An exonuclease V homologue is expressed predominantly during early megasporogenesis in apomictic Brachiaria brizantha.

MAIN CONCLUSION: An exonuclease V homologue from apomictic Brachiaria brizantha is expressed and localized in nucellar cells at key moments when these cells differentiate to give rise to unreduced gametophytes. Brachiaria is a genus of forage grasses with economical and agricultural importance to Brazil. Brachiaria reproduces by aposporic apomixis, in which unreduced embryo sacs, derived from nucellar cells, other than the megaspore mother cell (MMC), are formed. The unreduced embryo sacs produce an embryo without fertilization resulting in clones of the mother plant. Comparative gene expression analysis in ovaries of sexual and apomictic Brachiaria spp. revealed a sequence from B. brizantha that showed a distinct pattern of expression in ovaries of sexual and apomictic plants. In this work, we describe a gene named BbrizExoV with strong identity to exonuclease V (Exo V) genes from other grasses. Sequence analysis in signal prediction tools showed that BbrizExoV might have dual localization, depending on the translation point. A longer form to the nucleus and a shorter form which would be directed to the chloroplast. This is also the case for monocot sequences analyzed from other species. The long form of BbrizExoV protein localizes to the nucleus of onion epidermal cells. Analysis of ExoV proteins from dicot species, with exception of Arabidopsis thaliana ExoVL protein, showed only one localization. Using a template-based AlphaFold 2 modelling approach the structure of BbrizExoV in complex with metal and ssDNA was predicted based on the holo structure of the human counterpart. Features predicted to define ssDNA binding but a lack of sequence specificity are shared between the human enzyme and BbrizExoV. Expression analyses indicated the precise site and timing of transcript accumulation during ovule development, which coincides with the differentiation of nucelar cells to form the typical aposporic four-celled unreduced gametophyte. A putative function for this protein is proposed based on its homology and expression pattern.

Lecture capture affects student learning behaviour.

Lecture capture (the real-time recording of live lectures) has become commonplace in higher education. It is popular with students who like the associated flexibility and believe that lecture recordings improve their grades. Here, we performed a survey (n = 694, 53% of the cohort) and set up focus groups (2 focus groups, 15 participants) to explore biological sciences students' perceptions of how lecture capture impacts their study behaviour when recordings are provided for every lecture and are made available to students without restriction. The participants in our study were convinced that lecture capture improved their learning, and many students noted that they were dependent on the recordings, thinking that without them, they would not be able to achieve good grades. Students reported that they spend a considerable amount of time watching recordings and making verbatim notes, leaving them little time for independent study. For many, lecture capture seems to reinforce the view that memorisation equals learning, a view that may be reinforced by knowledge-focussed assessment formats. For most students, lecture capture did not affect self-reported live lecture attendance patterns. However, about one-third of the participants reported skipping more classes, and the same participants were more likely to postpone catching up on missed lectures. The outcomes of our study suggest that lecture capture provision may negatively affect some students' attendance and study behaviour, and thus, we suggest more needs to be done to mitigate against this.

A special issue of Essays in Biochemistry on current educational developments in molecular bioscience.

The 4th joint UK Biochemical Society and Federation of European Biochemical Societies (FEBS) education event, 'Evolving Molecular Bioscience Education' took place online on May 27 and 28, 2021. The event, continuing the biennial series, comprised the invited speakers' talks, group discussions and other participants' pre-recorded flash presentations. Although the UK dominated, there were also speakers and participants from other European countries and other continents. This special issue includes a varied collection of articles written by the speakers and other participants.

The transcriptome of the novel dinoflagellate Oxyrrhis marina (Alveolata: Dinophyceae): response to salinity examined by 454 sequencing.

BACKGROUND: The heterotrophic dinoflagellate Oxyrrhis marina is increasingly studied in experimental, ecological and evolutionary contexts. Its basal phylogenetic position within the dinoflagellates make O. marina useful for understanding the origin of numerous unusual features of the dinoflagellate lineage; its broad distribution has lent O. marina to the study of protist biogeography; and nutritive flexibility and eurytopy have made it a common lab rat for the investigation of physiological responses of marine heterotrophic flagellates. Nevertheless, genome-scale resources for O. marina are scarce. Here we present a 454-based transcriptome survey for this organism. In addition, we assess sequence read abundance, as a proxy for gene expression, in response to salinity, an environmental factor potentially important in determining O. marina spatial distributions. RESULTS: Sequencing generated ~57 Mbp of data which assembled into 7, 398 contigs. Approximately 24% of contigs were nominally identified by BLAST. A further clustering of contigs (at ≥ 90% identity) revealed 164 transcript variant clusters, the largest of which (Phosphoribosylaminoimidazole-succinocarboxamide synthase) was composed of 28 variants displaying predominately synonymous variation. In a genomic context, a sample of 5 different genes were demonstrated to occur as tandem repeats, separated by short (~200-340 bp) inter-genic regions. For HSP90 several intergenic variants were detected suggesting a potentially complex genomic arrangement. In response to salinity, analysis of 454 read abundance highlighted 9 and 20 genes over or under expressed at 50 PSU, respectively. However, 454 read abundance and subsequent qPCR validation did not correlate well - suggesting that measures of gene expression via ad hoc analysis of sequence read abundance require careful interpretation. CONCLUSION: Here we indicate that tandem gene arrangements and the occurrence of multiple transcribed gene variants are common and indicate potentially complex genomic arrangements in O. marina. Comparison of the reported data set with existing O. marina and other dinoflagellates ESTs indicates little sequence overlap likely as a result of the relatively limited extent of genome scale sequence data currently available for the dinoflagellates. This is one of the first 454-based transcriptome surveys of an ancestral dinoflagellate taxon and will undoubtedly prove useful for future comparative studies aimed at reconstructing the origin of novel features of the dinoflagellates.

Developing transferable skills through embedding reflection in the science curriculum.

A longstanding challenge for educators in Higher Education is the need to prepare students for their career journey after graduation. While theoretical foundations are needed, students should be able to apply knowledge in new contexts and be able to demonstrate and evidence life- and employability-skills valuable to employers. Many degrees provide students with the opportunity to develop transferable skills, for instance through giving presentations and working in teams. Nevertheless, students are not always able to reflect on their skills development, and on the connection between theory, practice and their learning. Authentic assessments can create links between theory and practice preparing students for the workplace. However, it is common to see the product of a particular activity being assessed, and not the process through which the product was produced. This may encourage students to value the end product over skills development, and therefore not appreciate how their University experiences prepare them for the workplace. Science students can struggle with self-reflection, and therefore may find it difficult to articulate and evidence skills during job applications. We present different ways to foster self-reflection when transferable skills are embedded and assessed in the curriculum. However, we claim that the process of reflection should be taught and supported and new ways of assessing students are needed to help them develop their ability to self-reflect.

A structured reflective process supports student awareness of employability skills development in a science placement module.

Placements are often an extra-curricular activity of a science degree. This study reports on the outcomes of a final-year credit-bearing 6-week placement module that was specifically designed to develop and enhance students' employability skills. A key element of this module was that the student placements were not only evaluated from a science perspective, but also evaluated with an emphasis on meaningful reflection and evaluation of employability skills development. Students recorded their levels of confidence in skills before, during and after the placement via an Online Reflective Log, as part of a module's summative assessment. The results showed that taking part in the placement and conducting their own independent research helped students to make connections between their scientific knowledge, otherwise constrained within the walls of the undergraduate science laboratory, and the wider impact of their research on society. Another theme that emerged concerned career choices and aspirations, and the placement experiences either confirmed prior choices or opened new horizons. The Online Reflective Log helped students to feel supported by their university supervisor who were at a distance. Feedback on their tasks prompted students to reflect on the scientific and personal skills while being engaged in scientific activities during placement. Students agreed that they had further developed their employability skills during the placement and acknowledged that it was challenging to acquire evidence of skill development. However, students appreciated the usefulness of this reflection in relation to their future career development.

Correction to: Highlights of the IUBMB education session at the 20th IUPAB congress, 45th Annual SBBf Meeting, and 50th Annual SBBq Meeting.

[This corrects the article DOI: 10.1007/s12551-021-00887-6.].

Students tell us what good written feedback looks like.

Feedback can be an important element of learning, but only if students engage with it. Students are only likely to engage with feedback that they find useful. This study aimed to identify characteristics of written feedback perceived by students as effective. We used a mixed-method approach, integrating quantitative and qualitative data that were collected through the analysis of feedback that was identified by students as good, a student questionnaire, as well as interviews and a focus group exploring students' views on what good feedback looks like. Although the results show that length and composition of 'good' feedback can be extremely variable, some common characteristics could be identified, leading to a set of recommendations for staff marking written assessments. According to students, good feedback should be detailed and specific, and it should tell students how they can improve. Students also find it important that feedback is honest and constructive. In addition, positive reinforcement was identified as important by the focus group, although few examples of good written feedback on the assignment contained any direct praise. Surprisingly, feedforward which might help students in other modules did not feature highly in students' perceptions of good feedback, possibly indicating a focus by students on improving the current assignment rather than on future assignments.

