Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Refining the application of direct embryogenesis in sugarcane: effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency

A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.     

A proton nuclear magnetic resonance assignment and secondary structure determination of recombinant human thioredoxin

Two-dimensional 1H NMR spectroscopy has been applied to a structural analysis of the reduced form of a recombinant human thioredoxin, a ubiquitous dithiol oxidoreductase recently isolated from an immunocompetent lymphoblastoid cell line. The sequential assignment of the spectrum, including all proline residues, has been accomplished by using experiments to demonstrate through-bond and through-space connectivities. The secondary structure has been determined by a qualitative interpretation of nuclear Overhauser effects, NH exchange data, and 3JHN alpha coupling constants. The secondary structure was found to be similar to that of the X-ray structure of Escherichia coli thioredoxin, consisting of a mixed five-stranded beta-sheet surrounded by four alpha-helices. The assignment and structural characterization of human thioredoxin was facilitated by the increased resolution and sensitivity afforded by a magnetic field strength of 600 MHz and required the use of two temperatures and two pH conditions to resolve ambiguities caused by a duplication of resonances. This duplication, extending from Phe-41 to Val-59, and including Lys-3-Ile-5, Val-24, Val-25, Asn-39, and Ile-101-Glu-103, appears to be due to heterogeneity arising from the presence or absence of the N-terminal methionine.     

Identifying Mozambique's most critical areas for plant conservation: An evaluation of protected areas and Important Plant Areas

Successful protected area networks must represent biodiversity across taxonomic groups. However, too often plant species are overlooked in conservation planning, and the resulting protected areas may, as a result, fail to encompass the most important sites for plant diversity. The Mozambique Tropical Important Plant Areas project sought to promote the conservation of Mozambique's flora through the identification of Important Plant Areas (IPAs). Here, we use the Weighted Endemism including Global Endangerment (WEGE) index to identify the richest areas for rare and endemic plants in Mozambique and subsequently evaluate how well represented these hotspots are within the current protected area and IPA networks. We also examine the congruence between IPA and protected areas to identify opportunities for strengthening the conservation of plants in Mozambique. We found that high WEGE scores, representing areas rich in endemic/near-endemic and threatened species, predict the presence of IPAs in Mozambique, but do not predict the presence of protected areas. We also find that there is limited overlap between IPAs and protected areas in Mozambique. We demonstrate how IPAs could be an important tool for ensuring priority sites for plant diversity are included within protected area network expansions, particularly following the adoption of the “30 by 30” target agreed within the post-2020 Convention on Biological Diversity framework, with great potential for this method to be replicated elsewhere in the global tropics.     

Exogenous ATP-stimulated calcium uptake in isolated rat intestinal epithelial cells

ATP in the extracellular medium is known to stimulate Ca uptake into avian intestinal epithelial cells. We have now demonstrated a similar effect of ATP in mammalian intestinal epithelial cells and have further characterized this effect. Exogenous ATP increased 45Ca uptake 2-6 fold in isolated rat small intestinal epithelial cells, with a maximal effect at 1 mM and an ED50 of 290 microM. A strict structural requirement for nucleotide-stimulated 45Ca uptake was observed. ADP was much less effective than ATP and gamma-thio-ATP, and 5'-AMP, cyclic AMP, adenosine, non-adenine nucleotides, non-hydrolyzable ATP analogs and ATP analogs with ring substitutions at the 8 position were inactive. Prenylamine (100 microM) completely inhibited ATP-stimulated 45Ca uptake, while verapamil (100 microM) had only a small effect. In the intact intestine, ATP increased short-circuit current (Isc) when added to the mucosal side of the tissue. This effect was reduced by 10 microM and abolished by 100 microM prenylamine. The effect of ATP on Isc was markedly reduced in Cl-free solutions and in reduced-Ca solutions. Serosal and mucosal addition of the nonhydrolyzable ATP analog, beta, gamma-methylene-ATP, and serosal addition of ATP had little or no effect on Isc. The similarities between the effects of ATP in isolated cells and in the intact intestine suggest that the effect of ATP on Isc may be at least partially mediated through stimulation of Ca uptake into the epithelial cells.     

Helminth infracommunities in Litoria genimaculata (Amphibia: Anura) from Birthday Creek, and upland rainforest stream in Northern Queensland, Australia

The helminth fauna of Litoria genimaculata, a rainforest frog from northern Queensland, was quantified from 53 adult male frogs collected at monthly intervals between April 1990 and March 1991. The helminth fauna of this species was depauperate (6 species: Mesocoelium sp., Parapolystoma bulliense, Austraplectana sp., Onchocercidae gen. sp., Cosmocerca sp. and an unidentified nematode larva). The most commonly encountered species was P. bulliense, but the intestinal infracommanity was dominated by the digenean Mesocoelium sp. Fifty-five per cent of frogs were infected with only 1 helminth species and only 1 frog had more than 2 species, resulting in low diversity values. These results support previous studies which indicate that amphibians have depauperate helminth communities.     

Aragonite infill in overgrown conceptacles of coralline Lithothamnion spp. (Hapalidiaceae,Hapalidiales, Rhodophyta): new insights in biomineralization and phylomineralogy

New empirical and quantitative data in the study of calcium carbonate biomineralization and an expanded coralline psbA framework for phylomineralogy are provided for crustose coralline red algae. Scanning electron microscopy (SEM) and energy dispersive spectrometry (SEM‐EDS) pinpointed the exact location of calcium carbonate crystals within overgrown reproductive conceptacles in rhodolith‐forming Lithothamnion species from the Gulf of Mexico and Pacific Panama. SEM‐EDS and X‐ray diffraction (XRD) analysis confirmed the elemental composition of these calcium carbonate crystals to be aragonite. After spore release, reproductive conceptacles apparently became overgrown by new vegetative growth, a strategy that may aid in sealing the empty conceptacle chamber, hence influencing the chemistry of the microenvironment and in turn promoting aragonite crystal growth. The possible relevance of various types of calcium carbonate polymorphs present in the complex internal structure and skeleton of crustose corallines is discussed. This is the first study to link SEM, SEM‐EDS, XRD, Microtomography and X‐ray microscopy data of aragonite infill in coralline algae with phylomineralogy. The study contributes to the growing body of literature characterizing and speculating about how the relative abundances of carbonate biominerals in corallines may vary in response to changes in atmospheric pCO₂, ocean acidification, and global warming.     

Heat capacity changes for protein-peptide interactions in the ribonuclease S system.

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. We have substituted the wild-type residue Met-13 with six other hydrophobic residues ranging in size from alanine to phenylalanine and have determined the thermodynamic parameters associated with binding of these analogues to S-protein by titration calorimetry in the temperature range 5-25 degrees C. The heat capacity change (delta Cp) associated with binding was obtained from a global analysis of the temperature dependences of the free energies and enthalpies of binding. The delta Cp's were not correlated in any simple fashion with the nonpolar surface area (delta Anp) buried upon binding.