Diversifying laboratory assessment modes broadens engagement with practical competencies in life science students.

Laboratory practicals in life science subjects are traditionally assessed by written reports that reflect disciplinary norms for documenting experimental activities. However, the exclusive application of this assessment has the potential to engage only a narrow range of competencies. In this study, we explored how multiple modes of laboratory assessment might affect student perceptions of learned skills in a life science module. We hypothesized that while a mixture of assessments may not impact student summative performance, it might positively influence student perceptions of different skills that varied assessments allowed them to practice. This was informed by universal design for learning and teaching for understanding frameworks. In our study, in a third-year Bioscience program, written reports were complemented with group presentations and online quizzes via Moodle. Anonymous surveys evaluated whether this expanded portfolio of assessments promoted awareness of, and engagement with, a broader range of practical competencies. Aspects that influenced student preferences in assessment mode included time limitations, time investment, ability to practice new skills, links with lecture material, and experience of assessment anxiety. In particular, presentations were highlighted as promoting collaboration and communication and the quiz as an effective means of diversifying assessment schedules. A key takeaway from students was that while reports were important, an overreliance on them was detrimental. This study suggests that undergraduate life science students can benefit significantly from a holistic assessment strategy that complements reports with performance-based approaches that incorporate broader competencies and allow for greater student engagement and expression in undergraduate modules.NEW & NOTEWORTHY This study suggests that undergraduate life science students can benefit significantly from a holistic assessment strategy that complements reports with performance-based approaches that incorporate broader competencies and allow for greater student engagement and expression in undergraduate modules.

Efflux, Signaling and Warfare in a Polymicrobial World.

The discovery void of antimicrobial development has occurred at a time when the world has seen a rapid emergence and spread of antimicrobial resistance, the 'perfect storm' as it has often been described. While the discovery and development of new antibiotics has continued in the research sphere, the pipeline to clinic has largely been fed by derivatives of existing classes of antibiotics, each prone to pre-existing resistance mechanisms. A novel approach to infection management has come from the ecological perspective whereby microbial networks and evolved communities already possess small molecular capabilities for pathogen control. The spatiotemporal nature of microbial interactions is such that mutualism and parasitism are often two ends of the same stick. Small molecule efflux inhibitors can directly target antibiotic efflux, a primary resistance mechanism adopted by many species of bacteria and fungi. However, a much broader anti-infective capability resides within the action of these inhibitors, borne from the role of efflux in key physiological and virulence processes, including biofilm formation, toxin efflux, and stress management. Understanding how these behaviors manifest within complex polymicrobial communities is key to unlocking the full potential of the advanced repertoires of efflux inhibitors.

Developing student codesigned immersive virtual reality simulations for teaching of challenging concepts in molecular and cellular biology.

Molecular biology theory represents a critical scaffold, which underpins multiple disciplines within life sciences education. However, it is well-documented that undergraduate students can struggle to achieve deeper understanding of key concepts and/or their application. One challenging, contributory aspect is the "invisible" nature of molecular biology processes compounded by critical 3D spatial orientations of the principal components and their interactions. Molecular theory specifically requires students to construct accurate, mental spatial models to develop their understanding. However, much of the traditional teaching and examination of such theory is limited to 2D representations. Technology-enhanced, complementary teaching and examination approaches, which engage students with spatial aspects of theoretical concepts, offer an exciting opportunity to support student learning in this area. In this study, we have explored the integration of an immersive virtual reality simulation based on a challenging molecular biology concept within an existing module taught at University College Cork. A mixed methods approach, grounded in learning theory, was undertaken to assess the student user and learning experience. The consensus response from students was one of enhanced learning, understanding, engagement, and motivation. Student partnership in the process of simulation design and integration was key to delivering the fully integrated experience.

Osteointegration, antimicrobial and antibiofilm activity of orthopaedic titanium surfaces coated with silver and strontium-doped hydroxyapatite using a novel blasting process.

