AIRFLOW PATTERNS AROUND SOME EARLY SEED PLANT OVULES AND CUPULES: IMPLICATIONS CONCERNING EFFICIENCY IN WIND POLLINATION

Aerodynamic analyses showing characteristic airflow patterns and the potential for wind-mediated pollination are presented for models of Paleozoic (Carboniferous) ovules and ovulate cupules (i.e., Genomosperma kidstoni, G. latens, Salpingostoma dasu, Physostoma elegans, Eurystoma angulare, and Stamnostoma huttonense). Lobes on ovules and cupules are shown to produce localized regions of turbulent flow with a concomitant reduction in airflow velocity. Data based upon models that mimic the characteristics of windborne pollen (= pseudopollen) show that these regions of turbulent flow correspond to those in which suspended pseudopollen impact with ovule and/or cupule surfaces. These data have bearing on a sequence of ovule morphologies purported to show the evolution of the integument by the progressive reduction in length of “preintegumentary” lobes and their acropetal fusion. As the preintegumentary lobes of the models studied consolidate around the megasporangium, regions of turbulent flow and high pseudopollen impact become localized around the pollen chamber or salpinx. The general morphologic trend envisioned for the evolution of the ovule is seen to be associated with an aerodynamic streamlining and an increased potential for wind-mediated pollination. Data for hair-bearing ovules and for ovulate cupules are discussed within the context of possible selective pressures favouring streamlining.     

COMPARATIVE PALEOBIOCHEMISTRY OF SOME FOSSIL AND EXTANT FAGACEAE

A Fagus-like leaf fossil (cuticular compression) with an attached fruit, differing from any known Fagus species (fossil or extant) or other fagoid taxa, has been discovered from the Miocene Clarkia Lake deposits of northern Idaho. Because of its unusual morphology (especially the fruit) the fossil taxon has been described as a new genus and species, Pseudofagus idahoensis Smiley and Huggins. The successful previous use of paleobiochemistry in studies of fossil taxa from the Miocene Succor Creek Flora of Oregon suggested that chemical data might help clarify the taxonomic affinities of Pseudofagus. Indeed, examination of the chemistry of the fossil, Pseudofagus idahoensis, and comparison with extant Fagus species and related fagoid genera indicate that: 1) based on steroid chemistry, Pseudofagus idahonesis does belong in the Fagaceae; 2) like all extant species of Fagus, the fossil lacks the tannin component, ellagic acid, which separates it from other extant fagoid genera, and 3) its simple flavonoid pigment profile places it closest to the extant North American Fagus grandifolia or the European/Eurasian Fagus sylvatica. However, the exclusive presence of an isorhamnetin (3'-methoxyquercetin) 3-0-glycoside, onocerane, and 5α-cholestane imparts a species-specific chemical character to Pseudofagus idahoensis, which also sets it apart from extant species of Fagus. While the chemistry does not decide the taxonomic level to be accorded to the fossil, it certainly supports, along with morphology and anatomy, the distinctness of Pseudofagus and its proposed relationships within the Fagaceae.     

The guanosine 3',5'-bis(diphosphate) (ppGpp) cycle. Comparison of synthesis and degradation of guanosine 3',5'-bis(diphosphate) in various bacterial systems.

4-(3-Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl-14C]acetyl label. The reactive coenzyme analogue inactivates alcohol dehydrogenase from Bacillus stearothermophilus by forming a covalent enzyme-coenzyme compound. The inactivation kinetics as well as the spectral properties of the modified enzyme after treatment with sodium hyposulphite suggest that the analogue is bound at the coenzyme binding site. B. stearothermophilus alcohol dehydrogenase modified with 14C-labelled coenzyme analogue and subseqeuntly carboxymethylated with unlabelled iodoacetic acid was digested with trypsin. The radioactive peptide was isolated and sequenced in parallel with the corresponding peptide similarly isolated from unmodified enzyme that had instead been carboxymethylated with iodo[14C]acetic acid. Amino acid and sequence analysis show that Cys-38 of the B. stearothermophilus alcohol dehydrogenase was modified by the reactive coenzyme analogue. This residue is homologous to Cys-43 in yeast alcohol dehydrogenase and Cys-46 in the horse liver enzyme but, unlike the latter two, Cys-38 is not reactive towards iodoacetate in the native bacterial enzyme.     

Purification and some properties of a neutral protease from human leukocyte granules and its comparison with pancreatic elastase.

1. A cationic protease has been purified from the granule fraction of blood-donor leukocytes by a preparative method including precipitation by acetone and chromatography on Bio-Gel A 1.5 m, CM-Sephadex C-50 and Sephadex G-G-75. 2. The pH optimum against denatured bovine hemoglobin is 7.4. Gel chromatography indicated a molecular weight close to 23 000. 3. This neutral protease (EC 3.4.-.-) is able to split the synthetic esters Z-Ala-NPh and AcAla3OMe, its activity on the former substrate being 2.2 times greater than that of pancreatic elastase, on the latter the same. It differs crucially from pancreatic elastase in having small elastinolytic activity. 4. In cationic disk electrophoresis, neutral protease resolves into three protein bands with lower mobility than lysozyme: all bands exhibit esterolytic activity against 2-acetoxy-3-naphthoic acid o-toluidide, strongly suggesting that they represent isoenzymes. 5. The enzyme is completely inhibited by iPr2P-F, partially so by soybean trypsin inhibitor and Trasylol. Cysteine, EDTA and TosLysCH2Cl have no effect. 6. During chromatography on CM-Sephadex C-50 a more positively charged enzyme(s) was identified. This had hemoglobinolytic activity at pH 7.4 but only a small esterolytic effect on Z-Ala-NPh; it showed only traces of activity against AcAla3OMe.     

Structural basis of successive adenosine modifications by the conserved ribosomal methyltransferase KsgA

Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.     

Disentangling the assembly mechanisms of ant cuticular bacterial communities of two Amazonian ant species sharing a common arboreal nest

Bacteria living on the cuticle of ants are generally studied for their protective role against pathogens, especially in the clade of fungus‐growing ants. However, little is known regarding the diversity of cuticular bacteria in other ant host species, as well as the mechanisms leading to the composition of these communities. Here, we used 16S rRNA gene amplicon sequencing to study the influence of host species, species interactions and the pool of bacteria from the environment on the assembly of cuticular bacterial communities on two phylogenetically distant Amazonian ant species that frequently nest together inside the roots system of epiphytic plants, Camponotus femoratus and Crematogaster levior. Our results show that (a) the vast majority of the bacterial community on the cuticle is shared with the nest, suggesting that most bacteria on the cuticle are acquired through environmental acquisition, (b) 5.2% and 2.0% of operational taxonomic units (OTUs) are respectively specific to Ca. femoratus and Cr. levior, probably representing their respective core cuticular bacterial community, and (c) 3.6% of OTUs are shared between the two ant species. Additionally, mass spectrometry metabolomics analysis of metabolites on the cuticle of ants, which excludes the detection of cuticular hydrocarbons produced by the host, were conducted to evaluate correlations among bacterial OTUs and m/z ion mass. Although some positive and negative correlations are found, the cuticular chemical composition was weakly species‐specific, suggesting that cuticular bacterial communities are prominently environmentally acquired. Overall, our results suggest the environment is the dominant source of bacteria found on the cuticle of ants.     

Model-based design and control of a small-scale integrated continuous end-to-end mAb platform

A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.     

Bone phenotype of P2X4 receptor knockout mice: implication of a P2X7 receptor mutation?

Transgenic and knockout animal models are widely used to investigate the role of receptors, signaling pathways, and other peptides and proteins. Varying results are often published on the same model from different groups, and much effort has been put into understanding the underlying causes of these sometimes conflicting results. Recently, it has been shown that a P2X4R knockout model carries a so-called passenger mutation in the P2X7R gene, potentially affecting the interpretation of results from studies using this animal model. We therefore report this case to raise awareness about the potential pitfalls using genetically modified animal models, especially within P2 receptor research. Although purinergic signaling has been recognized as an important contributor to the regulation of bone remodeling, the process that maintains the bone quality during life, little is known about the role of the P2X4 receptor (P2X4R) in regulation of bone remodeling in health and disease. To address this, we analyzed the bone phenotype of P2rx4tm1Rass (C57BL/6J) knockout mice and corresponding wildtype using microCT and biomechanical testing. Overall, we found that the P2X4R knockout mice displayed improved bone microstructure and stronger bones in an age- and gender-dependent manner. While cortical BMD, trabecular BMD, and bone volume were higher in the 6-month-old females and 3-month-old males, this was not the case for the 3-month-old females and the 6-month-old males. Bone strength was only affected in the females. Moreover, we found that P2X4R KO mice carried the P2X7 receptor 451P wildtype allele, whereas the wildtype mice carried the 451L mutant allele. In conclusion, this study suggests that P2X4R could play a role in bone remodeling, but more importantly, it underlines the potential pitfalls when using knockout models and highlights the importance of interpreting results with great caution. Further studies are needed to verify any specific effects of P2X4R on bone metabolism.

     

Systematics and evolutionary history of butterflies in the “Taygetis clade” (Nymphalidae: Satyrinae: Euptychiina): Towards a better understanding of Neotropical biogeography

The so-called “Taygetis clade” is a group of exclusively Neotropical butterflies classified within Euptychiina, one of the largest subtribes in the subfamily Satyrinae. Since the distribution of the ten genera belonging to this group ranges throughout the entire Neotropics, from lowlands to lower montane habitats, it offers a remarkable opportunity to study the region’s biogeographic history as well as different scenarios for speciation in upland areas. We inferred a robust and well-sampled phylogeny using DNA sequences from four genes (4035 bp in total) using maximum parsimony and Bayesian inference. We estimated divergence times using the Bayesian relaxed clock method calibrated with node ages from previous studies. Ancestral ranges of distribution were estimated using the dispersal–extinction–cladogenesis (DEC) model as implemented in the program Lagrange. We propose several taxonomic changes and recognize nine well-supported natural genera within the “Taygetis clade”: Forsterinaria (subsuming Guaianaza syn. nov.), Parataygetis, Posttaygetis, Harjesia (excluding Harjesia griseola and Harjesia oreba), Pseudodebis (including Taygetomorpha syn. nov.,), Taygetina (subsuming Coeruleotaygetis syn. nov., Harjesia oreba comb. nov., Taygetis weymeri comb. nov. and Taygetis kerea comb. nov.), Taygetis (excluding Taygetis ypthima, Taygetis rectifascia, Taygetis kerea and Taygetis weymeri), and two new genera, one containing Harjesia griseola, and the other Taygetis ypthima and Taygetis rectifascia. The group diversified mainly during late Miocene to Pliocene, coinciding with the period of drastic changes in landscape configuration in the Neotropics. Major dispersals inferred from the Amazon basin towards northwestern South America, the Atlantic forests and the eastern slope of the Andes have mostly shaped the evolution and diversification of the group. Furthermore, expansion of larval dietary repertoire might have aided net diversification in the two largest genera in the clade, Forsterinaria and Taygetis.