Early dynamics of photosynthetic Lhcf2 and Lhcf15 transcription and mRNA stabilities in response to herbivory-related decadienal in Phaeodactylum tricornutum.

Abiotic and biotic stresses widely reduce light harvesting complex (LHC) gene expression in higher plants and algae. However, control mechanisms and functions of these changes are not well understood. During herbivory, marine diatom species release oxylipins that impair grazer reproduction and serve as signaling molecules to nearby undamaged diatoms. To examine LHC mRNA regulation by oxylipin exposure, the diatom Phaeodactylum tricornutum was treated with a sublethal concentration of trans,trans-2,4-decadienal (DD) during the light cycle. Transcriptome analyses revealed extensive suppression of LHC mRNAs and a smaller set of up-regulated LHC mRNAs at 3 h. For two divergently regulated LHCF antennae family mRNAs, in vivo 4-thiouracil metabolic labeling was used to distinguish synthesis and degradation rates. Within 3 h of DD exposure, Lhcf2 mRNA levels and transcription were strongly suppressed and its mRNA half-life decreased. In contrast, Lhcf15 mRNA mainly accumulated between 3-9 h, its transcription increased and its mRNA was highly stabilized. Hence, DD-treated cells utilized transcriptional and mRNA stability control mechanisms which were likely major factors in the differing Lhcf2 and Lhcf15 expression patterns. Widespread LHC mRNA regulation and possible effects on photosynthesis may contribute to enhanced fitness in cells impacted by herbivory and other stresses.

Current topics in red cell biology: report on the Red Cell Special Interest Group meeting held at NHS Blood and Transplant Bristol on 30 October 2015.

The Red Cell Special Interest Group (SIG) meeting, hosted by the British Blood Transfusion Society, provides an annual forum for the presentation of UK- and European-based red cell research. The 2015 meeting was held on Friday 30 October at the National Health Service Blood & Transplant (NHSBT) facility in Filton, Bristol and provided an exciting and varied programme on the themes of erythropoiesis, malaria biology and pathophysiology and red cells properties in stress and disease. Ten speakers presented on these topics over the course of one day. The meeting was well attended by over 90 delegates. Posters were presented during the lunch break, and abstracts from the posters are published at the end of this issue.

Multiple stages of information processing are modulated during acute bouts of exercise.

Acute bouts of aerobic physical exercise can modulate subsequent cognitive task performance and oscillatory brain activity measured with electroencephalography (EEG). Here, we investigated the sequencing of these modulations of perceptual and cognitive processes using scalp recorded EEG acquired during exercise. Twelve participants viewed pseudo-random sequences of frequent non-target stimuli (cars), infrequent distractors (obliquely oriented faces) and infrequent targets that required a simple detection response (obliquely oriented faces, where the angle was different than the infrequent distractors). The sequences were presented while seated on a stationary bike under three conditions during which scalp recorded EEG was also acquired: rest, low-intensity exercise, and high-intensity exercise. Behavioral target detection was faster during high-intensity exercise compared to both rest and low-intensity exercise. An event-related potential (ERP) analysis of the EEG data revealed that the mean amplitude of the visual P1 component evoked by frequent non-targets measured at parietal-occipital electrodes was larger during low-intensity exercise compared to rest. The P1 component evoked by infrequent targets also peaked earlier during low-intensity exercise compared to rest and high-intensity exercise. The P3a ERP component evoked by infrequent distractors measured at parietal electrodes peaked significantly earlier during both low- and high-intensity exercise when compared to rest. The modulation of the visual P1 and the later P3a components is consistent with the conclusion that exercise modulates multiple stages of neural information processing, ranging from early stage sensory processing (P1) to post-perceptual target categorization (P3a).

Experience with a third generation recombinant factor VIII concentrate (Advate) for immune tolerance induction in patients with haemophilia A.

The development of an inhibitor represents one of the most challenging complications in patients with haemophilia A. Optimal management is immune tolerance induction (ITI), typically through the administration of high doses of factor VIII (FVIII) concentrate. Among 12 patients who underwent ITI using Advate, a third-generation recombinant FVIII product that is free of animal and human protein additives, tolerance was achieved in nine (75%), including seven of 10 patients (70%) with high-titre inhibitors. ITI is ongoing in two patients and not yet successful; immune tolerance failed in the third patient. The median time to success was 4.0 months for group as a whole and for patients with high-titre inhibitors. Treatment was well tolerated, and no adverse events were observed. Advate was found to be equivalent to other FVIII products with regard to both ITI success rates and the incidence of adverse effects when used in these immune tolerance regimens.

Walter Heiligenberg: the jamming avoidance response and beyond.

Walter Heiligenberg (1938-1994) was an exceptionally gifted behavioral physiologist who made enormous contributions to the analysis of behavior and to our understanding of how the brain initiates and controls species-typical behavioral patterns. He was distinguished by his rigorous analytical approach used in both behavioral studies and neuroethological investigations. Among his most significant contributions to neuroethology are a detailed analysis of the computational rules governing the jamming avoidance response in weakly electric fish and the elucidation of the principal neural pathway involved in neural control of this behavior. Based on his work, the jamming avoidance response is perhaps the best-understood vertebrate behavior pattern in terms of the underlying neural substrate. In addition to this pioneering work, Heiligenberg stimulated research in a significant number of other areas of ethology and neuroethology, including: the quantitative assessment of aggressivity in cichlid fish; the ethological analysis of the stimulus-response relationship in the chirping behavior of crickets; the exploration of the neural and endocrine basis of communicatory behavior in weakly electric fish; the study of cellular mechanisms of neuronal plasticity in the adult fish brain; and the phylogenetic analysis of electric fishes using a combination of morphology, electrophysiology, and mitochondrial sequence data.

Are the electroencephalograms mainly rhythmic? Assessment of periodicity in wide-band time series.

To test the hypotheses that (i). electroencephalograms (EEGs) are largely made up of oscillations at many frequencies and (ii). that the peaks in the power spectra represent oscillations, we applied a new method, called the period specific average (PSA) to a wide sample of EEGs. Both hypotheses can be rejected. Although the principal peaks in the two spectra agree most of the time, quite often a peak in the power spectrum accompanies no periodicity peak and some periodicity peaks have no power spectral peak. The Fourier spectrum is not a reliable indication of rhythms. EEG samples from patients during waking, sleeping and seizure states, and volunteer healthy subjects doing cognitive tasks quite often show no significant rhythms, on an arbitrary, common sense definition. When clear rhythms are seen, they involve one or two, rarely up to four or five simultaneous non-harmonically related frequencies. Rhythms are special cases; most of the power spectrum most of the time is nonrhythmic. "Good" rhythms usually have quite narrow peaks, with frequency modulation of <5%, strengths of >2.5 up to >10 times the expectation from chance, and they often show fine structure by being quite local and brief. Most rhythms are quasisinusoidal but others are sharp-cornered recurrent events with <50% duty cycle. In the face of wide variability, we do not report any systematic differences in periodicity among EEGs from different parts of the brain or different brain states or species; it will take many more exemplars of each state, species or brain part to establish characteristic features. The PSA method may be the best so far proposed to demonstrate and quantify periodicity in wide-band time series with noise, but it has serious limitations. Discussion leads to the conclusion that it is time for a new paradigm or metaphor for brain waves.

Manipulation of avidity to improve effectiveness of adoptively transferred CD8(+) T cells for melanoma immunotherapy in human MHC class I-transgenic mice.

The adoptive transfer of tumor-reactive CD8(+) T cells into tumor-bearing hosts provides an attractive alternative to vaccination-based active immunotherapy of melanoma. The development of techniques that result in the preferential expansion of tumor-reactive T cells is therefore of great importance. In this study, we report the generation of HLA-A*0201-restricted CD8(+) T cell populations that recognize either tyrosinase(369-376) or gp100(209-217) from tolerant human class I MHC-transgenic mice by using single amino acid-substituted variant peptides. Low peptide concentration or restimulation with the parent peptide was used to enhance the functional avidity, defined by stimulation of IFN-gamma accumulation, and cross-reactivity of the resulting T cell populations. We found a direct correlation between the ability of a T cell population to respond in vitro to low concentrations of the precise peptide expressed on the tumor and its ability to delay the outgrowth of B16 melanoma after adoptive transfer. Surprisingly, we found that some T cells that exhibited high functional avidity and were effective in controlling tumor outgrowth exhibited low structural avidity, as judged by MHC-tetramer staining. Our results establish strategies for the development and selection of CD8(+) T cell populations that persist despite peripheral tolerance, and that can control melanoma outgrowth. Furthermore, they support the use of human MHC class I-transgenic mice as a preclinical model for developing effective immunotherapies that can be rapidly extended into therapeutic settings.

Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization.

Preparation of large quantities of RNA molecules of a defined sequence is a prerequisite for biophysical analysis, and is particularly important to the determination of high-resolution structure by X-ray crystallography. We describe improved methods for the production of multimilligram quantities of homogeneous tRNAs, using a combination of chemical synthesis and enzymatic approaches. Transfer RNA half-molecules with a break in the anticodon loop were chemically synthesized on a preparative scale, ligated enzymatically, and cocrystallized with an aminoacyl-tRNA synthetase, yielding crystals diffracting to 2.4 A resolution. Multimilligram quantities of tRNAs with greatly reduced 3' heterogeneity were also produced via transcription by T7 RNA polymerase, utilizing chemically modified DNA half-molecule templates. This latter approach eliminates the need for large-scale plasmid preparations, and yields synthetase cocrystals diffracting to 2.3 A resolution at much lower RNA:protein stoichiometries than previously required. These two approaches developed for a tRNA-synthetase complex permit the detailed structural study of "atomic-group" mutants.

Immune responses to the HLA-A*0201-restricted epitopes of tyrosinase and glycoprotein 100 enable control of melanoma outgrowth in HLA-A*0201-transgenic mice.

Many of the Ags recognized by human melanoma-reactive CTL are derived from proteins that are also expressed in melanocytes. The possibility of self-tolerance to these epitopes has led to questions about their utility for antitumor immunotherapy. To investigate the issue, we established a preclinical model based on transgenic mice expressing a recombinant HLA-A*0201 molecule and B16 melanoma transfected to express this molecule. HLA-A*0201-restricted epitopes from the melanocyte differentiation proteins (MDP) tyrosinase and gp100 are expressed in both tumor cells and melanocytes, and the former is associated with self-tolerance. However, adoptive transfer of tyrosinase or gp100-reactive CTL developed from tolerant mice delayed tumor outgrowth, as did immunization with MDP peptide-pulsed dendritic cells. Protection was enhanced by the use of peptide ligands containing conservative substitutions that were cross-reactive with the original Ags. These data establish that CTL populations reactive against MDP-derived self-Ags can be activated to mount effective antitumor immunity and strongly support their continued development for tumor immunotherapy in humans.

Event-related potentials in an invertebrate: crayfish emit 'omitted stimulus potentials'.

Electrical signs of neural activity correlated with stimuli or states include a subclass called event-related potentials. These overlap with, but can often be distinguished from, simple stimulus-bound evoked potentials by their greater dependence on endogenous (internal state) factors. Studied mainly in humans, where they are commonly associated with cognition, they are considered to represent objective signs of moderately high-level brain processing. We tested the hypothesis that invertebrates lack such signs by looking in the crayfish Procambarus clarkii for a class of OFF-effects shown in humans to index expectancy. Disproving the hypothesis, we find, using chronic, implanted preparations, that a good omitted stimulus potential is reliably present. The system learns in a few cycles of a regularly repeated light flash to expect one on schedule. Omitted stimulus potentials are found in the protocerebrum, the circumesophageal connective and in the optic tract - perhaps arising in the retina, as in vertebrates. These potentials can be very local and can include loci with and without direct visual evoked potentials in response to each flash. In some loci, the omitted stimulus potential has a slow wave component, in others only a spike burst. Omitted stimulus potentials are more endogenous than visual evoked potentials, with little dependence on flash or ambient light intensity or on train duration. They vary little in size at different times of the day, but abruptly fail to appear if the ambient light is cut off. They can occur during walking, eating or the maintained defense posture but are diminished by 'distraction' and are often absent from an inert crayfish until it is aroused. We consider this form of apparent expectation of a learned rhythm (a property that makes it 'cognitive' in current usage), to be one of low level, even though some properties suggest endogenous factors. The flashes in a train have an inhibitory effect on a circuit that quickly 'learns' the stimulus interval so that the omitted stimulus potential, ready to happen after the learned interval, is prevented by each flash, until released by a missing stimulus.