Poor integration of orthopaedic devices with the host tissue owing to aseptic loosening and device-associated infections are two of the leading causes of implant failure, which represents a significant problem for both patients and the healthcare system. Novel strategies have focused on silver to combat antimicrobial infections as an alternative to drug therapeutics. In this study, we investigated the impact of increasing the % substitution (12% wt) of silver and strontium in hydroxyapatite (HA) coatings to enhance antimicrobial properties and stimulate osteoblasts, respectively. Additionally, we prepared a binary substituted coating containing both silver and strontium (AgSrA) at 12% wt as a comparison. All coatings were deposited using a novel blasting process, CoBlast, onto biomedical grade titanium (V). Surface physicochemical properties, cytocompatibility and antimicrobial functionality were determined. The anticolonising properties of the coatings were screened using Staphylococcus aureus ATCC 1448, and thereafter, the AgA coating was evaluated using clinically relevant strains. Strontium-doped surfaces demonstrated enhanced osteoblast viability; however, a lower inhibition of biofilm formation was observed compared with the other surfaces. A co-substituted AgSrA surface did not show enhanced osteoblast or anticolonising properties compared with the SrA and AgA surfaces, respectively. Due to its superior anticolonising performance in preliminary studies, AgA was chosen for further studies. The AgA coated surfaces demonstrated good antibacterial activity (eluted and immobilised ion) against methicillin-resistant S. aureus followed by methicillin-sensitive Staphylococcus aureus clinical isolates; however, the AgA surface displayed poor impact against Staphylococcus epidermidis. In conclusion, herein, we demonstrate that HA can be substituted with a range of ions to augment the properties of HA coatings on orthopaedic devices, which offer promising potential to combat orthopaedic device-associated infections and enhance device performance.

The Effects of the Marine-Derived Polysaccharides Laminarin and Chitosan on Aspects of Colonic Health in Pigs Challenged with Dextran Sodium Sulphate.

This study examined the effects of dietary supplementation with laminarin or chitosan on colonic health in pigs challenged with dextran sodium sulphate (DSS). Weaned pigs were assigned to: (1) a basal diet (n = 22); (2) a basal diet + laminarin (n = 10); and (3) a basal diet + chitosan (n = 10). On d35, the basal group was split, creating four groups: (1) the basal diet (control); (2) the basal diet + DSS; (3) the basal diet + laminarin + DSS; and (4) the basal diet + chitosan + DSS. From d39-42, the pigs were orally challenged with DSS. On d44, colonic tissue/digesta samples were collected. The basal DSS group had reduced growth, higher pathology score and an increased expression of MMP1, IL13 and IL23 compared with the controls (p < 0.05); these parameters were similar between the DSS-challenged groups (p > 0.05). In the basal DSS group, the relative abundance of beneficial taxa including Prevotella and Roseburia were reduced while Escherichia/Shigella were increased, compared with the controls (p < 0.05). The relative abundance of Escherichia/Shigella was reduced and the molar proportions of acetate were increased in the laminarin DSS group compared with the basal DSS group (p < 0.01), suggesting that laminarin has potential to prevent pathogen proliferation and enhance the volatile fatty acid profile in the colon in a porcine model of colitis.

Grazing Cow Behavior's Association with Mild and Moderate Lameness.

Accelerometer-based mobility scoring has focused on cow behaviors such as lying and walking. Accuracy levels as high as 91% have been previously reported. However, there has been limited replication of results. Here, measures previously identified as indicative of mobility, such as lying bouts and walking time, were examined. On a research farm and a commercial farm, 63 grazing cows' behavior was monitored in four trials (16, 16, 16, and 15 cows) using leg-worn accelerometers. Seventeen good mobility (score 0), 23 imperfect mobility (score 1), and 22 mildly impaired mobility (score 2) cows were monitored. Only modest associations with activity, standing, and lying events were found. Thus, behavior monitoring appears to be insufficient to discern mildly and moderately impaired mobility of grazing cows.