Identification of novel aspartic proteases from Strongyloides ratti and characterisation of their evolutionary relationships, stage-specific expression and molecular structure.

BACKGROUND: Aspartic proteases are known to play an important role in the biology of nematode parasitism. This role is best characterised in blood-feeding nematodes, where they digest haemoglobin, but they are also likely to play important roles in the biology of nematode parasites that do not feed on blood. In the present work, we investigate the evolution and expression of aspartic proteases in Strongyloides ratti, which permits a unique comparison between parasitic and free-living adult forms within its life-cycle. RESULTS: We identified eight transcribed aspartic protease sequences and a further two genomic sequences and compared these to homologues in Caenorhabditis elegans and other nematode species. Phylogenetic analysis demonstrated a complex pattern of gene evolution, such that some S. ratti sequences had a one-to-one correspondence with orthologues of C. elegans but that lineage-specific expansions have occurred for other aspartic proteases in these two nematodes. These gene duplication events may have contributed to the adaptation of the two species to their different lifestyles. Among the set of S. ratti aspartic proteases were two closely-related isoforms that showed differential expression during different life stages: ASP-2A is highly expressed in parasitic females while ASP-2B is predominantly found in free-living adults. Molecular modelling of the ASP-2 isoforms reveals that their substrate specificities are likely to be very similar, but that ASP-2B is more electrostatically negative over its entire molecular surface than ASP-2A. This characteristic may be related to different pH values of the environments in which these two isoforms operate. CONCLUSIONS: We have demonstrated that S. ratti provides a powerful model to explore the genetic adaptations associated with parasitic versus free-living life-styles. We have discovered gene duplication of aspartic protease genes in Strongyloides and identified a pair of paralogues differentially expressed in either the parasitic or the free-living phase of the nematode life-cycle, consistent with an adaptive role for aspartic proteases in the evolution of nematode parasitism.

S-nitrosylation of syntaxin 1 at Cys(145) is a regulatory switch controlling Munc18-1 binding.

Exocytosis is regulated by NO in many cell types, including neurons. In the present study we show that syntaxin 1a is a substrate for S-nitrosylation and that NO disrupts the binding of Munc18-1 to the closed conformation of syntaxin 1a in vitro. In contrast, NO does not inhibit SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein] receptor} complex formation or binding of Munc18-1 to the SNARE complex. Cys(145) of syntaxin 1a is the target of NO, as a non-nitrosylatable C145S mutant is resistant to NO and novel nitrosomimetic Cys(145) mutants mimic the effect of NO on Munc18-1 binding in vitro. Furthermore, expression of nitrosomimetic syntaxin 1a in living cells affects Munc18-1 localization and alters exocytosis release kinetics and quantal size. Molecular dynamic simulations suggest that NO regulates the syntaxin-Munc18 interaction by local rearrangement of the syntaxin linker and H3c regions. Thus S-nitrosylation of Cys(145) may be a molecular switch to disrupt Munc18-1 binding to the closed conformation of syntaxin 1a, thereby facilitating its engagement with the membrane fusion machinery.

Microarray analysis of gender- and parasite-specific gene transcription in Strongyloides ratti.

The molecular mechanisms by which parasitic nematodes reproduce and have adapted to life within a host are unclear. In the present study, microarray analysis was used to explore differential transcription among the different stages and sexes of Strongyloides ratti, a parasitic nematode of brown rats. Specifically, gender-biased transcription between free-living females and free-living males, and parasitic-biased transcription between parasitic females and free-living females was determined. Of the estimated 3,688 distinct transcripts represented on the microarray, 743 (20%) exhibited male-biased transcription of >1.4-fold (2(0.5)), 689 (19%) female-biased transcription, 418 (11%) parasitic-biased transcription and 305 (8%) free-living-biased transcription. Among those transcripts that exhibited the highest levels of differential transcription, an orthologue of major sperm protein was identified in males, distinct aspartic protease orthologues in either parasitic or in free-living females, and orthologues of hsp-17 chaperone in parasitic females. These 3,688 transcripts were separated into 12 clusters, such that the pattern of transcription between life-stages and biological replicates was similar among transcripts within a cluster and dissimilar between clusters. Using annotation inferred from Caenorhabditis elegans, gene ontology terms over-represented in one or more clusters were identified and showed that female-biased transcription was associated with genes involved in reproductive processes and larval development, male-biased transcription was linked to genes involved in metabolism, and free-living-biased transcription related to genes involved in the regulation of body fluids and response to external stimulus. The association of gene ontology with parasite-biased transcription was less clear. The present findings for S. ratti provide a basis for a detailed exploration of differentially regulated molecules and might assist in the search for novel drug or vaccine targets in parasitic nematodes.

