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A pericellular basket is a presynaptic configuration of numerous axonal boutons outlining a target neuron soma and its proximal dendrites. Recent studies show neurochemical diversity of pericellular baskets and suggest that neurotransmitter usage together with the dense, soma-proximal boutons may permit strong input effects on different time scales. Here we review the development, distribution, neurochemical phenotypes, and possible functions of pericellular baskets. As an example, we highlight pericellular baskets formed by projections of certain Pet1/Fev neurons of the serotonergic raphe nuclei. We propose that pericellular baskets represent convergence sites of competition or facilitation between neurotransmitter systems on downstream circuitry, especially in limbic brain regions, where pericellular baskets are widespread. Study of these baskets may enhance our understanding of monoamine regulation of memory, social behavior, and brain oscillations.

High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients’ leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future., Preclinical molecular models are useful that mimic a patient´s response to targeted therapy. Here, the authors establish an in vivo inducible RNAi-mediated gene silencing system in patient-derived xenograft models of acute leukemia to identify individual vulnerabilities and therapeutic targets.

Transposable elements (TEs) promote genetic innovation but also threaten genome stability. Despite multiple layers of host defence, TEs actively shape mammalian-specific developmental processes, particularly during pre-implantation and extra-embryonic development and at the maternal–fetal interface. Here, we review how TEs influence mammalian genomes both directly by providing the raw material for genetic change and indirectly via co-evolving TE-binding Kruppel-associated box zinc finger proteins (KRAB-ZFPs). Throughout mammalian evolution, individual activities of ancient TEs were co-opted to enable invasive placentation that characterizes live-born mammals. By contrast, the widespread activity of evolutionarily young TEs may reflect an ongoing co-evolution that continues to impact mammalian development. In this Review, Senft and Macfarlan discuss the diverse ways by which transposable elements (TEs) contribute to mammalian development and evolution, including direct contributions through TE-derived regulatory elements, RNAs and proteins, as well as indirect effects through the evolution of a TE repression system, the Kruppel-associated box zinc finger proteins (KRAB-ZFPs).

In mammals, the placenta mediates maternal–fetal nutrient and waste exchange and acts in an immunomodulatory way to facilitate maternal–fetal tolerance. The placenta is highly diverse across mammalian species, yet the molecular mechanisms that distinguish the placenta of human from other mammals are not fully understood. Using an interspecies transcriptomic comparison of human, macaque, and mouse late-gestation placentae, we identified hundreds of genes with lineage-specific expression—including dozens that are placentally enriched and potentially related to pregnancy. We further annotated the enhancers for different human tissues using epigenomic data and demonstrate that the placenta and chorion are unique in that their enhancers display the least conservation. We identified numerous lineage-specific human placental enhancers and found they highly overlap with specific families of endogenous retroviruses (ERVs), including MER21A, MER41A/B, and MER39B that were previously linked to immune response and placental function. Among these ERV families, we further demonstrate that MER41A/B insertions create dozens of lineage-specific serum response factor-binding loci in human, including one adjacent to FBN2, a placenta-specific gene with increased expression in humans that produces the peptide hormone placensin to stimulate glucose secretion and trophoblast invasion. Overall, our results demonstrate the prevalence of lineage-specific placental enhancers which are frequently associated with ERV insertions and likely facilitate the lineage-specific evolution of the mammalian placenta.

The visibility of cephalopod chromatophore organs is regulated dynamically by rosettes of obliquely striated radial muscles that dilate or relax the diameter of a central pigmented sacculus in 100-300 ms. Each of the several dozen muscles has a flared proximal end that adheres tightly to its pigmented sacculus and an extremely elongated distal end which branches into single fibrils that anchor into the dermis. This geometry provides ample opportunity for overlap of the many muscles from neighboring chromatophores. The temporal activity of these muscles has been believed to be patterned exclusively by monosynaptic projections from sets of efferent motor axons originating in the chromatophore lobes of the suboesophageal brain. Based on historical observations that distal radial muscles from some chromatophores appear to extend closely to muscles from other chromatophores, we asked whether radial muscles actually make specialized contacts. Using 3D electron microscopy of Doryteuthis pealeii mantle skin, we discovered tight putatively functional muscle-to-muscle contacts between radial muscles from different chromatophores, including elaborate sets of axonal processes located adjacent to those myo-myo junctions. These detailed ultrastructural findings demonstrate auxiliary anatomical routes for radial muscle activation and suggest plausible mechanisms whereby local physical synchronization and axo-axonic processing in the periphery can contribute to chromatophore pattern dynamics such as "passing cloud."

Brainstem median raphe (MR) neurons expressing the serotonergic regulator genePet1send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors. SomePet1neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 (Tph2) encoding the rate-limiting enzyme for serotonin [5-hydroxytryptamine (5-HT)] synthesis, but abundant for vesicular glutamate transporter type 3 (Vglut3) encoding a synaptic vesicle-associated glutamate transporter. Genetic fate maps show these nonclassical, putatively glutamatergicPet1neurons in the MR arise embryonically from the same progenitor cell compartment—hindbrain rhombomere 2 (r2)—as serotonergic TPH2+MRPet1neurons. Well established is the distribution of efferents en masse from r2-derived,Pet1-neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2-Pet1Tph2-highversus r2-Pet1Vglut3-high. Using male and female mice, we found r2-Pet1axonal boutons segregated anatomically largely by serotonin+versus VGLUT3+identity. The former present in the suprachiasmatic nucleus, paraventricular nucleus of the thalamus, and olfactory bulb; the latter are found in the hippocampus, cortex, and septum. Thus r2-Pet1Tph2-highand r2-Pet1Vglut3-highneurons likely regulate distinct brain regions and behaviors. Some r2-Pet1boutons encased interneuron somata, forming specialized presynaptic “baskets” of VGLUT3+or VGLUT3+/5-HT+identity; this suggests that some r2-Pet1Vglut3-highneurons may regulate local networks, perhaps with differential kinetics via glutamate versus serotonin signaling. Fibers from otherPet1neurons (non-r2-derived) were observed in many of these same baskets, suggesting multifaceted regulation. Collectively, these findings inform brain organization and new circuit nodes for therapeutic considerations.SIGNIFICANCE STATEMENTOur findings match axonal bouton neurochemical identity with distant cell bodies in the brainstem raphe. The results are significant because they suggest that disparate neuronal subsystems derive fromPet1+precursor cells of the embryonic progenitor compartment rhombomere 2 (r2). Of these r2-Pet1neuronal subsystems, one appears largely serotonergic, as expected given expression of the serotonergic regulator PET1, and projects to the olfactory bulb, thalamus, and suprachiasmatic nucleus. Another expresses VGLUT3, suggesting principally glutamate transmission, and projects to the hippocampus, septum, and cortex. Some r2-Pet1boutons—those that are VGLUT3+or VGLUT3+/5-HT+co-positive—comprise “baskets” encasing interneurons, suggesting that they control local networks perhaps with differential kinetics via glutamate versus serotonin signaling. Results inform brain organization and circuit nodes for therapeutic consideration.

Aims Changes in metabolism are known to contribute to tumour phenotypes. If and how metabolic alterations in brain tumours contribute to patient outcome is still poorly understood. Epigenetics impact metabolism and mitochondrial function. The aim of this study is a characterisation of metabolic features in molecular subgroups of isocitrate dehydrogenase mutant (IDHmut) and isocitrate dehydrogenase wildtype (IDHwt) gliomas. Methods We employed DNA methylation pattern analyses with a special focus on metabolic genes, large-scale metabolism panel immunohistochemistry (IHC), qPCR-based determination of mitochondrial DNA copy number and immune cell content using IHC and deconvolution of DNA methylation data. We analysed molecularly characterised gliomas (n = 57) for in depth DNA methylation, a cohort of primary and recurrent gliomas (n = 22) for mitochondrial copy number and validated these results in a large glioma cohort (n = 293). Finally, we investigated the potential of metabolic markers in Bevacizumab (Bev)-treated gliomas (n = 29). Results DNA methylation patterns of metabolic genes successfully distinguished the molecular subtypes of IDHmut and IDHwt gliomas. Promoter methylation of lactate dehydrogenase A negatively correlated with protein expression and was associated with IDHmut gliomas. Mitochondrial DNA copy number was increased in IDHmut tumours and did not change in recurrent tumours. Hierarchical clustering based on metabolism panel IHC revealed distinct subclasses of IDHmut and IDHwt gliomas with an impact on patient outcome. Further quantification of these markers allowed for the prediction of survival under anti-angiogenic therapy. Conclusion A mitochondrial signature was associated with increased survival in all analyses, which could indicate tumour subgroups with specific metabolic vulnerabilities.

Forisomes are large mechanoprotein complexes found solely in legumes such as Medicago truncatula. They comprise several "sieve element occlusion by forisome" (SEO-F) subunits, with MtSEO-F1 as the major structure-forming component. SEO-F proteins possess three conserved domains -an N-terminal domain (SEO-NTD), a potential thioredoxin fold, and a C-terminal domain (SEO-CTD)- but structural and biochemical data are scarce and little is known about the contribution of these domains to forisome assembly. To identify key amino acids involved in MtSEO-F1 dimerization and complex formation, we investigated protein-protein interactions by bimolecular fluorescence complementation and the analysis of yeast two-hybrid and random mutagenesis libraries. We identified a SEO-NTD core region as the major dimerization site, with abundant hydrophobic residues and rare charged residues suggesting dimerization is driven by the hydrophobic effect. We also found that ~45% of the full-length MtSEO-F1 sequence must be conserved for higher-order protein assembly, indicating that large interaction surfaces facilitate stable interactions, contributing to the high resilience of forisome bodies. Interestingly, the removal of 62 amino acids from the C-terminus did not disrupt forisome assembly. This is the first study unraveling interaction sites and mechanisms within the MtSEO-F1 protein at the level of dimerization and complex formation.

Two gold(III) complexes, square-planar [Au(DPP)Cl2]+(1) and distorted square-pyramidal [Au(DMP)Cl3] (2) (where DMP = 2,9-dimethyl-1,10-phenanthroline and DPP = 4,7-diphenyl-1,10-phenanthroline), were studied by different experimental methods. Their stability in water and in buffer solution (25 mM Hepes, 30 mM NaCl, pH = 7.2) was investigated by UV-Vis spectroscopy while their redox stability is confirmed by CV. Substitution reactions between complexes 1 and 2, and biologically relevant ligands, such as thiourea (Tu), guanosine-5′-monophosphate (5′-GMP), glutathione (GSH) and L-methionine (L-Met), were studied by a stopped-flow technique, under the pseudo-first order conditions as a function of ligand concentration and temperature. According to the values of the activation parameters, all studied reactions followed an associative substitution mechanism. DNA binding studies of complexes 1 and 2 were performed by UV-Vis and fluorescence spectroscopy and viscosity measurements, as well as interactions with bovine serum albumin (BSA). Density functional theory (DFT) was implemented in order to analyse the wave function of the optimized structures to get better insight into the binding interactions between the inert ligands and gold(III) center. The experimental results of binding studies with DNA and BSA were simulated and compared by performing a molecular docking study. All results demonstrate the strong connection between the reactivity of the complexes toward biologically important targets and their structural and electronic characteristics. The cytotoxic activity of complexes 1 and 2 against different cell lines (MDA-MB-231, HCT-116, and HaCaT) was evaluated 24 and 72 h after treatments. The results indicate reduced viability of cell lines in a time- and dose-dependent manner.

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.

