Metabarcoding of marine zooplankton: prospects, progress and pitfalls

Metabarcoding (large-scale taxonomic identification of complex samples via analysis of one or few orthologous DNA regions, called barcodes) is revolutionizing analysis of biodiversity of marine zooplankton assemblages. Metabarcoding relies on high-throughput DNA sequencing (HTS) technologies, which yield millions of DNA sequences in parallel and allow large-scale analysis of environmental samples. Metabarcoding studies of marine zooplankton have used various regions of nuclear small- (18S) and large-subunit (28S) rRNA, which allow accurate classification of novel sequences and reliable amplification with consensus primers, but- due to their relatively conserved nature- may underestimate species diversity in a community. To discriminate species, more variable genes are needed. A limited number of metabarcoding studies have used mitochondrial cytochrome oxidase I (COI), which ensures detection of species-level diversity, but may require group-specific primers and thus result in inconsistent amplification success rates. Reference databases with sequences for accurately-identified species are critically needed to allow taxonomic designation of molecular operational taxonomic units (MOTU) and comparison with previous studies of zooplankton diversity. Potential and promising applications of metabarcoding include rapid detection of impacts of climate change, monitoring and assessment of ecosystem health, calculation of biotic indices, characterization of food webs and detection of introduced, non-indigenous species

Integrating conventional microscopy and molecular analysis to analyse the abundance and distribution of four Calanus congeners in the North Atlantic

Analysis of the demographic structure of Calanus species in the North Atlantic presents particular difficulties due to the overlapping spatial distributions of four main congeneric species (Calanus finmarchicus, Calanus helgolandicus, Calanus glacialis and Calanus hyperboreus). These species have similar morphologies, making microscopic discrimination only possible between some of the species at late copepodite or adult stages. However, molecular techniques now offer the possibility of screening significant numbers of specimens and unambiguously identifying them to species, regardless of developmental stage. Unfortunately, the processing rate of specimens by molecular methods is still too low to offer a realistic alternative to microscopy for analysis of samples from large field surveys. Here, we outline and test an approach involving the use of molecular methodology in conjunction with conventional microscopy to assess the species assignment of developmental stage abundances of Calanus congeners. Our study has highlighted many important methodological issues. First, it cannot be assumed that the species composition is homogeneous across the development stages; applying proportional species composition of adults to morphologically undistinguishable earlier development stages can result in error. The second important conclusion is that prosome length may be a highly unreliable discriminator of C. finmarchicus and C. glaciali

A Single-Arm, Proof-Of-Concept Trial of Lopimune (Lopinavir/Ritonavir) as a Treatment for HPV-Related Pre-Invasive Cervical Disease

BACKGROUND: Cervical cancer is the most common female malignancy in the developing nations and the third most common cancer in women globally. An effective, inexpensive and self-applied topical treatment would be an ideal solution for treatment of screen-detected, pre-invasive cervical disease in low resource settings. METHODS: Between 01/03/2013 and 01/08/2013, women attending Kenyatta National Hospital's Family Planning and Gynaecology Outpatients clinics were tested for HIV, HPV (Cervista®) and liquid based cervical cytology (LBC -ThinPrep®). HIV negative women diagnosed as high-risk HPV positive with high grade squamous intraepithelial lesions (HSIL) were examined by colposcopy and given a 2 week course of 1 capsule of Lopimune (CIPLA) twice daily, to be self-applied as a vaginal pessary. Colposcopy, HPV testing and LBC were repeated at 4 and 12 weeks post-start of treatment with a final punch biopsy at 3 months for histology. Primary outcome measures were acceptability of treatment with efficacy as a secondary consideration. RESULTS: A total of 23 women with HSIL were treated with Lopimune during which time no adverse reactions were reported. A maximum concentration of 10 ng/ml of lopinavir was detected in patient plasma 1 week after starting treatment. HPV was no longer detected in 12/23 (52.2%, 95%CI: 30.6-73.2%). Post-treatment cytology at 12 weeks on women with HSIL, showed 14/22 (63.6%, 95%CI: 40.6-82.8%) had no dysplasia and 4/22 (18.2%, 95%CI: 9.9-65.1%) were now low grade demonstrating a combined positive response in 81.8% of women of which 77.8% was confirmed by histology. These data are supported by colposcopic images, which show regression of cervical lesions. CONCLUSIONS: These results demonstrate the potential of Lopimune as a self-applied therapy for HPV infection and related cervical lesions. Since there were no serious adverse events or detectable post-treatment morbidity, this study indicates that further trials are clearly justified to define optimal regimes and the overall benefit of this therapy. TRIAL REGISTRATION: ISRCTN Registry 48776874

Using protein engineering to accelerate implementation of continuous biocatalysis for API synthesis

Biocatalysis is an invaluable tool for the synthesis of many active pharmaceutical ingredients1,2. Enzymatic reactions are frequently performed in batch, but continuous biocatalysis is gaining interest in industry3, not least because it would allow better integration of chemical and enzymatic reaction steps4. However, selecting a suitable reactor configuration for continuous biocatalysis is often challenging due to the common limitations of enzymes, such as poor kinetic constants towards industrially relevant substrates, substrate/product inhibition, the need for an aqueous reaction environment and/or pH control. For this reason, we have recently developed a tool5 that presents a variety of reactor configurations, combining continuously stirred tank reactors (CSTRs) and continuous plug-flow reactors (CPFRs), to overcome the aforementioned limitations and facilitate reactor selection. However, the continued development of protein engineering technologies6,7 has revealed one of the strongest advantages of biocatalysis over chemocatalysis, namely that the properties of the catalyst can be modified to remove limitations and better suit industrial production processes.8,9 In this presentation we highlight how protein engineering, guided by process-specific targets, can streamline process development. For example, reducing product inhibition by protein engineering could allow operation in a single CPFR where the possibility of complete conversion greatly simplifies downstream processing, improving the tolerance of enzymes towards organic solvents could allow water-free operation without mass transfer limitations between phases and improving pH tolerance could remove the need for acid/base addition to combat pH changes during operation. Such changes to the catalyst could greatly facilitate process development in an industry where reduction of time-to-market is often critical. (1) Pollard, D. J.; Woodley, J. M. Trends Biotechnol. 2007, 25, 66–73. (2) Devine, P. N.; Howard, R. M.; Kumar, R.; Thompson, M. P.; Truppo, M. D.; Turner, N. J. Nat. Rev. Chem. 2018, 2, 409–421. (3) Tamborini, L.; Fernandes, P.; Paradisi, F.; Molinari, F. Trends Biotechnol. 2018, 36, 73–88. (4) Rudroff, F.; Mihovilovic, M. D.; Gröger, H.; Snajdrova, R.; Iding, H.; Bornscheuer, U. T. Nat. Catal. 2018, 1, 12–22. (5) Lindeque, R. M.; Woodley, J. M. Catalysts 2019, 9, 262. (6) Arnold, F. H. Angew. Chem. Int. Ed. 2018, 57, 4143–4148. (7) Yang, K. K.; Wu, Z.; Arnold, F. H. arXiv:1811.10775 2018. (8) Woodley, J. M. Curr. Opin. Chem. Biol. 2013, 17, 310–316. (9) Woodley, J. M. Phil. Trans. R. Soc. A 2017, 376

Microplastic ingestion ubiquitous in marine turtles

Despite concerns regarding the environmental impacts of microplastics, knowledge of the incidence and levels of synthetic particles in large marine vertebrates is lacking. Here, we utilize an optimized enzymatic digestion methodology, previously developed for zooplankton, to explore whether synthetic particles could be isolated from marine turtle ingesta. We report the presence of synthetic particles in every turtle subjected to investigation (n = 102) which included individuals from all seven species of marine turtle, sampled from three ocean basins (Atlantic [ATL]: n = 30, four species; Mediterranean (MED): n = 56, two species; Pacific (PAC): n = 16, five species). Most particles (n = 811) were fibres (ATL: 77.1% MED: 85.3% PAC: 64.8%) with blue and black being the dominant colours. In lesser quantities were fragments (ATL: 22.9%: MED: 14.7% PAC: 20.2%) and microbeads (4.8%; PAC only; to our knowledge the first isolation of microbeads from marine megavertebrates). Fourier transform infrared spectroscopy (FT‐IR) of a subsample of particles (n = 169) showed a range of synthetic materials such as elastomers (MED: 61.2%; PAC: 3.4%), thermoplastics (ATL: 36.8%: MED: 20.7% PAC: 27.7%) and synthetic regenerated cellulosic fibres (SRCF; ATL: 63.2%: MED: 5.8% PAC: 68.9%). Synthetic particles being isolated from species occupying different trophic levels suggest the possibility of multiple ingestion pathways. These include exposure from polluted seawater and sediments and/or additional trophic transfer from contaminated prey/forage items. We assess the likelihood that microplastic ingestion presents a significant conservation problem at current levels compared to other anthropogenic threats

Isolation of microplastics in biota-rich seawater samples and marine organisms.

notes: PMCID: PMC3970126types: Journal Article; Research Support, Non-U.S. Gov'tThis is an open access article that is freely available in ORE or from the publisher's web site. Please cite the published version.Microplastic litter is a pervasive pollutant present in aquatic systems across the globe. A range of marine organisms have the capacity to ingest microplastics, resulting in adverse health effects. Developing methods to accurately quantify microplastics in productive marine waters, and those internalized by marine organisms, is of growing importance. Here we investigate the efficacy of using acid, alkaline and enzymatic digestion techniques in mineralizing biological material from marine surface trawls to reveal any microplastics present. Our optimized enzymatic protocol can digest >97% (by weight) of the material present in plankton-rich seawater samples without destroying any microplastic debris present. In applying the method to replicate marine samples from the western English Channel, we identified 0.27 microplastics m(-3). The protocol was further used to extract microplastics ingested by marine zooplankton under laboratory conditions. Our findings illustrate that enzymatic digestion can aid the detection of microplastic debris within seawater samples and marine biota.Natural Environment Research Council (NERC

The growing need to assess the kinetic stability of enzymes

1. Bommarius, A.S., Paye, M.F. (2013) Chem. Soc. Rev., 42, 6534. 2. Sheldon, R.A., Woodley, J.M. (2018) Chem. Rev., 118, 801. 3. Bhagia, S., Dhir, R., Kumar, R., Wyman, C.E. (2018) Sci. Rep., 8, 1350. 4. Thomas, C., Geer, D. (2011) Biotechnol. Lett., 33, 443. 5. Rehn, G., Toftgaard Pedersen, A., Woodley, J.M. (2016) J. Mol. Catal. B Enym., 134, 331

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation

Communication skills for medical/dental students at the University of Pretoria: lessons learnt from a two-year study using a forum theatre method

Background: This study describes the lessons learnt from using a novel method for teaching communication skills to second-year medical/dental students.Methods: Medical and drama teachers designed this action research project to serve the educational interests of secondyear medical/dental and drama students. The drama students enacted problematic doctor-patient scenarios for their forum theatre course. The interactive enactment was done for groups of 60-70 medical/dental students. The latter interrupted the actors to suggest improved communication skills. The drama students then re-enacted the scenarios, incorporating the improvements. The medical/dental students\' knowledge of communication skills was assessed before the enactment, three weeks later, and again four months after that. Their semi-structured feedback was analysed thematically. In the next year, the feedback was used to improve the methodology for the new second-year students. Results: In both years, the medical/dental students\' knowledge showed a statistically significant improvement after the enactment, and this was sustained for four months. In year 1, the feedback revolved around language problems and disrespectful attitudes. In year 2, visual cue cards of the communication skills were displayed during the act, and the drama students emphasised these rather than attitudinal problems. However, feedback showed that caricaturing the doctors\' attitudes still detracted attention from the desired focus on communication skills. Conclusions: Although the forum theatre method can transfer knowledge of communication skills, the focus of the acting should be on the demonstration of inappropriate communication skills rather than inappropriate attitudes. One limitation of this study is that assessment was limited to knowledge and did not progress to skills. South African Family Practice Vol. 47(6) 2005: 60-6