Beyond heuristics: CFD-based novel multiparameter scale-up for geometrically disparate bioreactors demonstrated at industrial 2kL-10kL scales.

The timely delivery of the most up-to-date medicines and drug products is essential for patients throughout the world. Successful scaling of the bioreactors used within the biopharmaceutical industry plays a large part in the quality and time to market of these products. Scale and topology differences between vessels add a large degree of complication and uncertainty within the scaling process. Currently, this approach is primarily achieved through extensive experimentation and facile empirical correlations, which can be costly and time consuming while providing limited information. The work undertaken in the current study demonstrates a more robust and complete approach using computational fluid dynamics (CFD) to provide potent multiparameter scalability, which only requires geometric and material properties before a comprehensive and detailed solution can be generated. The CFD model output parameters that can be applied in the scale-up include mass transfer rates, mixing times, shear rates, gas hold-up values, and bubble residence times. The authors examined three bioreactors with variable geometries and were able to validate them based on single-phase and multiphase experiments. Furthermore, leveraging the resulting CFD output information enabled the authors to successfully scale-up from a known 2kL to a novel and disparate 5kL single-use bioreactor in the first attempted cell culture. This multiparameter scaling approach promises to ultimately lead to a reduction in the time to market providing patients with earlier access to the most groundbreaking medicines.

Transforming PPh3 into bidentate phosphine ligands at Ru-Zn heterobimetallic complexes.

The reaction of [Ru(PPh3)3Cl2] with excess ZnMe2 led to P-C/C-H bond activation and P-C/C-C bond formation to generate a chelating diphenylphosphinobenzene ligand as well as a cyclometallated (diphenylphosphino)biphenyl group in the final product of the reaction, [Ru(dppbz)(PPh2(biphenyl)')(ZnMe)] (1; dppbz = 1,2-bis(diphenylphosphino)benzene); PPh2(biphenyl)' = cyclometallated PPh2(biphenyl). The mechanism of reaction was studied and C-C coupling to give a bidentate 2,2'-bis(diphenylphosphino)biphenyl (BIPHEP) ligand was suggested to be one of the key steps of the process. This was confirmed by the reaction of [Ru(BIPHEP)(PPh3)HCl] with ZnMe2, which also gave 1. An analogous set of steps took place upon addition of ZnMe2 to [Ru(rac-BINAP)(PPh3)HCl] (rac-BINAP = racemic(2,2'-bis(diphenylphosphino)-1,1'-binaphthyl) to give [Ru(dppbz)(PPh2(binaphthyl)')ZnMe] (3). H2 and the C-H bond of PhC[triple bond, length as m-dash]CH added across the Ru-Zn bond of 1, and also reversed the phosphine cyclometallation, to give [Ru(dppbz)(Ph2P(biphenyl))(H)2(H)(ZnMe)] (4) and [Ru(dppbz)(Ph2P(biphenyl))(C[triple bond, length as m-dash]CPh)2(H)(ZnMe)] (5) respectively.

Dominance of the genus Polaromonas in the microbial ecology of an Intermittently Aerated Sequencing Batch Reactor (IASBR) treating dairy processing wastewater under varying aeration rates.

In this Research Communication we investigate potential correlations between key bacterial groups and nutrient removal efficiency in an Intermittently Aerated Sequencing Batch Reactor (IASBR) treating synthetic dairy processing wastewater. Reactor aeration rates of 0·6 and 0·4 litre per minute (LPM) were applied to an 8 l laboratory scale system and the relative impacts on IASBR microbial community structure and orthophosphate (PO4-P) and ammonium (NH4-N) removal efficiencies compared. Aeration at 0·6 LPM over several sludge retention times (SRTs) resulted in approximately 92% removal efficiencies for both PO4-P and NH4-N. Biomass samples subjected to next-generation sequencing (NGS), 16S rRNA profiling revealed a concomitant enrichment of Polaromonas under 0·6 LPM conditions, up to ~50% relative abundance within the reactor biomass. The subsequent shift in reactor aeration to 0·4 LPM, over a period of 3 SRTs, resulted in markedly reduced nutrient removal efficiencies for PO4-P (50%) and NH4-N (45%). An 85·7% reduction in the genus level relative abundance of Polaromonas was observed under 0·4 LPM aeration conditions over the same period.

DairyWater: striving for sustainability within the dairy processing industry in the Republic of Ireland.

This Review describes the objectives and methodology of the DairyWater project as it aims to aid the Irish dairy processing industry in achieving sustainability as it expands. With the abolition of European milk quotas in March 2015, the Republic of Ireland saw a surge in milk production. The DairyWater project was established in anticipation of this expansion of the Irish dairy sector in order to develop innovative solutions for the efficient management of water consumption, wastewater treatment and the resulting energy use within the country's dairy processing industry. Therefore, the project can be divided into three main thematic areas: dairy wastewater treatment technologies and microbial analysis, water re-use and rainwater harvesting and environmental assessment. In order to ensure the project remains as relevant as possible to the industry, a project advisory board containing key industry stakeholders has been established. To date, a number of large scale studies, using data obtained directly from the Irish dairy industry, have been performed. Additionally, pilot-scale wastewater treatment (intermittently aerated sequencing batch reactor) and tertiary treatment (flow-through pulsed ultraviolet system) technologies have been demonstrated within the project. Further details on selected aspects of the project are discussed in greater detail in the subsequent cluster of research communications.

Evaluation of dairy processing wastewater biotreatment in an IASBR system: Aeration rate impacts on performance and microbial ecology.

Dairy processing generates large volumes of wastewater that require extensive nutrient remediation prior to discharge. Significant commercial opportunities exist therefore for cost-effective biotechnologies capable of achieving this requirement. In this study the authors evaluated the use of intermittently aerated sequencing batch reactors, (IASBRs), as a single-tank biotreatment system for co-removal of COD, nitrogen and phosphorus from synthetic dairy processing wastewater. Variation of the IASBR aeration rates, (0.8, 0.6 and 0.4 L/min), had significant impacts on the respective nutrient removal efficiencies and underlying microbial diversity profiles. Aeration at 0.6 L/min was most effective and resulted in >90% co-removal of orthophosphate and ammonium. 16S rRNA based pyrosequencing of biomass DNA samples revealed the family Comamonadaceae was notably enriched (>80% relative abundance) under these conditions. In silico predictive metabolic modelling also identified Comamonadaceae as the major contributor of several known genes for nitrogen and phosphorus assimilation (nirK, nosZ, norB, ppK, ppX and phbC).

Draft Genome Sequence of Pseudomonas putida CA-3, a Bacterium Capable of Styrene Degradation and Medium-Chain-Length Polyhydroxyalkanoate Synthesis.

Pseudomonas putida strain CA-3 is an industrial bioreactor isolate capable of synthesizing biodegradable polyhydroxyalkanoate polymers via the metabolism of styrene and other unrelated carbon sources. The pathways involved are subject to regulation by global cellular processes. The draft genome sequence is 6,177,154 bp long and contains 5,608 predicted coding sequences.

Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3.

Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed.

Aquimarina amphilecti sp. nov., isolated from the sponge Amphilectus fucorum.

A Gram-stain-negative, rod-shaped, orange-coloured, catalase- and oxidase-positive, non-motile bacterium, designated strain 92V(T), was isolated from the marine sponge Amphilectus fucorum, collected from Lough Hyne, County Cork, Ireland. 16S rRNA gene sequence analysis revealed that strain 92V(T) clustered with members of the family Flavobacteriaceae, the closest member being Aquimarina latercula NCIMB 1399(T), with a gene sequence similarity of 97.5%. Strain 92V(T) required seawater for growth with optimal growth occurring at 25 °C, at pH 6-7 and with 3% (w/v) NaCl. MK-6 was the sole respiratory quinone present and the major fatty acids were iso-C(17 : 0) 3-OH, iso-C(15 : 0), iso-C(17 : 1)ω9c and iso-C(15 : 0) 3-OH. The DNA G+C content was 36.1 mol%. Combined phenotypic differences and phylogenetic analysis indicate that strain 92V(T) represents a novel species of the genus Aquimarina, for which the name Aquimarina amphilecti sp. nov. is proposed. The type strain is 92V(T) ( = NCIMB 14723(T) = DSM 25232(T)).

GacS-dependent regulation of polyhydroxyalkanoate synthesis in Pseudomonas putida CA-3.

To date, limited reports are available on the regulatory systems exerting control over bacterial synthesis of the biodegradable polyester group known as polyhydroxyalkanoates (PHAs). In this study, we performed random mini-Tn5 mutagenesis of the Pseudomonas putida CA-3 genome and screened transconjugants on nitrogen-limited medium for reduced PHA accumulation phenotypes. Disruption of a GacS sensor kinase in one such mutant was found to eliminate medium-chain-length PHA production in Pseudomonas putida CA-3. Recombinant expression of wild-type gacS from a pBBRgacS vector fully restored PHA accumulation capacity in the mutant strain. PCR-based screening of the P. putida CA-3 genome identified gene homologues of the GacS/GacA-rsm small RNA (sRNA) regulatory cascade with 96% similarity to published P. putida genomes. However, reverse transcription-PCR (RT-PCR) analyses revealed active transcription of the rsmY and rsmZ sRNAs in gacS-disrupted P. putida CA-3, which is atypical of the commonly reported Gac/Rsm regulatory cascade. Quantitative real-time RT-PCR analyses of the phaC1 synthase responsible for polymer formation in P. putida CA-3 indicated no statistically significant difference in transcript levels between the wild-type and gacS-disrupted strains. Subsequently, SDS-PAGE protein analyses of these strains identified posttranscriptional control of phaC1 synthase as a key aspect in the regulation of PHA synthesis by P. putida CA-3.

Regulation of phenylacetic acid uptake is σ54 dependent in Pseudomonas putida CA-3.

BACKGROUND: Styrene is a toxic and potentially carcinogenic alkenylbenzene used extensively in the polymer processing industry. Significant quantities of contaminated liquid waste are generated annually as a consequence. However, styrene is not a true xenobiotic and microbial pathways for its aerobic assimilation, via an intermediate, phenylacetic acid, have been identified in a diverse range of environmental isolates. The potential for microbial bioremediation of styrene waste has received considerable research attention over the last number of years. As a result the structure, organisation and encoded function of the genes responsible for styrene and phenylacetic acid sensing, uptake and catabolism have been elucidated. However, a limited understanding persists in relation to host specific regulatory molecules which may impart additional control over these pathways. In this study the styrene degrader Pseudomonas putida CA-3 was subjected to random mini-Tn5 mutagenesis and mutants screened for altered styrene/phenylacetic acid utilisation profiles potentially linked to non-catabolon encoded regulatory influences. RESULTS: One mutant, D7, capable of growth on styrene, but not on phenylacetic acid, harboured a Tn5 insertion in the rpoN gene encoding σ54. Complementation of the D7 mutant with the wild type rpoN gene restored the ability of this strain to utilise phenylacetic acid as a sole carbon source. Subsequent RT-PCR analyses revealed that a phenylacetate permease, PaaL, was expressed in wild type P. putida CA-3 cells utilising styrene or phenylacetic acid, but could not be detected in the disrupted D7 mutant. Expression of plasmid borne paaL in mutant D7 was found to fully restore the phenylacetic acid utilisation capacity of the strain to wild type levels. Bioinformatic analysis of the paaL promoter from P. putida CA-3 revealed two σ54 consensus binding sites in a non-archetypal configuration, with the transcriptional start site being resolved by primer extension analysis. Comparative analyses of genomes encoding phenylacetyl CoA, (PACoA), catabolic operons identified a common association among styrene degradation linked PACoA catabolons in Pseudomonas species studied to date. CONCLUSIONS: In summary, this is the first study to report RpoN dependent transcriptional activation of the PACoA catabolon paaL gene, encoding a transport protein essential for phenylacetic acid utilisation in P. putida CA-3. Bioinformatic analysis is provided to suggest this regulatory link may be common among styrene degrading Pseudomonads.

The contribution of 'omic'-based approaches to the study of enhanced biological phosphorus removal microbiology.

The role that microorganisms play in the biological removal of phosphate from wastewater streams has received sustained interest since its initial observation over 30 years ago. Recent advances in 'omic'-based approaches have greatly advanced our knowledge in this field and facilitated a refinement of existing enhanced biological phosphate removal (EBPR) models, which were primarily based on culture-dependent approaches that had predominantly been used to investigate the process. This minireview will focus on the recent advances made in our overall understanding of the EBPR process resulting from the use of 'omic'-based methodologies.

Effect of heterologous expression of phaG [(R)-3-hydroxyacyl-ACP-CoA transferase] on polyhydroxyalkanoate accumulation from the aromatic hydrocarbon phenylacetic acid in Pseudomonas species.

Five Pseudomonas strains capable of growth with the aromatic carboxylic acid phenylacetic acid were investigated with a view to improving PHA accumulation. The overexpression of (R)-3-hydroxyacyl-ACP-CoA transferase (PhaG) from Pseudomonas putida CA-3 increased PHA accumulation in only one of the five strains tested, namely Pseudomonas jessenii C8. Recombinant P. jessenii C8 harbouring the phaG gene showed a 4.1-fold increase (9.6-39% cell dry weight) in PHA accumulation when grown on phenylacetic acid (15 mM) compared with the wild-type strain. This is the highest reported level of PHA accumulation from phenylacetic acid. This is also the first time the heterologous expression of phaG has resulted in improved PHA accumulation from an aromatic carbon source. The growth patterns of the wild type and recombinant strains were very similar, with no significant differences observed in carbon and nitrogen utilization.

Cloning and functional characterization of the styE gene, involved in styrene transport in Pseudomonas putida CA-3.

A 1.5-kb region immediately downstream of the styABCD operon involved in styrene degradation in Pseudomonas putida CA-3 has been cloned. Sequence analysis revealed a 1,296-bp open reading frame, designated styE, and BLAST P database comparisons of the deduced StyE amino acid sequence revealed 33 to 98% identity with several membrane-associated ATPase-dependent kinase proteins involved in the active transport of aromatic hydrocarbons across bacterial membranes and also with FadL, an outer membrane protein necessary for the uptake of long-chain fatty acids in Escherichia coli. Transcription of styE is styrene dependent, and the gene is cotranscribed with the styABCD structural genes. StyE appears to be membrane associated, with a corresponding 45.9-kDa band being identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane preparations from styrene-grown cells. P. putida CA-3 cells in which the styE gene had been interrupted were no longer capable of growth on styrene. In contrast, overexpression of styE in P. putida CA-3 resulted in a 4.2-fold increase in styrene monooxygenase activity compared with wild-type cells grown on styrene, with a concomitant 8-fold increase in styA mRNA transcript levels. Experiments with the classic, ATPase inhibitor vanadate revealed that growth of wild-type cells on styrene was inhibited at a concentration of 1 mM, while 1.75 mM was required to achieve a similar effect in the StyE overexpression strain. Growth of either strain on citrate was not inhibited in the presence of up to 7 mM vanadate. These findings suggest a role for StyE in the active transport of styrene in Pseudomonas putida CA-3 and identify styrene transport as a potentially limiting factor with respect to mRNA transcript levels and associated enzymatic activity of the styrene degradative pathway.