1.Plants exhibit impressive genetic and chemical diversity, not just between species but also within species, and the importance of plant intraspecific variation for structuring ecological communities is well known. When there is variation at the local population level, this can create a spatially-heterogeneous habitat for specialized herbivores potentially leading to non-random distribution of individuals across host-plants. 2.Plant variation can affect herbivores directly and indirectly via a third species, resulting in variable herbivore growth rates across different host plants. Herbivores also exhibit within-species variation, with some genotypes better adapted to some plant variants than others. 3.We genotyped aphids collected across two years from a field site containing ~200 patchily-distributed host plants that exhibit high chemical diversity. The distribution of aphid genotypes, their ant mutualists, and other predators was assessed across the plants. 4.We present evidence that the local distribution of aphid (Metopeurum fuscoviride) genotypes across host-plant individuals is associated with variation in the plant volatiles (chemotypes) and non-volatile metabolites (metabotypes) of their host plant tansy (Tanacetum vulgare). Furthermore, these interactions in the field were influenced by plant-host preferences of aphid-mutualist ants. 5.Our results emphasize that plant intraspecific variation can structure ecological communities not only at the species level but also at the genetic level within species, and that this effect can be enhanced through indirect interactions with a third species.

Reactivity assays previously suggested that two quinol-containing MRI contrast agent sensors for H2O2, [Mn(H2qp1)(MeCN)]2+ and [Mn(H4qp2)Br2], could also catalytically degrade superoxide. Subsequently, [Zn(H2qp1)(OTf)]+ was found to use the redox activity of the H2qp1 ligand to catalyze the conversion of O2˙− to O2 and H2O2, raising the possibility that the organic ligand, rather than the metal, could serve as the redox partner for O2˙− in the manganese chemistry. Here, we use stopped-flow kinetics and cryospray-ionization mass spectrometry (CSI-MS) analysis of the direct reactions between the manganese-containing contrast agents and O2˙− to confirm the activity and elucidate the catalytic mechanism. The obtained data are consistent with the operation of multiple parallel catalytic cycles, with both the quinol groups and manganese cycling through different oxidation states during the reactions with superoxide. The choice of ligand impacts the overall charges of the intermediates and allows us to visualize complementary sets of intermediates within the catalytic cycles using CSI-MS. With the diquinolic H4qp2, we detect Mn(iii)-superoxo intermediates with both reduced and oxidized forms of the ligand, a Mn(iii)-hydroperoxo compound, and what is formally a Mn(iv)-oxo species with the monoquinolate/mono-para-quinone form of H4qp2. With the monoquinolic H2qp1, we observe a Mn(ii)-superoxo ↔ Mn(iii)-peroxo intermediate with the oxidized para-quinone form of the ligand. The observation of these species suggests inner-sphere mechanisms for O2˙− oxidation and reduction that include both the ligand and manganese as redox partners. The higher positive charges of the complexes with the reduced and oxidized forms of H2qp1 compared to those with related forms of H4qp2 result in higher catalytic activity (kcat ∼ 108 M−1 s−1 at pH 7.4) that rivals those of the most active superoxide dismutase (SOD) mimics. The manganese complex with H2qp1 is markedly more stable in water than other highly active non-porphyrin-based and even some Mn(ii) porphyrin-based SOD mimics., Manganese complexes with polydentate quinol-containing ligands are found to catalyze the degradation of superoxide through inner-sphere mechanisms. The redox activity of the ligand stabilizes higher-valent manganese species.

Two adolescent boys presented with acute acneiform eruptions in the setting of recent dupilumab administration. Subsequent investigation via direct scraping of pustules revealed live Demodex mite colonization of the face. These adolescent patients represent a population not commonly associated with Demodex folliculitis, and we theorize their baseline commensal Demodex mite population may have increased as a consequence of dupilumab-induced, focused immunomodulation. We recommend that pediatricians consider Demodex potentially etiologic in patients presenting with new onset acneiform or rosacea-like dermatoses in patients treated with dupilumab.

CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.

Background Tansy plants (Tanacetum vulgare L.) are known for their high intraspecific chemical variation, especially of volatile organic compounds (VOC) from the terpenoid compound group. These VOCs are closely involved in plant-insect interactions and, when profiled, can be used to classify plants into groups known as chemotypes. Tansy chemotypes have been shown to influence plant-aphid interactions, however, to date no information is available on the response of different tansy chemotypes to simultaneous herbivory by more than one insect species. Results Using a multi-cuvette system, we investigated the responses of five tansy chemotypes to feeding by sucking and/or chewing herbivores (aphids and caterpillars; Metopeurum fuscoviride Stroyan and Spodoptera littoralis Boisduval). Herbivory by caterpillars following aphid infestation led to a plant chemotype-specific change in the patterns of terpenoids stored in trichome hairs and in VOC emissions. The transcriptomic analysis of a plant chemotype represents the first de novo assembly of a transcriptome in tansy and demonstrates priming effects of aphids on a subsequent herbivory. Overall, we show that the five chemotypes do not react in the same way to the two herbivores. As expected, we found that caterpillar feeding increased VOC emissions, however, a priori aphid infestation only led to a further increase in VOC emissions for some chemotypes. Conclusions We were able to show that different chemotypes respond to the double herbivore attack in different ways, and that pre-treatment with aphids had a priming effect on plants when they were subsequently exposed to a chewing herbivore. If neighbouring chemotypes in a field population react differently to herbivory/dual herbivory, this could possibly have effects from the individual level to the group level. Individuals of some chemotypes may respond more efficiently to herbivory stress than others, and in a group environment these “louder” chemotypes may affect the local insect community, including the natural enemies of herbivores, and other neighbouring plants.

Visual Abstract, Brain networks underlying states of social and sensory alertness are normally adaptive, influenced by serotonin and dopamine (DA), and abnormal in neuropsychiatric disorders, often with sex-specific manifestations. Underlying circuits, cells, and molecules are just beginning to be delineated. Implicated is a subtype of serotonergic neuron denoted Drd2-Pet1, distinguished by expression of the type-2 DA receptor (Drd2) gene, inhibited cell-autonomously by DRD2 agonism in slice, and, when constitutively silenced in male mice, affects levels of defensive and exploratory behaviors (Niederkofler et al., 2016). Unknown has been whether DRD2 signaling in these Pet1 neurons contributes to their capacity for shaping defensive behaviors. To address this, we generated mice in which Drd2 gene sequences were deleted selectively in Pet1 neurons. We found that Drd2Pet1-CKO males, but not females, demonstrated increased winning against sex-matched controls in a social dominance assay. Drd2Pet1-CKO females, but not males, exhibited blunting of the acoustic startle response, a protective, defensive reflex. Indistinguishable from controls were auditory brainstem responses (ABRs), locomotion, cognition, and anxiety-like and depression-like behaviors. Analyzing wild-type Drd2-Pet1 neurons, we found sex-specific differences in the proportional distribution of axonal collaterals, in action potential (AP) duration, and in transcript levels of Gad2, important for GABA synthesis. Drd2Pet1-CKO cells displayed sex-specific differences in the percentage of cells harboring Gad2 transcripts. Our results suggest that DRD2 function in Drd2-Pet1 neurons is required for normal defensive/protective behaviors in a sex-specific manner, which may be influenced by the identified sex-specific molecular and cellular features. Related behaviors in humans too show sex differences, suggesting translational relevance.

Upon mitochondrial dysfunction, CHOP acts as a rheostat that attenuates prolonged stress and delays the onset of cardiomyopathy., In response to disturbed mitochondrial gene expression and protein synthesis, an adaptive transcriptional response sharing a signature of the integrated stress response (ISR) is activated. We report an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBPβ, and ATF4. We show that CHOP acts as a rheostat that attenuates prolonged ISR, prevents unfavorable metabolic alterations, and postpones the onset of mitochondrial cardiomyopathy. Upon mitochondrial dysfunction, CHOP interaction with C/EBPβ is needed to adjust ATF4 levels, thus preventing overactivation of the ATF4-regulated transcriptional program. Failure of this interaction switches ISR from an acute to a chronic state, leading to early respiratory chain deficiency, energy crisis, and premature death. Therefore, contrary to its previously proposed role as a transcriptional activator of mitochondrial unfolded protein response, our results highlight a role of CHOP in the fine-tuning of mitochondrial ISR in mammals.

H3K4me1 methyltransferases MLL3 (KMT2C) and MLL4 (KMT2D) are critical for enhancer activation, cell differentiation and development. However, roles of MLL3/4 enzymatic activities and MLL3/4-mediated enhancer H3K4me1 in these processes remain unclear. Here, we report that constitutive elimination of both MLL3 and MLL4 enzymatic activities leads to gastrulation failure and early embryonic lethality in mice. However, selective elimination of MLL3/4 enzymatic activities in embryonic, but not extraembryonic, lineages leaves gastrulation largely intact. Consistently, embryonic stem cells (ESCs) lacking MLL3/4 enzymatic activities can differentiate towards the three embryonic germ layers but show aberrant differentiation to extraembryonic endoderm and trophectoderm. The failure in extraembryonic endoderm differentiation can be attributed to markedly reduced enhancer-binding of the lineage-determining transcription factor GATA6. Furthermore, we show that MLL3/4-catalyzed H3K4me1 is largely dispensable for enhancer activation during ESC differentiation. Together, our findings suggest a lineage-selective, but enhancer activation-independent, role of MLL3/4 methyltransferase activities in early embryonic development and embryonic stem cell differentiation.

In mammals, the placenta mediates maternal-fetal nutrient and waste exchange and provides immunomodulatory actions that facilitate maternal-fetal tolerance. The placenta is highly diversified among mammalian species, yet the molecular mechanisms that distinguish the placenta of human from other mammals are not fully understood. Using an interspecies transcriptomic comparison of human, macaque, and mouse term placentae, we identified hundreds of genes with lineage-specific expression – including dozens that are placentally-enriched and potentially related to pregnancy. We further annotated the enhancers for different human tissues using epigenomic data and demonstrate that the placenta and chorion are unique in that their enhancers display the least conservation. We identified numerous lineage-specific human placental enhancers, and found they are highly overlapped with specific families of endogenous retroviruses (ERVs), including MER21A, MER4A/B and MER39B that were previously linked to immune response and placental function. Among these ERV families, we further demonstrate that MER41 insertions create dozens of lineage-specific Serum Response Factor (SRF) binding loci in human, including one adjacent to FBN2, a placenta-specific gene with increased expression in humans that produces the peptide hormone placensin to stimulate glucose secretion and trophoblast invasion. Our results demonstrate the prevalence of lineage-specific human placental enhancers which are frequently associated with ERV insertions and likely facilitated the lineage-specific evolution of the mammalian placenta.

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons – the most molecularly distinct subtype – possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability., Maintenance and quality control of the mitochondrial respiratory chain complexes responsible for bulk energy production are unclear. Here, the authors show that the mitochondrial protease ClpXP is required for the rapid turnover of the core N-module of respiratory complex I, which happens independently of other modules in the complex.

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons – the most molecularly distinct subtype – possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

International audience; Honeydew is a sugar-rich resource excreted by many hemipteran species and is a key food source for other insect species such as ants and parasitoid wasps. Here, we evaluated the nutritional value of 14 honeydews excreted by 13 aphid species for the generalist aphid parasitoid Lysiphlebus testaceipes to test a series of hypotheses concerning variation in the nutritional value of honeydew. There was a positive correlation between the body sugar content of honeydew-fed parasitoids and their longevity. This information is valuable for biological control researchers because it demonstrates that the nutritional state of honeydew-fed parasitoids in the wild can indicate their fitness, independently of the honeydew source they have fed on. Although the carbohydrate content and longevity of L. testaceipes differed greatly among the different honeydews, we did not find a significant effect of aphid or host plant phylogeny on these traits. This result suggests that honeydew is evolutionarily labile and may be particularly subject to ecological selection pressures. This becomes apparent when considering host aphid suitability: Schizaphis graminum, one of the most suitable and commonly used hosts of L. testaceipes, produced honeydew of the poorest quality for the parasitoid whereas Uroleucon sonchi, one of the few aphids tested that cannot be parasitized by L. testaceipes, excreted the honeydew with the highest nutritional value. These data are consistent with the hypothesis that hemipterans are subject to selection pressure to minimize honeydew quality for the parasitoids that attack them.

