Industrial Scale Isolation,Structural and Spectroscopic Characterization of Epiisopiloturine from Pilocarpus microphyllus Stapf Leaves: A Promising Alkaloid against Schistosomiasis

This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)-Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), ¹H and ¹³C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution.     

Armored with skin and bone: A combined histological and μCT-study of the exceptional integument of the Antsingy leaf chameleon Brookesia perarmata (Angel, 1933)

Madagascar's endemic ground-dwelling leaf chameleons (Brookesiinae: Brookesia Gray, 1865 + Palleon Glaw, et al., Salamandra, 2013, 49, pp. 237–238) form the sister taxon to all other chameleons (i.e., the Chamaeleoninae). They possess a limited ability of color change, a rather dull coloration, and a nonprehensile tail assisting locomotion in the leaf litter on the forest floor. Most Brookesia species can readily be recognized by peculiar spiky dorsolateral projections (“Rückensäge”), which are caused by an aberrant vertebral structure and might function as body armor to prevent predation. In addition to a pronounced Rückensäge, the Antsingy leaf chameleon Brookesia perarmata (Angel, 1933) exhibits conspicuous, acuminate tubercle scales on the lateral flanks and extremities, thereby considerably enhancing the overall armored appearance. Such structures are exceptional within the Chamaeleonidae and despite an appreciable interest in the integument of chameleons in general, the morphology of these integumentary elements remains shrouded in mystery. Using various conventional and petrographic histological approaches combined with μCT-imaging, we reveal that the tubercle scales consist of osseous, multicusped cores that are embedded within the dermis. Based on this, they consequently can be interpreted as osteoderms, which to the best of our knowledge is the first record of such for the entire Chamaeleonidae and only the second one for the entire clade Iguania. The combination of certain aspects of tissue composition (especially the presence of large, interconnected, and marrow-filled cavities) together with the precise location within the dermis (being completely enveloped by the stratum superficiale), however, discriminate the osteoderms of B. perarmata from those known for all other lepidosaurs.     

Affective communication in rodents: ultrasonic vocalizations as a tool for research on emotion and motivation

Mice and rats emit and perceive calls in the ultrasonic range, i.e., above the human hearing threshold of about 20 kHz: so-called ultrasonic vocalizations (USV). Juvenile and adult rats emit 22-kHz USV in aversive situations, such as predator exposure and fighting or during drug withdrawal, whereas 50-kHz USV occur in appetitive situations, such as rough-and-tumble play and mating or in response to drugs of abuse, e.g., amphetamine. Aversive 22-kHz USV and appetitive 50-kHz USV serve distinct communicative functions. Whereas 22-kHz USV induce freezing behavior in the receiver, 50-kHz USV lead to social approach behavior. These opposite behavioral responses are paralleled by distinct patterns of brain activation. Freezing behavior in response to 22-kHz USV is paralleled by increased neuronal activity in brain areas regulating fear and anxiety, such as the amygdala and periaqueductal gray, whereas social approach behavior elicited by 50-kHz USV is accompanied by reduced activity levels in the amygdala but enhanced activity in the nucleus accumbens, a brain area implicated in reward processing. These opposing behavioral responses, together with distinct patterns of brain activation, particularly the bidirectional tonic activation or deactivation of the amygdala elicited by 22-kHz and 50-kHz USV, respectively, concur with a wealth of behavioral and neuroimaging studies in humans involving emotionally salient stimuli, such as fearful and happy facial expressions. Affective ultrasonic communication therefore offers a translational tool for studying the neurobiology underlying socio-affective communication. This is particularly relevant for rodent models of neurodevelopmental disorders characterized by social and communication deficits, such as autism and schizophrenia.     

When should a DDH experiment be mandatory in microbial taxonomy?

DNA–DNA hybridizations (DDH) play a key role in microbial species discrimination in cases when 16S rRNA gene sequence similarities are 97 % or higher. Using real-world 16S rRNA gene sequences and DDH data, we here re-investigate whether or not, and in which situations, this threshold value might be too conservative. Statistical estimates of these thresholds are calculated in general as well as more specifically for a number of phyla that are frequently subjected to DDH. Among several methods to infer 16S gene sequence similarities investigated, most of those routinely applied by taxonomists appear well suited for the task. The effects of using distinct DDH methods also seem to be insignificant. Depending on the investigated taxonomic group, a threshold between 98.2 and 99.0 % appears reasonable. In that way, up to half of the currently conducted DDH experiments could safely be omitted without a significant risk for wrongly differentiated species.     

Genetic and phylogenetic evidence for horizontal gene transfer among ecologically disparate groups of marine Vibrio

Vibrio represents a diverse bacterial genus found in different niches of the marine environment, including numerous genera of marine sponges (phylum Porifera), inhabiting different depths and regions of benthic seas, that are potentially important in driving adaptive change among Vibrio spp. Using 16S rRNA gene sequencing, a previous study showed that sponge‐derived (SD) vibrios clustered with their mainstream counterparts present in shallow, coastal ecosystems, suggesting a genetic relatedness between these populations. Sequences from the topA, ftsZ, mreB, rpoD, rctB and toxR genes were used to investigate the degree of relatedness existing between these two separate populations by examining their phylogenetic and genetic disparity. Phylogenies were constructed from the concatenated sequences of the six housekeeping genes using maximum‐parsimony, maximum‐likelihood and neighbour‐joining algorithms. Genetic recombination was evaluated using the incongruence length difference test, Split decomposition and measuring overall compatibility of sites. This combined technical approach provided evidence that SD Vibrio strains are largely genetically homologous to their shallow‐water counterparts. Moreover, the analyses conducted support the existence of extensive horizontal gene transfer between these two groups, supporting the idea of a single panmictic population structure among vibrios from two seemingly distinct, marine environments.     

Mating system variation in Veronica (Plantaginaceae): inferences from pollen/ovule ratios and other reproductive traits

The pollen–ovule ratio (P/O) is commonly used to estimate the mode of sexual reproduction in flowering plants. In previous studies, a clear correspondence has been detected between this character and the degree of autogamy. We here investigate variation in this character and its expected correlates in the genus Veronica (Plantaginaceae). Pollen–ovule ratios of 45 species representing eleven percent of all the species in the genus were investigated and compared with results from crossing experiments from previous studies. In addition, multiple populations of 17 of the 45 studied species were sampled and a controlled‐environment experiment was conducted to evaluate the extent of intraspecific variation. Moreover, relationships between P/O and other primary and secondary reproductive characters of the Veronica flower were investigated in relation to a phylogenetic hypothesis in order to determine the phylogenetic constraints on reproductive characters. The differences in P/O among species correspond well to the diversity of mating systems in Veronica and correlate well with other floral characters such as corolla size. These characters together seem to allow a powerful and fast tool to infer mating systems. However, causes for intraspecific variation of P/O, such as different cytotypes, ecotypes or different growth conditions, need to be considered.     

Mechanism of Bacterial Oligosaccharyltransferase: IN VITRO QUANTIFICATION OF SEQUON BINDING AND CATALYSIS*

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the −2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.     

Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion

HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env''s fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag''s ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells.