29). The woodchuck has been used as a mammalian model for research on HBV, including the pathogenesis of acute and chronic HBV infection, and for preclinical evaluation of the safety and efficacy of candidate antiviral drugs and therapeutic immunomodulators for the treatment of chronic HBV infection (29) and prevention of HCC (47).All woodchucks chronically infected with WHV as neonates develop HCC, and the median time for tumor appearance is 24 months of age (34, 47). After identification of HCC, the median survival time of woodchucks is 6 months, a situation similar to that for patients with HCC. In addition, WHV-induced hepatocarcinogenesis shows strong similarity to HBV-induced carcinogenesis in humans (34, 47). These features of HCC that are associated with persistent hepatitis virus infection make the woodchuck model unique compared to other animal models, in which HCC is induced by a chemical carcinogen or by transplantation of established tumor cell lines into immune-deficient or immune-compatible hosts. Woodchucks with large liver tumors that acquire malignant characteristics in a stepwise process similar to HCC in humans are an attractive and suitable model for the preclinical evaluation of new treatment strategies for HBV-induced HCC in humans (47).The antitumoral efficacy of a SFV vector expressing high levels of IL-12 (SFV-enhIL-12) was investigated in six woodchucks with established chronic WHV infection and primary HCC. The results demonstrate that SFV-delivered IL-12 expression produced a dose-dependent, partial tumor remission that was associated with a general activation of cellular immune responses against HCC. The antitumoral activity, in addition to an antiviral activity against WHV, and the favorable safety profile in woodchucks suggest that a therapeutic approach based on SFV-enhIL-12 may represent a treatment strategy for HCC in patients with chronic HBV infection, but the overall results also indicate that this approach needs further improvement for inducing a complete tumor remission.     

Occurrence of <Emphasis Type="Italic">TRGV-BJ</Emphasis> hybrid gene in SV40-transformed fibroblast cell lines

Illegitimate V(D)J-recombination in lymphoid malignancies involves rearrangements in immunoglobulin or T-cell receptor genes, and these rearrangements may play a role in oncogenic events. High frequencies of TRGV-BJ hybrid gene (rearrangement between the TRB and TRG loci at 7q35 and 7p14-15, respectively) have been detected in lymphocytes from patients with ataxia telangiectasia (AT), and also in patients with lymphoid malignancies. Although the TRGV-BJ gene has been described only in T-lymphocytes, we previously detected the presence of TRGV-BJ hybrid gene in the genomic DNA extracted from SV40-transformed AT5BIVA fibroblasts from an AT patient. Aiming to determine whether the AT phenotype or the SV40 transformation could be responsible for the production of the hybrid gene by illegitimate V(D)J-recombination, DNA samples were extracted from primary and SV40-transformed (normal and AT) cell lines, following Nested-PCR with TRGV- and TRBJ-specific primers. The hybrid gene was only detected in SV40-transformed fibroblasts (AT-5BIVA and MRC-5). Sequence alignment of the cloned PCR products using the BLAST program confirmed that the fragments corresponded to the TRGV-BJ hybrid gene. The present results indicate that the rearrangement can be produced in nonlymphoid cells, probably as a consequence of the genomic instability caused by the SV40-transformation, and independently of ATM gene mutation.     

Improper excess light energy dissipation in Arabidopsis results in a metabolic reprogramming

Background  

Plant performance is affected by the level of expression of PsbS, a key photoprotective protein involved in the process of feedback de-excitation (FDE), or the qE component of non-photochemical quenching, NPQ.     

Universal Artifacts Affect the Branching of Phylogenetic Trees,Not Universal Scaling Laws

Background

The superficial resemblance of phylogenetic trees to other branching structures allows searching for macroevolutionary patterns. However, such trees are just statistical inferences of particular historical events. Recent meta-analyses report finding regularities in the branching pattern of phylogenetic trees. But is this supported by evidence, or are such regularities just methodological artifacts? If so, is there any signal in a phylogeny?

Methodology

In order to evaluate the impact of polytomies and imbalance on tree shape, the distribution of all binary and polytomic trees of up to 7 taxa was assessed in tree-shape space. The relationship between the proportion of outgroups and the amount of imbalance introduced with them was assessed applying four different tree-building methods to 100 combinations from a set of 10 ingroup and 9 outgroup species, and performing covariance analyses. The relevance of this analysis was explored taking 61 published phylogenies, based on nucleic acid sequences and involving various taxa, taxonomic levels, and tree-building methods.

Principal Findings

All methods of phylogenetic inference are quite sensitive to the artifacts introduced by outgroups. However, published phylogenies appear to be subject to a rather effective, albeit rather intuitive control against such artifacts. The data and methods used to build phylogenetic trees are varied, so any meta-analysis is subject to pitfalls due to their uneven intrinsic merits, which translate into artifacts in tree shape. The binary branching pattern is an imposition of methods, and seldom reflects true relationships in intraspecific analyses, yielding artifactual polytomies in short trees. Above the species level, the departure of real trees from simplistic random models is caused at least by two natural factors –uneven speciation and extinction rates; and artifacts such as choice of taxa included in the analysis, and imbalance introduced by outgroups and basal paraphyletic taxa. This artifactual imbalance accounts for tree shape convergence of large trees.

Significance

There is no evidence for any universal scaling in the tree of life. Instead, there is a need for improved methods of tree analysis that can be used to discriminate the noise due to outgroups from the phylogenetic signal within the taxon of interest, and to evaluate realistic models of evolution, correcting the retrospective perspective and explicitly recognizing extinction as a driving force. Artifacts are pervasive, and can only be overcome through understanding the structure and biological meaning of phylogenetic trees.Catalan Abstract in Translation S1.     

Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks

Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.     

Consequences of Lineage-Specific Gene Loss on Functional Evolution of Surviving Paralogs: ALDH1A and Retinoic Acid Signaling in Vertebrate Genomes

Genome duplications increase genetic diversity and may facilitate the evolution of gene subfunctions. Little attention, however, has focused on the evolutionary impact of lineage-specific gene loss. Here, we show that identifying lineage-specific gene loss after genome duplication is important for understanding the evolution of gene subfunctions in surviving paralogs and for improving functional connectivity among human and model organism genomes. We examine the general principles of gene loss following duplication, coupled with expression analysis of the retinaldehyde dehydrogenase Aldh1a gene family during retinoic acid signaling in eye development as a case study. Humans have three ALDH1A genes, but teleosts have just one or two. We used comparative genomics and conserved syntenies to identify loss of ohnologs (paralogs derived from genome duplication) and to clarify uncertain phylogenies. Analysis showed that Aldh1a1 and Aldh1a2 form a clade that is sister to Aldh1a3-related genes. Genome comparisons showed secondarily loss of aldh1a1 in teleosts, revealing that Aldh1a1 is not a tetrapod innovation and that aldh1a3 was recently lost in medaka, making it the first known vertebrate with a single aldh1a gene. Interestingly, results revealed asymmetric distribution of surviving ohnologs between co-orthologous teleost chromosome segments, suggesting that local genome architecture can influence ohnolog survival. We propose a model that reconstructs the chromosomal history of the Aldh1a family in the ancestral vertebrate genome, coupled with the evolution of gene functions in surviving Aldh1a ohnologs after R1, R2, and R3 genome duplications. Results provide evidence for early subfunctionalization and late subfunction-partitioning and suggest a mechanistic model based on altered regulation leading to heterochronic gene expression to explain the acquisition or modification of subfunctions by surviving ohnologs that preserve unaltered ancestral developmental programs in the face of gene loss.     

The insulin polymorphism -23Hph increases the risk for type 1 diabetes mellitus in the Romanian population

The insulin -23Hph and IGF2 Apa polymorphisms were genotyped in Romanian patients with T1DM (n = 204), T2DM (n = 215) or obesity (n = 200) and normoponderal healthy subjects (n = 750). The genotypes of both polymorphisms were distributed in concordance with Hardy-Weinberg equilibrium in all groups. The -23Hph AA genotype increased the risk for T1DM (OR: 3.22, 95%CI: 2.09-4.98, p < 0,0001), especially in patients without macroalbuminuria (OR: 4.32, 95%CI: 2.54-7.45, p < 0,0001). No other significant association between the alleles or genotypes of insulin -23Hph and IGF2 Apa and diabetes or obesity was identified.     

Characterization of a membrane protein folding motif, the Ser zipper, using designed peptides

Polar residues play important roles in the association of transmembrane helices and the stabilities of membrane proteins. Although a single Ser residue in a transmembrane helix is unable to mediate a strong association of the helices, the cooperative interactions of two or more appropriately placed serine hydroxyl groups per helix has been hypothesized to allow formation of a "serine zipper" that can stabilize transmembrane helix association. In particular, a heptad repeat Sera Xxx Xxx Leud Xxx Xxx Xxx (Xxx is a hydrophobic amino acid) appears in both antiparallel helical pairs of polytopic membrane proteins as well as the parallel helical dimerization motif found in the murine erythropoietin receptor. To examine the intrinsic conformational preferences of this motif independent of its context within a larger protein, we synthesized a peptide containing three copies of a SeraLeud heptad motif. Computational results are consistent with the designed peptide adopting either a parallel or antiparallel structure, and conformational search calculations yield the parallel dimer as the lowest energy configuration, which is also significantly more stable than the parallel trimer. Analytical ultracentrifugation indicated that the peptide exists in a monomer-dimer equilibrium in dodecylphosphocholine micelles. Thiol disulfide interchange studies showed a preference for forming parallel dimers in micelles. In phospholipid vesicles, only the parallel dimer was formed. The stability of the SerZip peptide was studied in vesicles prepared from phosphatidylcholine (PC) lipids of different chain length: POPC (C16:0C18:1 PC) and DLPC (C12:0PC). The stability was greater in POPC, which has a good match between the length of the hydrophobic region of the peptide and the bilayer length. Finally, mutation to Ala of the Ser residues in the SerZip motif gave rise to a relatively small decrease in the stability of the dimer, indicating that packing interactions rather than hydrogen-bonding provided the primary driving force for association.