Biodiversity patterns and conservation of the coastal forests of Eastern Africa

The earth is in the midst of a sixth major extinction crisis with biodiversity highly threatened by climate change at global scales. Biodiversity is crucial to humans due to the ecosystem functioning and services it provides, and therefore the conservation of biodiversity is paramount to human prosperity in in the future. This PhD thesis uses amphibians to examine biodiversity patterns across the Coastal Forests of Eastern Africa (CFEA), a global biodiversity hotspot. The CFEA comprises of a network of tiny fragmented forest patches thought to be the remnants of a once widespread tropical forest that spanned across tropical Africa from West to East prior to major tectonic activity in the Miocene. The gradual aridification of the African continent since then, combined with significant climate and sea level oscillations during the Pliocene and Pleistocene contributed to the natural fragmentation of the CFEA, but human impacts have severely accelerated the pace of forest loss. The work in this thesis integrates field work, taxonomy, morphology, molecular techniques and spatial data to measure biodiversity across the CFEA, explain its distribution and provide a conceptual framework in which this data can be usefully applied for future conservation efforts. A broad scale DNA barcoding project forms the basis of all work in the thesis, assimilating museum specimens and archived spatial records with newly collected data from recent fieldwork to create the most thorough inventory of the CFEA amphibians currently known. Chapter 1 focuses on the use of Next Generation Sequencing (NGS) data for five widespread species clades to estimate phylogeny and population structure, genetic distances between populations, and explain these patterns using long-term environmental data. Chapter 2 maps the spatial distribution of evolutionary history measured from phylogenetic branch lengths for multiple intraspecific lineages within species, highlighting places which appear to be refugia and examining their environmental correlates and conservation. Chapter 3 uses close to the full assemblage of Tanzania and Kenya (55 species) to categorize the types of endemism present, distinguishing areas that are ‘museums’ supporting ancient diversity (paleo-endemism) from ‘cradles’ that support recently evolved diversity (neo-endemism). Chapter 4 combines morphological, genetic and spatial data to describe a new endemic species of treefrog, Hyperolius ruvuensis, from a highly threatened reserve in coastal Tanzania. A synthesis chapter summarises the work in the thesis and outlines new directions for CFEA conservation

Compounds enhancing human sperm motility identified using a high-throughput phenotypic screening platform

STUDY QUESTION: Can a high-throughput screening (HTS) platform facilitate male fertility drug discovery? SUMMARY ANSWER: An HTS platform identified a large number of compounds that enhanced sperm motility. WHAT IS KNOWN ALREADY: Several efforts to find small molecules modulating sperm function have been performed but none have used high-throughput technology. STUDY DESIGN, SIZE, DURATION: Healthy donor semen samples were used and samples were pooled (3–5 donors per pool). Primary screening was performed singly; dose–response screening was performed in duplicate (using independent donor pools). PARTICIPANTS/MATERIALS, SETTING, METHODS: Spermatozoa isolated from healthy donors were prepared by density gradient centrifugation and incubated in 384-well plates with compounds (6.25 μM) to identify those compounds with enhancing effects on motility. Approximately 17 000 compounds from the libraries, ReFRAME, Prestwick, Tocris, LOPAC, CLOUD and MMV Pathogen Box, were screened. Dose–response experiments of screening hits were performed to confirm the enhancing effect on sperm motility. Experiments were performed in a university setting. MAIN RESULTS AND THE ROLE OF CHANCE: From our primary single concentration screening, 105 compounds elicited an enhancing effect on sperm motility compared to dimethylsulphoxide-treated wells. Confirmed enhancing compounds were grouped based on their annotated targets/target classes. A major target class, phosphodiesterase inhibitors, were identified, in particular PDE10A inhibitors as well as number of compounds not previously known to enhance human sperm motility, such as those related to GABA signalling. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although this approach provides data about the activity of the compound, it is only a starting point. For example, further substantive experiments are necessary to provide a more comprehensive picture of each compound’s activity, the effect on the kinetics of the cell populations and subpopulations, and their potential mechanisms of action. Compounds have been tested with prepared donor spermatozoa, incubated under non-capacitating conditions, and only incubated with compounds for a relatively short period of time. Therefore, the effect of compounds under different conditions, for example in whole semen, for longer incubation times, or using samples from patient groups, may be different and require further study. All experiments were performed in vitro. WIDER IMPLICATIONS OF THE FINDINGS: This phenotypic screening assay identified a large number of compounds that increased sperm motility. In addition to furthering our understanding of human sperm function, for example identifying new avenues for discovery, we highlight potential compounds as promising start-point for a medicinal chemistry programme for potential enhancement of male fertility. Moreover, with disclosure of the results of screening, we present a substantial resource to inform further work in the field. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Bill and Melinda Gates Foundation, Scottish Funding Council and Scottish Universities Life Science Alliance. C.L.R.B. is Editor for RBMO. C.L.R.B. receives funding from Chief Scientists Office (Scotland), ESHRE and Genus PLC, consulting fees from Exscientia and lecture fees from Cooper Surgical and Ferring. S.M.d.S. is an Associate Editor of Human Reproduction, and an Associate Editor of Reproduction and Fertility. S.M.d.S. receives funding from Cooper Surgical and British Dietetic Society. No other authors declared a COI