The cellular response to external stress signals and DNA damage depends on the activity of ubiquitin ligases (E3s), which regulate numerous cellular processes, including homeostasis, metabolism and cell cycle progression. E3s recognize, interact with and ubiquitylate protein substrates in a temporally and spatially regulated manner. The topology of the ubiquitin chains dictates the fate of the substrates, marking them for recognition and degradation by the proteasome or altering their subcellular localization or assembly into functional complexes. Both genetic and epigenetic alterations account for the deregulation of E3s in cancer. Consequently, the stability and/or activity of E3 substrates are also altered, in some cases leading to downregulation of tumour-suppressor activities and upregulation of oncogenic activities. A better understanding of the mechanisms underlying E3 regulation and function in tumorigenesis is expected to identify novel prognostic markers and to enable the development of the next generation of anticancer therapies. This Review summarizes the oncogenic and tumour-suppressor roles of selected E3s and highlights novel opportunities for therapeutic intervention.

Five new gold(III) complexes, with general formula [Au(N N)Cl2]+ for complexes 1–3 and [Au(N N)2]3+ for complexes 4–5 (where N N is 3-((2-((5-phenyl-1H-pyrazol-3-yl)methoxy)naphthalene-3-yloxy)methyl)-5-phenyl-1H-pyrazole – L1, (3-((2-((5-phenyl-1H-pyrazol-3-yl)methoxy) phenoxy)methyl)-5-phenyl-1H-pyrazole – L2, (3-((2-((5-naphthalen-2-yl)-1H-pyrazol-3-yl)methoxy)phenoxy)methyl)-5-(naphthalen-3-yl)-1H-pyrazole – L3), were synthesized and characterized by elemental analysis, 1H NMR, IR, UV–Vis, ESI-MS and conductometry. Also, ligand L2 was characterized by X-ray analysis. Stability of complexes in water and in Hepes buffer was confirmed by UV–Vis. Kinetics and mechanism of the substitution reactions of 1–3 with guanosine-5′-monophosphate (5′-GMP), glutathione (GSH) and. l -Methionine ( l -Met) were studied by stopped-flow technique. Obtained results have shown that complex 1 is the most reactive, while the reactivity of the nucleophiles decreases in order: GSH > 5′-GMP > l -Met. Calculated values of the entropy of activation support an associative mechanism. Redox stability of complexes 1–3 was investigated in the presence of the same biomolecules by cyclic voltametry. Obtained voltammograms showed reduction of gold(III) up to gold(0). DNA binding studies in the presence of ethidium bromide (EB) and 2-(4-hydroxyphenyl)-5-[5-(4-methylpipera-zine-1yl)-benzimidazo-2-yl]-benzimidazole (HOE) were performed by UV–Vis, fluorescence spectroscopy and viscosity measurements, in order to assess the binding mode. The results showed that gold(III) complexes interact with calf-thymus (CT-DNA) via covalently binding mode rather than via intercalation. Also, all complexes shown high values of binding constants for the interactions with bovine serum albumin (BSA) and human serum albumin (HSA). Furthermore, the binding studies with CT-DNA and BSA/HSA were supported by molecular docking. The strong connection between structure and reactivity of gold(III) complexes toward biologically important molecules was confirmed.

1. Spatially distributed resources can lead to the formation of metapopulations, where individual subpopulations are often small and can experience frequent local extinction events followed by recolonisation. An example of terrestrial metapopulations are specialised phytophagous insects on their patchily distributed host plants. 2. The present study investigated the population dynamics of a specialised aphid (Metopeurum fuscoviride) on its patchily distributed host plant (Tanacetum vulgare) and associated community of mutualistic ants and predators in a small-scale field site. Furthermore, aphid habitat differences (plant size, C/N ratio, location and surrounding vegetation) were quantified, and seasonal timing and precipitation were considered. 3. Seasonal timing and precipitation both had effects on aphid colonisation, extinction events and aphid colony persistence. Towards the end of the season, and after higher precipitation, aphid colonisation events decreased and extinction events increased. Plant size and location as well as aphid within-field dispersal determined the spatio-temporal distribution of aphid colonies. 4. Mutualistic ants (Lasius niger and Myrmica rubra) increased the chance of establishment of aphid colonies. However, when M. rubra was tending, aphid colony persistence was reduced. Aphid persistence and extinction were dependent on aphid abundance, as a higher colony size reduced the probability of extinction by predation. 5. The results emphasise the importance of dispersal limitation, population growth and the presence of mutualists when studying the spatio-temporal dynamics of tansy aphids, particularly in a small-scale field site.

FMRFamide is a neuropeptide that is widely distributed in invertebrates and known to be involved in many physiological functions. Previously we noted marked differences in expression of thefmrfgene in the stellate ganglion ofDoryteuthis pealei*compared to the central nervous system. In this study we aimed to examen the brain systems ofDoryteuthis pealei*for the presence and distribution offmrf-expressing cells and fiber networks. Late squid embryos and hatchlings were examined by in situ hybridization and immunohistochemistry in whole mounts and tissue sections. All central lobes contained limited numbers of scattered neurons expressingfmrf, but the FMRFamide-containing fiber systems were abundant and extensive, mostly present in the neuropil of lobes. Main clusters of neurons were located in the magnocellular and chromatophore lobes of the posterior subesophageal mass (PSM), and in dorsal aspects of the basal lobe (BL). Dense FMRFamide-immunoreactive fibers were particularly seen in the optic lobe (OL), medial and posterior supraesophageal masses (MSM and SPM) often with a commissural organization. The data show that the central lobes ofDoryteuthis pealeihatchlings have a matured FMRFamide system organized in a limited number of centers, but with widely distributed efferents. This suggests that FMRFamide neurons are already functionally engaged in the late embryo. The localization indicates that control of chromatophores and fin movement are amongst these functions.

Cytochrome c oxidase (CcO) catalyzes the reduction of dioxygen to water utilizing a heterobinuclear active site composed of a heme moiety and a mononuclear copper center coordinated to three histidine residues, one of which is covalently cross-linked to a tyrosine residue via a post-translational modification (PTM). Although this tyrosine—histidine moiety has functional and structural importance, the pathway behind this net oxidative C—N bond coupling is still unknown. A novel route employing an iron(III) meso-substituted isoporphyrin derivative, isoelectronic with Cmpd-I ((Por(•+))Fe(IV)=O), is for the first time proposed to be a key intermediate in the Tyr-His cofactor biogenesis. Newly synthesized iron(III) meso-substituted isoporphyrins were prepared with azide, cyanide, and substituted imidazole functionalities, by adding nucleophiles to an iron(III) π-dication species formed via addition of trifluoroacetic acid to F(8)Cmpd-I (F(8) = (tetrakis(2,6-difluorophenyl)porphyrinate)). Isoporphyrin derivatives were characterized at cryogenic temperatures via ESI-MS and UV—vis, (2)H NMR, and EPR spectroscopies. Addition of 1,3,5-trimethoxybenzene or 4-methoxyphenol to the imidazole-substituted isoporphyrin led to formation of the organic product containing the imidazole coupled to aromatic substrate via a new C—N bond, as detected via cryo-ESI-MS. Experimental evidence for the formation of an imidazole-substituted isoporphyrin and its promising reactivity to form the imidazole—phenol coupled product yields viability to the herein proposed pathway behind the PTM (i.e., biogenesis) leading to the key covalent Tyr-His cross-link in CcO.

Purpose: Clinically aggressive meningiomas (MGMs) are rare but treatment-resistant tumors in need for more effective therapies. Because tumor-infiltrating T lymphocytes (TILs) are essential for successful immunotherapy, we assessed TIL numbers and their activation status in primary (p-) and recurrent (r-) meningiomas and their impact on survival. Experimental Design: Presence of TILs was analyzed in 202 clinically well-annotated cases (n = 123 pMGMs and n = 79 rMGMs) focusing on higher-grade meningiomas [n = 97 World Health Organization (WHO) °II, n = 62 WHO°III]. TILs were quantified by a semiautomated analysis on whole-tissue sections stained by multicolor immunofluorescence for CD3, CD8, FOXP3, and programmed cell death protein 1 (PD-1). Results: Median T-cell infiltration accounted for 0.59% TILs per total cell count. Although there were no significant WHO°-dependent changes regarding helper (CD3+CD8−FOXP3−) and cytotoxic (CD3+CD8+FOXP3−) TILs in pMGMs, higher number of cytotoxic TILs were associated with an improved progression-free survival (PFS) independent of prognostic confounders. rMGMs were characterized by lower numbers of TILs in general, helper, and cytotoxic TILs. The additional analysis of their activation status revealed that a proportion of PD-1+CD8+ TILs within the TIL population was significantly decreased with higher WHO grade and in rMGMs. Furthermore, lower proportions of PD-1+CD8+ TILs were associated with inferior PFS in multivariate analyses, arguing for PD-1 as activation rather than exhaustion marker. Conclusions: We identified higher numbers of CD3+CD8+FOXP3− TILs and proportions of PD-1–expressing CD3+CD8+FOXP3− TILs as novel biomarkers for better survival. These findings might facilitate the selection of patients who may benefit from immunotherapy and argue in favor of an intervention in primary rather than recurrent tumors.

Cascading effects in ecological systems acting across three or more trophic levels can be either of a resource‐based (bottom‐up) or natural enemy‐based (top‐down) nature. But, due to their complexity, these effects are often considered separately and their relative strength, acting simultaneously, remains unknown. In a semi‐natural field experiment using tansy (Tanacetum vulgare L.) and the specialised tansy‐aphid Metopeurum fuscoviride Stroyan as a model system, we compared the effects of four distinct plant chemotypes (i.e., bottom‐up), defined by the bouquet of their volatile terpenoids, on aphid population dynamics by manipulating the presence/absence of mutualistic ants and presence/absence of naturally occurring predators (i.e., top‐down). Predators reduced aphid abundance and colony survival but did not reduce initial growth rate due to a time‐lag until predators arrived on the plants. Ants directly benefited initial aphid growth rates and abundance, even in the absence of predators, but not the number of days an aphid colony persisted on the plant. Plant chemotype directly affected aphid growth rate and final abundances across the different plants and indirectly affected the abundances of tending ants and predators through effects on aphids. We found that tending ants were more abundant on one plant chemotype. Although ant abundance did not affect aphid population development, it became clear that ants had a preference towards aphids on certain chemotypes. However, a higher number of predators led to a lower number of aphids. The results confirm the importance of plant chemical variation, acting through multiple effects on many species in arthropod communities, and support results from field studies. In a natural population, with a diverse selection of host‐plant variants, aphid populations and their interacting species can therefore be structured at the level of an individual plant. Specialist aphids on patchily distributed host plants can exhibit metacommunity dynamics at very local scales. Plant within‐species variation within a local population is often ignored in metacommunity ecology, yet our work shows that this can have strong effects on insect–ant–natural enemy dynamics, and therefore, future research should incorporate this into current theory and experimental studies. A plain language summary is available for this article.

Dormant cancer cells often survive treatment and increase the risk for tumor relapse, associated with dismal prognosis. Two recent papers describe mechanisms used by the bone marrow niche to regulate leukemia dormancy. The findings provide a molecular basis for niche-targeting therapies that may enable elimination of dormant tumor cells.

Considerable progress has been made in understanding the contribution of E3 ubiquitin ligases to health and disease, including the pathogenesis of immunological disorders. Ubiquitin ligases exert exquisite spatial and temporal control over protein stability and function, and are thus crucial for the regulation of both innate and adaptive immunity. Given that immune responses can be both detrimental (autoimmunity) and beneficial (antitumor immunity), it is vital to understand how ubiquitin ligases maintain immunological homeostasis. Such knowledge could reveal novel mechanisms underlying immune regulation and identify new therapeutic approaches to enhance antitumor immunity and safeguard against autoimmunity.