Revisiting the concept of identifiable neurons.

Although eutely in nematodes was known, giant neurons in several taxa and unique motor neurons to leg muscles in decapod crustaceans, the idea that many animals have many identifiable neurons with relatively consistent dynamical properties and connections was only slowly established in the late 1960s and early 1970s. This has to be one of the important quiet revolutions in neurobiology. It stimulated a vast acquisition of specific information and led to some euphoria in the degree and pace of understanding activity of nervous systems and consequent behavior in terms of neuronal connections and properties. Some implications, problems and opportunities for new discovery are developed. The distribution of identifiable neurons among taxa and parts of the nervous system is not yet satisfactorily known. Their evolution may have been a case of several independent inventions. The degree of consistency has been quantified only in a few examples and the plasticity is little known. Identified neurons imply identifiable circuits but whether this extends to discrete systems, functionally definable, seems likely to have several answers in different animals or sites. Very limited attempts have been made to extend the concept to cases of two or ten or a hundred fully equivalent neurons, on all kinds of criteria. These attempts suggest a much smaller redundancy and vaster number of types of neurons than hitherto believed. Theory as well as empirical information has not yet interpreted the range of systems from those with small sets of relatively reliable neurons to those with large numbers of parallel, partially redundant units. The now classical notion of local circuits has to be extended to take account of and find roles for the plethora of integrative variables, of evidence for neural processing independent of spikes and classical synapses, of spatial configurations of terminal arbors and dendritic geometry, of modulators and transmitters, degrees of rhythmicity (regularity varying several orders of magnitude), and of synchrony. Adequate language and models need to go beyond 'circuits' in any engineering sense. Identifiable neurons can contribute to a broad spectrum of issues in neurobiology.

Odor input generates approximately 1.5 Hz and approximately 3 Hz spectral peaks in the helix pedal ganglion.

In 1999 we reported that odorants evoke in the Helix pedal ganglion (PG) activity patterns which are largely odorant-specific and related to the nature of odor and its behavioral output. Notably, some activities (for example, approximately 1.5 and approximately 3 Hz), nonspecific to odorants, were consistently evoked in PG. The present contribution goes farther in a deeper survey of the intrinsic and odorant-evoked activities of PG with special weight on the nonspecific fluctuations. We address the following questions. (i) What are the features of the activities? (ii) Are they comparable to the activities found in the motor systems of the other invertebrates? (iii) To what functions can they be related? Three main frequency components represented by power peaks at <1 Hz, 1-2 Hz and 2-8 Hz seem to feature the response activities of PG. (a) The aversive odorants induce odorant-specific patterns represented by peak power frequencies at <1 Hz. (b) The oscillation at approximately 1 Hz, which exists intrinsically in the Helix PG, can be specifically enhanced by appetitive odors. Activities induced in the procerebrum (PC), the visceral ganglion (VG) and PG by appetitive odorants, such as ethanol and apple, peak at 1.3-2 Hz, whereas those induced by aversive ones, such as formic acid and onion at <1 Hz. (c) The 2-8 Hz components always accompany the odorant-evoked activities of the PG either as the second or third strongest component, or in the form of conspicuous, long-lasting approximately 3 Hz oscillations. (d) The nonspecific odor-evoked 1-2 Hz and approximately 3 Hz activities, and the intrinsic approximately 1 Hz activity of the PG seem to be interrelated by a degree of mutual exclusion. We may therefore consider these activities as elementary, slow components that are involved in the processing of signals in this ganglion. It can be inferred from the findings in other invertebrates that the 1-3 Hz spontaneous discharge is strongly connected with motor activity that involves the feedback mechanism of the procerebro-cerebro-buccal or -procerebro-cerebro-pedal circuit. Our approach differs from most others reported so far in the following aspects: (i) use of gross steel electrodes for recording population activities; (ii) lengthy stimulation (10 min); (iii) long observation during and after stimulation; (iv) power spectral presentation of temporal evolution of activity patterns; (v) estimation of peak power frequency by Frequency-Amplitude Plot (FAP) (obtained from signals averaged in the frequency domain; a method based on systems theory).

Tertiary core rearrangements in a tight binding transfer RNA aptamer.

Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5'-44CAUUC48-3' is replaced by 5'-44AGGU48-3'. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 A cocrystal structure of the RNA aptamer-GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA-protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15-U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA-protein contacts.

Direct identification of human tumor-associated peptide antigens and a preclinical model to evaluate their use.

Although the arsenal of a healthy immune system includes both circulating antibodies and cellular components such as T cells, the latter seem to be particularly important in tumor immunology. Under normal conditions, the immune system does not react to the body's cells, which may be described as expressing "self" antigens on the cell surface. When a cell becomes cancerous, however, novel antigens are expressed on the cell surface. These novel "tumor" antigens are recognized as foreign by the body's immune system, and the cells that express them are destroyed or incapacitated. Whereas antibodies may react directly with protein antigens, T cells instead recognize peptide antigens presented by class I and class II molecules of the major histocompatibility complex (MHC). All cells normally break down proteins that they have made. The class I antigen-processing pathway has evolved to display peptides produced by this breakdown process as a way to provide information to cytotoxic T cells about what the cell is making. The display of new peptides as a result of infection or transformation can stimulate cytotoxic T cells to kill the cell. In addition, antigen-processing cells such as dendritic cells engulf dead or dying cells and degradeproteins into peptide fragments. These peptides are then displayed by the MHC class II molecules and presented to T helper cells, which augment the activity of the cytotoxic T cells. Cytotoxic T lymphocytes have recently been isolated from human tumors (especially melanoma) and are critical to the development of promising immunotherapeutic agents. As we shall discuss, these cells can recognize antigens that are common to tumors from different patients. We shall also explore how advances in instrumentation and the use of transgenic mice have increased our understanding of tumor-associated peptides to the point where we can begin to strive for a peptide-based therapeutic vaccine. The caveats for such therapy will also be addressed.

Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases.

The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops. E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair. To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48. Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15. The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases.

Self-tolerance to the murine homologue of a tyrosinase-derived melanoma antigen: implications for tumor immunotherapy.

The human tyrosinase-derived peptide YMDGTMSQV is presented on the surface of human histocompatibility leukocyte antigen (HLA)-A*0201(+) melanomas and has been suggested to be a tumor antigen despite the fact that tyrosinase is also expressed in melanocytes. To gain information about immunoreactivity and self-tolerance to this antigen, we established a model using the murine tyrosinase-derived homologue of this peptide FMDGTMSQV, together with transgenic mice expressing the HLA-A*0201 recombinant molecule AAD. The murine peptide was processed and presented by AAD similarly to its human counterpart. After immunization with recombinant vaccinia virus encoding murine tyrosinase, we detected a robust AAD-restricted cytotoxic T lymphocyte (CTL) response to FMDGTMSQV in AAD transgenic mice in which the entire tyrosinase gene had been deleted by a radiation-induced mutation. A residual response was observed in the AAD(+)tyrosinase(+) mice after activation under certain conditions. At least some of these residual CTLs in AAD(+)tyrosinase(+) mice were of high avidity and induced vitiligo upon adoptive transfer into AAD(+)tyrosinase(+) hosts. Collectively, these data suggest that FMDGTMSQV is naturally processed and presented in vivo, and that this presentation leads to substantial but incomplete self-tolerance. The relevance of this model to an understanding of the human immune response to tyrosinase is discussed.