Sperm Toolbox-A selection of small molecules to study human spermatozoa

Male contraceptive options and infertility treatments are limited, and almost all innovation has been limited to updates to medically assisted reproduction protocols and methods. To accelerate the development of drugs that can either improve or inhibit fertility, we established a small molecule library as a toolbox for assay development and screening campaigns using human spermatozoa. We have profiled all compounds in the Sperm Toolbox in several automated high-throughput assays that measure stimulation or inhibition of sperm motility or the acrosome reaction. We have assayed motility under non-capacitating and capacitating conditions to distinguish between pathways operating under these different physiological states. We also assayed cell viability to ensure any effects on sperm function are specific. A key advantage of our studies is that all compounds are assayed together in the same experimental conditions, which allows quantitative comparisons of their effects in complementary functional assays. We have combined the resulting datasets to generate fingerprints of the Sperm Toolbox compounds on sperm function. The data are included in an on-line R-based app for convenient querying.</p

Sperm Toolbox-A selection of small molecules to study human spermatozoa

Male contraceptive options and infertility treatments are limited, and almost all innovation has been limited to updates to medically assisted reproduction protocols and methods. To accelerate the development of drugs that can either improve or inhibit fertility, we established a small molecule library as a toolbox for assay development and screening campaigns using human spermatozoa. We have profiled all compounds in the Sperm Toolbox in several automated high-throughput assays that measure stimulation or inhibition of sperm motility or the acrosome reaction. We have assayed motility under non-capacitating and capacitating conditions to distinguish between pathways operating under these different physiological states. We also assayed cell viability to ensure any effects on sperm function are specific. A key advantage of our studies is that all compounds are assayed together in the same experimental conditions, which allows quantitative comparisons of their effects in complementary functional assays. We have combined the resulting datasets to generate fingerprints of the Sperm Toolbox compounds on sperm function. The data are included in an on-line R-based app for convenient querying.</p

Glycosylation increases active site rigidity leading to improved enzyme stability and turnover

Glycosylation is the most prevalent protein post‐translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet, the full impact of glycosylation on the protein structure–function relationship, especially in enzymes, is still limited. Here, we show that glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its turnover and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo‐HRP's thermal stability and promoted significant helical structure in the absence of haem (apo‐HRP). Glycosylation also resulted in a 10‐fold increase in enzymatic turnover towards o‐phenylenediamine dihydrochloride when compared to its nonglycosylated form. Utilising a naturally occurring site‐specific probe of active site flexibility (Trp117) in combination with red‐edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data show that glycosylation does not just have a passive effect on HRP stability but can exert long‐range effects that mediate the ‘native’ enzyme's activity and stability through changes in inherent dynamics

Effects of microbiota-directed foods in gnotobiotic animals and undernourished children

To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of fecal samples to characterize the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned, after standard treatment, to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes targeting weaning-phase bacterial taxa underrepresented in SAM and MAM microbiota were characterized in gnotobiotic mice and gnotobiotic piglets colonized with age- and growth-discriminatory bacteria. A randomized, double-blind controlled feeding study identified a lead MDCF that changes the abundances of targeted bacteria and increases plasma biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function in children with MAM

Quantitative estimates of glacial refugia for chimpanzees (Pan troglodytes) since the Last Interglacial (120,000 BP)

Paleoclimate reconstructions have enhanced our understanding of how past climates have shaped present-day biodiversity. We hypothesize that the geographic extent of Pleistocene forest refugia and suitable habitat fluctuated significantly in time during the late Quaternary for chimpanzees (Pan troglodytes). Using bioclimatic variables representing monthly temperature and precipitation estimates, past human population density data, and an extensive database of georeferenced presence points, we built a model of changing habitat suitability for chimpanzees at fine spatio-temporal scales dating back to the Last Interglacial (120,000 BP). Our models cover a spatial resolution of 0.0467° (approximately 5.19 km2 grid cells) and a temporal resolution of between 1000 and 4000 years. Using our model, we mapped habitat stability over time using three approaches, comparing our modeled stability estimates to existing knowledge of Afrotropical refugia, as well as contemporary patterns of major keystone tropical food resources used by chimpanzees, figs (Moraceae), and palms (Arecacae). Results show habitat stability congruent with known glacial refugia across Africa, suggesting their extents may have been underestimated for chimpanzees, with potentially up to approximately 60,000 km2 of previously unrecognized glacial refugia. The refugia we highlight coincide with higher species richness for figs and palms. Our results provide spatio-temporally explicit insights into the role of refugia across the chimpanzee range, forming the empirical foundation for developing and testing hypotheses about behavioral, ecological, and genetic diversity with additional data. This methodology can be applied to other species and geographic areas when sufficient data are available.Additional co-authors: Alfred K. Assumang, Emma Bailey, Mattia Bessone, Bartelijntje Buys, Joana S. Carvalho, Rebecca Chancellor, Heather Cohen, Emmanuel Danquah, Tobias Deschner, Zacharie N. Dongmo, Osiris A. Doumbé, Jef Dupain, Chris S. Duvall, Manasseh Eno-Nku, Gilles Etoga, Anh Galat-Luong, Rosa Garriga, Sylvain Gatti, Andrea Ghiurghi, Annemarie Goedmakers, Anne-Céline Granjon, Dismas Hakizimana, Josephine Head, Daniela Hedwig, Ilka Herbinger, Veerle Hermans, Sorrel Jones, Jessica Junker, Parag Kadam, Mohamed Kambi, Ivonne Kienast, Célestin Y. Kouakou, Kouamé P. N′Goran, Kevin E. Langergraber, Juan Lapuente, Anne Laudisoit, Kevin C. Lee, Nadia Mirghani, Deborah Moore, David Morgan, Emily Neil, Sonia Nicholl, Louis Nkembi, Anne Ntongho, Christopher Orbell, Lucy Jayne Ormsby, Liliana Pacheco, Alex K. Piel, Lilian Pintea, Andrew J. Plumptre, Aaron Rundus, Crickette Sanz, Volker Sommer, Tenekwetche Sop, Fiona A. Stewart, Jacqueline Sunderland-Groves, Nikki Tagg, Angelique Todd, Els Ton, Joost van Schijndel, Hilde VanLeeuwe, Elleni Vendras, Adam Welsh, José F. C. Wenceslau, Erin G. Wessling, Jacob Willie, Roman M. Wittig, Nakashima Yoshihiro, Yisa Ginath Yuh, Kyle Yurkiw, Christophe Boesch, Mimi Arandjelovic, Hjalmar Küh

Author Correction: Environmental variability supports chimpanzee behavioural diversity

The original version of the Supplementary Information associated with this Article included an incorrect Supplementary Data 1 file, in which three columns (L, M and P) had slightly different variable names from those written in the code. The HTML has been updated to include a corrected version of Supplementary Data 1; the correct version of Supplementary Data 1 can be found as Supplementary Information associated with this Correction.Additional co-authors: Mattia Bessone, Gregory Brazzola, Valentine Ebua Buh, Rebecca Chancellor, Heather Cohen, Charlotte Coupland, Bryan Curran, Emmanuel Danquah, Tobias Deschner, Dervla Dowd, Manasseh Eno-Nku, J. Michael Fay, Annemarie Goedmakers, Anne-Céline Granjon, Josephine Head, Daniela Hedwig, Veerle Hermans, Sorrel Jones, Jessica Junker, Parag Kadam, Mohamed Kambi, Ivonne Kienast, Deo Kujirakwinja, Kevin E. Langergraber, Juan Lapuente, Bradley Larson, Kevin C. Lee, Vera Leinert, Manuel Llana, Sergio Marrocoli, Amelia C. Meier, David Morgan, Emily Neil, Sonia Nicholl, Emmanuelle Normand, Lucy Jayne Ormsby, Liliana Pacheco, Alex Piel, Jodie Preece, Martha M. Robbins, Aaron Rundus, Crickette Sanz, Volker Sommer, Fiona Stewart, Nikki Tagg, Claudio Tennie, Virginie Vergnes, Adam Welsh, Erin G. Wessling, Jacob Willie, Roman M. Wittig, Yisa Ginath Yuh, Klaus Zuberbühler & Hjalmar S. Küh