The mitochondrial matrix protease CLPP plays a central role in the activation of the mitochondrial unfolded protein response (UPRmt) in Caenorhabditis elegans . Far less is known about mammalian UPRmt signaling, although similar roles were assumed for central players, including CLPP. To better understand the mammalian UPRmt signaling, we deleted CLPP in hearts of DARS2‐deficient animals that show robust induction of UPRmt due to strong dysregulation of mitochondrial translation. Remarkably, our results clearly show that mammalian CLPP is neither required for, nor it regulates the UPRmt in mammals. Surprisingly, we demonstrate that a strong mitochondrial cardiomyopathy and diminished respiration due to DARS2 deficiency can be alleviated by the loss of CLPP, leading to an increased de novo synthesis of individual OXPHOS subunits. These results question our current understanding of the UPRmt signaling in mammals, while introducing CLPP as a possible novel target for therapeutic intervention in mitochondrial diseases. ![][1] Loss of mammalian ClpP does not affect the mitochondrial unfolded protein response (UPRmt) under proteotoxic stress. Instead, loss of ClpP alleviates mitochondrial cardiomyopathy by partially rescuing the mitochondrial translation defect and reducing the level of potentially toxic peptides. [1]: /embed/graphic-1.gif

Presomitic mesoderm (PSM) cells are the precursors of the somites, which flank both sides of the neural tube and give rise to the musculo-skeletal system shaping the vertebrate body. WNT and FGF signaling control the formation of both the PSM and the somites and show a graded distribution with highest levels in the posterior PSM. We have used reporters for the mesoderm/PSM control genes T, Tbx6, and Msgn1 to investigate the differentiation of mouse ESCs from the naïve state via EpiSCs to PSM cells. Here we show that the activation of WNT signaling by CHIR99021 (CH) in combination with FGF ligand induces embryo-like PSM at high efficiency. By varying the FGF ligand concentration, the state of PSM cells formed can be altered. High FGF concentration supports posterior PSM formation, whereas low FGF generates anterior/differentiating PSM, in line with in vivo data. Furthermore, the level of Msgn1 expression depends on the FGF ligand concentration. We also show that Activin/Nodal signaling inhibits CH-mediated PSM induction in EpiSCs, without affecting T-expression. Inversely, Activin/Nodal inhibition enhances PSM induction by WNT/high FGF signaling. The ability to generate PSM cells of either posterior or anterior PSM identity with high efficiency in vitro will promote the investigation of the gene regulatory networks controlling the formation of nascent PSM cells and their switch to differentiating/somitic paraxial mesoderm.

The gene regulatory networks that coordinate hematopoietic commitment in the developing murine yolk-sac (YS) remain ill-defined. Here we report that the T-box transcription factor Eomesodermin (Eomes) is transiently expressed in mesodermal progenitors that generate virtually all YS hematopoietic and endothelial cells. Using an embryonic stem cell (ESC) differentiation system, we find that Eomes activity is essential for the production of primitive erythrocytes and definitive hematopoietic progenitors but dispensable for the development of endothelial cells. Bulk RNA-seq and single-cell-RNA-seq experiments demonstrate that in the absence of Eomes function Flk-1+ hematovascular mesoderm is specified but upon further differentiation it is diverted towards an endothelial rather than hematopoietic fate. Utilizing ESC reporter lines, we show that Eomes is expressed prior to both SCL and Runx1 during mesoderm patterning. Interestingly, Eomes activity is dispensable for the expression of SCL but is essential for the normal development of Runx1+ hemogenic endothelium (HE). ATAC-Seq experiments reveal that Eomes governs the accessibility of numerous hematopoietic enhancers that SCL normally utilizes to specify the hematopoietic fate. Finally, ChIP-seq experiments suggest that Eomes coordinates the development of hemogenic competent mesoderm in the context of Activin/Nodal and Tead-Yap signalling at mesodermal stages of development. Collectively, these experiments demonstrate that Eomes sits at the top of the transcriptional hierarchy, functioning upstream of Runx1 expression and SCL functional activity, to promote hemogenic competence of the YS mesodermal lineage. These results suggest that hemogenic competence is endowed earlier during murine embryogenesis than previously appreciated and have widespread implications for the generation of HE from pluripotent stem cell sources.

The essential roles played by Nodal and Bmp signalling during early mouse development have been extensively documented. Here we used conditional deletion strategies to investigate functional contributions made by Nodal, Bmp and Smad downstream effectors during primordial germ cell (PGC) development. We demonstrate that Nodal and its target gene Eomes provide early instructions during formation of the PGC lineage. We discovered that Smad2 inactivation in the visceral endoderm results in increased numbers of PGCs due to an expansion of the PGC niche. Smad1 is required for specification, whereas in contrast Smad4 controls the maintenance and migration of PGCs. Importantly we found that beside Blimp1, down-regulated phosphoSmad159 levels also distinguishes PGCs from their somatic neighbours so that emerging PGCs become refractory to Bmp signalling that otherwise promotes mesodermal development in the posterior epiblast. Thus balanced Nodal/Bmp signalling cues regulate germ cell versus somatic cell fate decisions in the early posterior epiblast.

Frontiers in computational neuroscience 12, 41 (2018). doi:10.3389/fncom.2018.00041, Published by Frontiers Research Foundation, Lausanne

Mitochondria are fundamental for cellular metabolism as they are both a source and a target of nutrient intermediates originating from converging metabolic pathways, and their role in the regulation of systemic metabolism is increasingly recognized. Thus, maintenance of mitochondrial homeostasis is indispensable for a functional energy metabolism of the whole organism. Here, we report that loss of the mitochondrial matrix protease CLPP results in a lean phenotype with improved glucose homeostasis. Whole-body CLPP-deficient mice are protected from diet-induced obesity and insulin resistance, which was not present in mouse models with either liver- or muscle-specific depletion of CLPP However, CLPP ablation also leads to a decline in brown adipocytes function leaving mice unable to cope with a cold-induced stress due to non-functional adaptive thermogenesis. These results demonstrate a critical role for CLPP in different metabolic stress conditions such as high-fat diet feeding and cold exposure providing tools to understand pathologies with deregulated Clpp expression and novel insights into therapeutic approaches against metabolic dysfunctions linked to mitochondrial diseases.

As I have already pointed out elsewhere (Senft 2008; 2010; 2014), the Gricean conversational maxims of Quality – “Try to make your contribution one that is true” – and Manner “Be perspicuous”, specifically “Avoid obscurity of expression” and “Avoid ambiguity” (Grice 1967; 1975; 1978) – are not observed by the Trobriand Islanders of Papua New Guinea, neither in forms of their ritualized communication nor in forms and ways of everyday conversation and other ordinary verbal interactions. The speakers of the Austronesian language Kilivila metalinguistically differentiate eight specific non-diatopical registers which I have called “situational-intentional” varieties. One of these varieties is called “biga sopa”. This label can be glossed as “joking or lying speech, indirect speech, speech which is not vouched for”. The biga sopa constitutes the default register of Trobriand discourse and conversation. This contribution to the workshop on philosophy and pragmatics presents the Trobriand Islanders’ indigenous typology of non-diatopical registers, especially elaborating on the concept of sopa, describing its features, discussing its functions and illustrating its use within Trobriand society. It will be shown that the Gricean maxims of quality and manner are irrelevant for and thus not observed by the speakers of Kilivila. On the basis of the presented findings the Gricean maxims and especially Grice’s claim that his theory of conversational implicature is “universal in application” is critically discussed from a general anthropological-linguistic point of view.

Investigating retinal specializations offers insights into eye functionality. Using retinal wholemount techniques, we investigated the distribution of retinal ganglion cells in the Little skate Leucoraja erinacea by (a) dye-backfilling into the optic nerve prior to retinal wholemounting; (b) Nissl-staining of retinal wholemounts. Retinas were examined for regional specializations (higher numbers) of ganglion cells that would indicate higher visual acuity in those areas. Total ganglion cell number were low compared to other elasmobranchs (backfilled: average 49,713 total ganglion cells, average peak cell density 1,315 ganglion cells mm-2 ; Nissl-stained: average 47,791 total ganglion cells, average peak cell density 1,319 ganglion cells mm-2 ). Ganglion cells fit into three size categories: small (5-20 µm); medium (20-30 µm); large: (≥ 30 µm), and they were not homogeneously distributed across the retina. There was a dorsally located horizontal visual streak with increased ganglion cell density; additionally, there were approximately three local maxima in ganglion cell distribution (potential areae centrales) within this streak in which densities were highest. Using computerized tomography (CT) and micro-CT, geometrical dimensions of the eye were obtained. Combined with ganglion cell distributions, spatial resolving power was determined to be between 1.21 and 1.37 cycles per degree. Additionally, photoreceptor sizes across different retinal areas varied; photoreceptors were longest within the horizontal visual streak. Variations in the locations of retinal specializations appear to be related to the animal's anatomy: shape of the head and eyes, position of eyes, location of tapetum, and shape of pupil, as well as the visual demands associated with lifestyle and habitat type.

The introduction of isolated genes or recombinant DNA into cultured cells is a key procedure in cell and molecular biological research. At present there are many methods for gene transfer. All of these methods have certain shortcomings. Most of them are strongly cell-type dependent (i.e., they can work only on certain cells), and the transfer efficiency is frequently unsatis- factorily low. Furthermore, these methods often produce un- desirable biological or chemical side-effects. Recently, a new physical method has been developed ( 1). The cell membrane can be temporarily permeabilized by exposing the cell to a pulse of high intensity electric field (2). This process is attributed to the creation of resealable pores in the cell mem- brane and is thus called “electroporation.” The electroporation method offers more advantages than most other methods. First, it is simple to use and is time efficient. It can also be used to inject a single cell or millions of cells. Second, because it is a physical method, electroporation is less dependent on cell type. Third, it has fewer harmful biological and chemical side effects. In the last several years, the electroporation method has been successfully used to introduce cloned genes into a wide variety of cells, including mammalian cell lines, isolated cells, plant cells, bacteria, and yeast (3, 4). In addition to gene transfer, electroporation has the potential for use as a micro-injection method to introduce molecules other than DNA, such as second messengers, kinases, and kinase in- hibitors, drugs, and antibodies into a large number of cells. We are currently working to improve this technology. Our approach is to experiment with different wave forms and pulse protocols and to test their effects on cell survival rate and transfection efficiency. Our earlier results had suggested that, by

In tumor cells, the ability to maintain viability over long time periods without proliferation is referred to as a state of dormancy. Maintenance of dormancy is controlled by numerous cellular and environmental factors, from immune surveillance and tumor-stroma interaction to intracellular signaling. Interference of dormancy (to an 'awaken' state) is associated with reduced response to therapy, resulting in relapse or in metastatic burst. Thus, maintaining a dormant state should prolong therapeutic responses and delay metastasis. Technical obstacles in studying tumor dormancy have limited our understanding of underlying mechanisms and hampered our ability to target dormant cells. In this review, we summarize the progress of research in the field of immunogenic, angiogenic, and cellular dormancy in diverse malignancies with particular attention to our current understanding in melanoma.

Glioblastoma (GBM) is the most common malignant primary brain tumor. Although clinical presentation and brain imaging might be suggestive, histopathological evaluation by means of a brain biopsy is routinely performed to establish the diagnosis. A serum marker indicative of GBM may simplify the diagnostic work-up of patients suspected to having a brain tumor. We prospectively examined 113 patients with newly diagnosed single supratentorial or infratentorial space-occupying brain lesions. Glial fibrillary acidic protein (GFAP) levels were determined from venous blood samples via a prototype ELISA assay prior to any invasive procedures. Serum levels of GFAP were correlated with histopathological findings and MRI parameters. GFAP values were significantly higher in GBM patients (n = 33) compared to all other tumors (p < 0.001). A GFAP serum concentration of ≥0.01 µg/L revealed a sensitivity of 85 % and a specificity of 70 % for differentiating GBM from other entities. By applying a GFAP cut-off point of 0.20 µg/L, specificity was maximized (99 %), but sensitivity dropped to 27 %. In GBM patients, serum GFAP values were significantly correlated with tumor volume. GBM patients with high GFAP levels showed more in vivo GFAP expression as well as more necrosis and perilesional edema compared to GBM patients having low or non-detectable GFAP levels. GFAP serum concentrations differentiated between patients with GBM and patients with cerebral mass lesions of other entities with a moderate diagnostic accuracy. Serum GFAP levels in GBM patients were positively correlated with tumor volume and histopathological tumor characteristics.