ribB and ribBA genes from Acidithiobacillus ferrooxidans: expression levels under different growth conditions and phylogenetic analysis.

Acidithiobacillus ferrooxidans is a Gram-negative, chemolithoautotrophic bacterium involved in metal bioleaching. Using the RNA arbitrarily primed polymerase chain reaction (RAP-PCR), we have identified several cDNAs that were differentially expressed when A. ferrooxidans LR was submitted to potassium- and phosphate-limiting conditions. One of these cDNAs showed similarity with ribB. An analysis of the A. ferrooxidans ATCC 23270 genome, made available by The Institute for Genomic Research, showed that the ribB gene was not located in the rib operon, but a ribBA gene was present in this operon instead. The ribBA gene was isolated from A. ferrooxidans LR and expression of both ribB and ribBA was investigated. Transcript levels of both genes were enhanced in cells grown in the absence of K2HPO4, in the presence of zinc and copper sulfate and in different pHs. Transcript levels decreased upon exposure to a temperature higher than the ideal 30 degrees C and at pH 1.2. A comparative genomic analysis using the A. ferrooxidans ATCC 23270 genome revealed similar putative regulatory elements for both genes. Moreover, an RFN element was identified upstream from the ribB gene. Phylogenetic analysis of the distribution of RibB and RibBA in bacteria showed six different combinations. We suggest that the presence of duplicated riboflavin synthesis genes in bacteria must provide their host with some benefit in certain stressful situations.

Structural flexibility in Trypanosoma brucei enolase revealed by X-ray crystallography and molecular dynamics.

Enolase is a validated drug target in Trypanosoma brucei. To better characterize its properties and guide drug design efforts, we have determined six new crystal structures of the enzyme, in various ligation states and conformations, and have carried out complementary molecular dynamics simulations. The results show a striking structural diversity of loops near the catalytic site, for which variation can be interpreted as distinct modes of conformational variability that are explored during the molecular dynamics simulations. Our results show that sulfate may, unexpectedly, induce full closure of catalytic site loops whereas, conversely, binding of inhibitor phosphonoacetohydroxamate may leave open a tunnel from the catalytic site to protein surface offering possibilities for drug development. We also present the first complex of enolase with a novel inhibitor 2-fluoro-2-phosphonoacetohydroxamate. The molecular dynamics results further encourage efforts to design irreversible species-specific inhibitors: they reveal that a parasite enzyme-specific lysine may approach the catalytic site more closely than crystal structures suggest and also cast light on the issue of accessibility of parasite enzyme-specific cysteines to chemically modifying reagents. One of the new sulfate structures contains a novel metal-binding site IV within the catalytic site cleft.

'Students-as-partners' scheme enhances postgraduate students' employability skills while addressing gaps in bioinformatics education.

Teaching bioinformatics is a longstanding challenge for educators who need to demonstrate to students how skills developed in the classroom may be applied to real world research. This study employed an action research methodology which utilised student-staff partnership and peer-learning. It was centred on the experiences of peer-facilitators, students who had previously taken a postgraduate bioinformatics module, and had applied knowledge and skills gained from it to their own research. It aimed to demonstrate to peer-receivers, current students, how bioinformatics could be used in their own research while developing peer-facilitators' teaching and mentoring skills. This student-centred approach was well received by the peer-receivers, who claimed to have gained improved understanding of bioinformatics and its relevance to research. Equally, peer-facilitators also developed a better understanding of the subject and appreciated that the activity was a rare and invaluable opportunity to develop their teaching and mentoring skills, enhancing their employability.

Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases.

Cysteine proteinases from larvae of the common bean weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae), were isolated by ion exchange affinity chromatography on a CM-Cellulose column and used to select mutant cystatins from a library made with the filamentous M13 phage display system. The library contained variant cystatins derived from the nematode Onchocerca volvulus cystatin through mutagenesis of loop 1, which contains the QVVAG motif that is involved in binding to proteinases. After three rounds of selection, the activity of variant cystatins against papain and cysteine proteinases from A. obtectus was assayed by ELISA. Two different variant cystatins (presenting amino acids DVVSA and NTSSA at positions 65-69) bound to A. obtectus cysteine proteinases more tightly than to papain. In contrast, the wild type had similar affinity for A. obtectus proteinases and for papain. These two selected variants cystatins have greater specificity towards A. obtectus cysteine proteinases than the original sequence and could represent good candidate genes for the production of transgenic plants resistant to this insect pest.

Structure and mechanism of action of a cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus with broad specificity phosphatase activity.

The crystal structure of Bacillus stearothermophilus PhoE (originally termed YhfR), a broad specificity monomeric phosphatase with a molecular mass of approximately 24 kDa, has been solved at 2.3 A resolution in order to investigate its structure and function. PhoE, already identified as a homolog of a cofactor-dependent phosphoglycerate mutase, shares with the latter an alpha/beta/alpha sandwich structure spanning, as a structural excursion, a smaller subdomain composed of two alpha-helices and one short beta-strand. The active site contains residues from both the alpha/beta/alpha sandwich and the sub-domain. With the exception of the hydrophilic catalytic machinery conserved throughout the cofactor-dependent phosphoglycerate mutase family, the active-site cleft is strikingly hydrophobic. Docking studies with two diverse, favored substrates show that 3-phosphoglycerate may bind to the catalytic core, while alpha-napthylphosphate binding also involves the hydrophobic portion of the active-site cleft. Combining a highly favorable phospho group binding site common to these substrate binding modes and data from related enzymes, a catalytic mechanism can be proposed that involves formation of a phosphohistidine intermediate on His10 and likely acid-base behavior of Glu83. Other structural factors contributing to the broad substrate specificity of PhoE can be identified. The dynamic independence of the subdomain may enable the active-site cleft to accommodate substrates of different sizes, although similar motions are present in simulations of cofactor-dependent phosphoglycerate mutases, perhaps favoring a more general functional role. A significant number of entries in protein sequence databases, particularly from unfinished microbial genomes, are more similar to PhoE than to cofactor-dependent phosphoglycerate mutases or to fructose-2,6-bisphosphatases. This PhoE structure will therefore serve as a valuable basis for inference of structural and functional characteristics of these proteins.

Insights into the catalytic mechanism of cofactor-independent phosphoglycerate mutase from X-ray crystallography, simulated dynamics and molecular modeling.

Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms. Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4A) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant. The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity. Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains. Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzyme's surface during certain stages of the catalytic process. Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters. Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme. Taken together, these results allow for a better understanding of the mechanism of action of iPGMs. Many of the results are relevant to a series of evolutionarily related enzymes. These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance.

Nucleotide sequence and phylogenetic analyses of the DNA polymerase gene of Anticarsia gemmatalis nucleopolyhedrovirus.

The DNA polymerase from Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) was identified and sequenced, and its amino acid sequence was compared with other viral DNA polymerases to identify conserved regions and to reconstruct a phylogenetic tree. The sequence analysis of the AgMNPV DNA polymerase gene revealed the presence of a 2976 nucleotides open reading frame (ORF) encoding a polypeptide of 991 amino acid residues with a predicted molecular mass of 114.7 kDa. Among the baculovirus DNA polymerase genes identified to date, the AgMNPV DNA polymerase gene shared maximum amino acid sequence identity with the DNA polymerase gene of Choristoneura fumiferana nucleopolyhedrovirus defective strain (CfDEFNPV) (94%). The alignment of 140 virus sequences, 23 of them from baculovirus, showed that, of the 10 conserved regions identified, 5 are exclusive to baculoviruses (R1, R5, R9, R6 and R10), only 2 of them (R6 and R10) previously described as such in the literature. Our analysis, based on their positions in the AgMNPV DNA polymerase model, suggests that R9 and R10 could interact with DNA. Phylogenetic analysis of DNA polymerase sequences places the enzyme from AgMNPV within the cluster containing the polymerases of Group I Nucleopolyhedrovirus and suggests that the AgMNPV DNA polymerase is more closely related to that of CfDEFNPV than to those of other baculoviruses.

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora.

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases.