Discovery of a Biotin Synthase That Utilizes an Auxiliary 4Fe-5S Cluster for Sulfur Insertion.

Biotin synthase (BioB) is a member of the Radical SAM superfamily of enzymes that catalyzes the terminal step of biotin (vitamin B7) biosynthesis, in which it inserts a sulfur atom in desthiobiotin to form a thiolane ring. How BioB accomplishes this difficult reaction has been the subject of much controversy, mainly around the source of the sulfur atom. However, it is now widely accepted that the sulfur atom inserted to form biotin stems from the sacrifice of the auxiliary 2Fe-2S cluster of BioB. Here, we bioinformatically explore the diversity of BioBs available in sequence databases and find an unexpected variation in the coordination of the auxiliary iron-sulfur cluster. After in vitro characterization, including the determination of biotin formation and representative crystal structures, we report a new type of BioB utilized by virtually all obligate anaerobic organisms. Instead of a 2Fe-2S cluster, this novel type of BioB utilizes an auxiliary 4Fe-5S cluster. Interestingly, this auxiliary 4Fe-5S cluster contains a ligated sulfide that we propose is used for biotin formation. We have termed this novel type of BioB, Type II BioB, with the E. coli 2Fe-2S cluster sacrificial BioB representing Type I. This surprisingly ubiquitous Type II BioB has implications for our understanding of the function and evolution of Fe-S clusters in enzyme catalysis, highlighting the difference in strategies between the anaerobic and aerobic world.

How many Mutations are needed to Evolve the Chemical Makeup of a Synthetic Cell?

The chemical evolution of a synthetic cell endowed with a synthetic amino acid as building block, analog to tryptophan, required the emergence of key mutations in genes involved in, inter alia, the general stress response, amino acid metabolism, stringent response, and chemotaxis. Understanding adaptation mechanisms to non-canonical biomass components will inform strategies for engineering synthetic metabolic pathways and cells.

PASQUALE - A long-term partnership to improve hand hygiene and capacity building in infection prevention and control in the Faranah region of Guinea.

Across the globe, hand hygiene (HH) is promoted to fight the spread of healthcare associated infections. Despite multiple ongoing HH campaigns and projects, the healthcare associated infection rates remain high especially in low- and middle-income countries. In the narrative overview presented here, we aim to share objectives, framework, successes and challenges of our long-term partnership in Guinea to offer guidance for other projects aiming to sustainably improve HH.

Does the body size, sex, and reproductive modes of leaf litter anurans affect the diversity of parasites?

We describe the composition of endoparasites associated with leaf litter anurans from an Atlantic Forest area, in southeastern Brazil. We tested if body size, sex, and reproductive modes of anuran hosts influence endoparasite abundance and richness. We sampled 583 individuals from 11 anuran species and recorded 1,600 helminths from 14 taxa. The helminths that infected the greatest number of anuran host species were the nematodes Cosmocerca parva (8 spp.), Physaloptera sp. (8 spp.), and Cosmocerca brasiliense (7 spp.), and the most abundant helminth species were Physaloptera sp. (14.6%), Cosmocerca brasiliense (13.7%) and Cosmocerca parva (12.6%). Both helminth abundance and richness were positively affected by anuran body size and dependence on water for reproduction. Larger hosts can contain a higher abundance of parasites because they may provide more physical space than smaller ones, or it can simply be a function of age. Besides, parasite species richness can be highly correlated with the amount of time a host spends in association with aquatic habitats, a conservative aspect of both parasite and host natural history. Within host species, there was a positive and significant influence of body size on helminth abundance. Haddadus binotatus females had greater helminth abundance than males, probably due to sex-related differences in behavior and/or in physiology. Our data suggest that reproductive modes could also influence helminth infection parameters in other anuran communities and should be considered in detail in future analyses.

Metabolic engineering of Escherichia coli for high-level production of free lipoic acid.

L-Lipoic acid (LA) is an important antioxidant with various industrial applications as a nutraceutical and therapeutic. Currently, LA is produced by chemical synthesis. Cell factory development is complex as LA and its direct precursors only occur naturally in protein-bound forms. Here we report a rationally engineered LA cell factory and demonstrate de novo free LA production from glucose for the first time in E. coli. The pathway represents a significant challenge as the three key enzymes, native Octanoyltransferase (LipB) and Lipoyl Synthase (LipA), and heterologous Lipoamidase (LpA), are all toxic to overexpress in E. coli. To overcome the toxicity of LipB, functional metagenomic selection was used to identify a highly active and non-toxic LipB and LipA from S. liquefaciens. Using high throughput screening, we balanced translation initiation rates and dual, orthogonal induction systems for the toxic genes, LipA and LpA. The optimized strain yielded 2.5 mg free LA per gram of glucose in minimal media, expressing carefully balanced LipB and LipA, Enterococcus faecalis LpA, and a truncated, native, Dihydrolipoyllysine-residue acetyltransferase (AceF) lipoylation domain. When the optimized cell factory strain was cultivated in a fed-batch fermentation, a titer of 87 mg/L free LA in the supernatant was reached after 48 h. This titer is ∼3000-fold higher than previously reported free LA titer and ∼8-fold higher than the previous best total, protein-bound LA titer. The strategies presented here could be helpful in designing, constructing and balancing biosynthetic pathways that harbor toxic enzymes with protein-bound intermediates or products.

Design of Sturm global attractors 1: Meanders with three noses, and reversibility.

We systematically explore a simple class of global attractors, called Sturm due to nodal properties, for the semilinear scalar parabolic partial differential equation (PDE) ut=uxx+f(x,u,ux) on the unit interval 0

On the hidden diversity and niche specialization of the microbial realm of subterranean estuaries.

Subterranean estuaries (STEs) modulate the chemical composition of continental groundwater before it reaches the coast, but their microbial community is poorly known. Here, we explored the microbial ecology of two neighbouring, yet contrasting STEs (Panxón and Ladeira STEs; Ría de Vigo, NW Iberian Peninsula). We investigated microbial composition (16S rRNA gene sequencing), abundance, heterotrophic production and their geochemical drivers. A total of 10,150 OTUs and 59 phyla were retrieved from porewater sampled during four surveys covering each STE seepage face. In both STEs, we find a very diverse microbial community composed by abundant cosmopolitans and locally restricted rare taxa. Porewater oxygen and dissolved organic matter are the main environmental predictors of microbial community composition. More importantly, the high variety of benthic microbiota links to biogeochemical processes of different elements in STEs. The oxygen-rich Panxón beach showed strong associations of the ammonium oxidizing archaea Nitrosopumilales with the heterotrophic community, thus acting as a net source of nitrogen to the coast. On the other hand, the prevailing anoxic conditions of Ladeira beach promoted the dominance of anaerobic heterotrophs related to the degradation of complex and aromatic compounds, such as Dehalococcoidia and Desulfatiglans, and the co-occurrence of methane oxidizers and methanogens.

Random placement models explain species richness and dissimilarity of frog assemblages within Atlantic Forest fragments.

Understanding the effects of random versus niche-based processes on biodiversity patterns is a central theme in ecology, and an important tool for predicting effects of habitat loss and fragmentation on biodiversity. We investigated the predictive power of random processes to explain species richness and species dissimilarity of amphibian assemblages in a fragmented tropical landscape of the Atlantic Forest of South America. We analyzed a large database of amphibian abundance and occupancy, sampled in 21 forest fragments ranging in size from 1.9 to 619 ha. We compared observed species richness and species dissimilarity with the outcomes of two null (random placement) models: 1- the traditional Coleman's area-based model and 2- an abundance-based model (based on the number of individuals observed in each fragment). We applied these models for all species combined, and separately for forest-dependent and habitat-generalist species. The abundance-based model fitted the observed species richness data better than the area-based model for all species, forest-dependent species, and generalist species. The area-based and the abundance-based models were also able to significantly explain species dissimilarity for all species and for generalists, but not for forest dependent species. The traditional area-based model assigned too many individuals to large fragments, thus failing to accurately explain species richness within patches across the landscape. Although niche-based processes may be important to structuring the regional pool of species in fragmented landscapes, our results suggest that part of the variation in species richness and species dissimilarity can be successfully explained by random placement models, especially for generalist species. Evaluating which factors cause variation in the number of individuals among patches should be a focus in future studies aiming to understand biodiversity patterns in fragmented landscapes.

Compreender os efeitos de processos aleatórios versus processos baseados em nicho nos padrões de biodiversidade é um tema central em ecologia e uma ferramenta importante para prever os efeitos da perda e fragmentação de habitat na biodiversidade. Nós investigamos o poder preditivo de processos aleatórios para explicar a riqueza e a dissimilaridade de espécies de assembleias de anfíbios em uma paisagem fragmentada tropical da Mata Atlântica da América do Sul. Analisamos um grande conjunto de dados de abundância e ocupação de anfíbios, amostrados em 21 fragmentos florestais com tamanhos de 1.9 a 619 ha. Comparamos a riqueza e a dissimilaridade de espécies observadas com os resultados de dois modelos nulos (posicionamento aleatório): 1- o modelo tradicional baseado em área de Coleman e 2 - um modelo baseado em abundância (com base no número de indivíduos observados em cada fragmento). Aplicamos esses modelos para todas as espécies combinadas e separadamente para espécies dependentes de floresta e espécies generalistas de habitat. O modelo baseado em abundância ajustou-se melhor aos dados observados de riqueza de espécies do que o modelo baseado em área para todas as espécies, espécies dependentes de floresta e espécies generalistas. Os modelos baseados em área e em abundância também foram capazes de explicar significativamente a dissimilaridade de espécies para todas as espécies e para generalistas, mas não para espécies dependentes de floresta. O modelo tradicional baseado em área atribuiu muitos indivíduos a grandes fragmentos, falhando assim em explicar com precisão a riqueza de espécies dentro de manchas na paisagem. Embora processos baseados em nicho possam ser importantes para estruturar o conjunto regional de espécies em paisagens fragmentadas, nossos resultados sugerem que parte da variação na riqueza e dissimilaridade de espécies pode ser explicada com sucesso por modelos de posicionamento aleatório, especialmente para espécies generalistas. Avaliar quais fatores causam variação no número de indivíduos entre manchas deve ser um foco em estudos futuros que visem compreender os padrões de biodiversidade em paisagens fragmentadas.

A comparative analysis of experienced uncertainties in relation to risk communication during COVID19: a four-country study.

BACKGROUND: During outbreaks, uncertainties experienced by affected communities can influence their compliance to government guidance on public health. Communicators and authorities are, hence, encouraged to acknowledge and address such uncertainties. However, in the midst of public health crises, it can become difficult to define and identify uncertainties that are most relevant to address. We analyzed data on COVID-19-related uncertainties from four socio-economic contexts to explore how uncertainties can influence people's perception of, and response to Risk Communication and Community Engagement (RCCE) strategies. RESULTS: This qualitative study, which adopts an interpretative approach, is based on data from a documentary review, key informant interviews (KII), and focus group discussions (FGD) with members of the general public and people with barriers to information from Germany, Guinea, Nigeria, and Singapore. Transcripts from the KII and FGD were coded and analyzed thematically. We interviewed a total of 155 KIs and conducted 73 FGD. Our analysis uncovered a divergence between uncertainties deemed relevant by stakeholders involved in policy making and uncertainties that people reportedly had to navigate in their everyday lives and which they considered relevant during the pandemic. We identified four types of uncertainties that seemed to have influenced people's assessment of the disease risk and their trust in the pandemic control strategies including RCCE efforts: epidemiological uncertainties (related to the nature and severity of the virus), information uncertainties (related to access to reliable information), social uncertainties (related to social behavior in times of heightened risk), and economic uncertainties (related to financial insecurities). CONCLUSION: We suggest that in future outbreaks, communicators and policy makers could improve the way in which affected communities assess their risk, and increase the trust of these communities in response efforts by addressing non-epidemiological uncertainties in RCCE strategies.

Integration of the elements involved in scientific publication.

The issue of publication of articles by researchers in the universe of scientific publications, has become over the last decades, increasingly asymmetrical among the parties involved (author, Author's institution, Development agencies, Magazine / Publisher, and referees). Here, I analyzed to what extent are all the parts involved in the process of scientific publication equally balanced, and the gains and losses among parts involved during the process. The analysis points that the gains and losses among parts involved are quite asymmetric. I conclude that there are advantages, disadvantages and costs that should be addressed at a global level to better balance the process of publication and that should have some adjustments to better balance of the parts involved in the process. All parts involved should search for a more equilibrated system of publishing, since, now, the system is strongly asymmetric among the parts involved in scientific publication. It is clear is that this is not a simple subject, but possible changes are quite simple and desirable.

Reassessing the Use of Undecanoic Acid as a Therapeutic Strategy for Treating Fungal Infections.

Treating fungal infections is challenging and frequently requires long-term courses of antifungal drugs. Considering the limited number of existing antifungal drugs, it is crucial to evaluate the possibility of repositioning drugs with antifungal properties and to revisit older antifungals for applications in combined therapy, which could widen the range of therapeutic possibilities. Undecanoic acid is a saturated medium-chain fatty acid with known antifungal effects; however, its antifungal properties have not been extensively explored. Recent advances indicate that the toxic effect of undecanoic acid involves modulation of fungal metabolism through its effects on the expression of fungal genes that are critical for virulence. Additionally, undecanoic acid is suitable for chemical modification and might be useful in synergic therapies. This review highlights the use of undecanoic acid in antifungal treatments, reinforcing its known activity against dermatophytes. Specifically, in Trichophyton rubrum, against which the activity of undecanoic acid has been most widely studied, undecanoic acid elicits profound effects on pivotal processes in the cell wall, membrane assembly, lipid metabolism, pathogenesis, and even mRNA processing. Considering the known antifungal activities and associated mechanisms of undecanoic acid, its potential use in combination therapy, and the ability to modify the parent compound structure, undecanoic acid shows promise as a novel therapeutic against fungal infections.

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes.

Directed evolution of stereo-, regio-, and chemoselective enzymes constitutes a unique way to generate biocatalysts for synthetically interesting transformations in organic chemistry and biotechnology. In order for this protein engineering technique to be efficient, fast, and reliable, and also of relevance to synthetic organic chemistry, methodology development was and still is necessary. Following a description of early key contributions, this review focuses on recent developments. It includes optimization of molecular biological methods for gene mutagenesis and the design of efficient strategies for their application, resulting in notable reduction of the screening effort (bottleneck of directed evolution). When aiming for laboratory evolution of selectivity and activity, second-generation versions of Combinatorial Active-Site Saturation Test (CAST) and Iterative Saturation Mutagenesis (ISM), both involving saturation mutagenesis (SM) at sites lining the binding pocket, have emerged as preferred approaches, aided by in silico methods such as machine learning. The recently proposed Focused Rational Iterative Site-specific Mutagenesis (FRISM) constitutes a fusion of rational design and directed evolution. On-chip solid-phase chemical gene synthesis for rapid library construction enhances library quality notably by eliminating undesired amino acid bias, the future of directed evolution?

Regio- and Stereoselective Steroid Hydroxylation at C7 by Cytochrome†P450 Monooxygenase Mutants.

Steroidal C7ß alcohols and their respective esters have shown significant promise as neuroprotective and anti-inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C-H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450-BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and ß stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil-Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high-resolution X-ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio- and stereoselectivity.

tRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii.

tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been determined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spectrometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii A new posttranscriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was discovered and localized to position 34. Moreover, data consistent with wyosine pathway modifications were obtained beyond the canonical tRNAPhe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our understanding of archaeal tRNA modification profiles and decoding strategies.IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii.

Climate change effects on population dynamics of three species of Amazonian lizards.

The scarcity of data on natural history and ecology of lizards still limits the understanding of population dynamics for many species. We attempt to evaluate possible effects of climate change on the population dynamics of three lizard species (Ameiva ameiva, Gonatodes humeralis and Norops fuscoauratus) in two Amazonian localities (Caxiuanã National Forest and Ducke Reserve). We calculated a tolerance index combining environmental thermal adequacy with the b-d model, which consider survival and reproductive rates to calculate population dynamics. Thus, we simulated population growth rates based on current and future environmental operative temperatures, considering an optimistic and a business-as-usual scenario of greenhouse gases emissions (GGE), and evaluate if the sensitivity of life history traits to population growth rate are likely to be trigged by climate change. Our results demonstrated that both populations of G. humeralis and the Ducke population of N. fuscoauratus may become locally extinct under both scenarios of GGE, while both populations of A. ameiva are likely to decrease, but without reaching a scenario of local extirpation. This study represents the first effort to evaluate the sensitivity of lizard populations and elasticity to climate change and demonstrate the geographic variability of these traits in three widespread and habitat-generalist species. We highlight the need of new studies focusing on species with different biological trait patterns, such as endemic distributions and habitat-specialists, to provide the theoretical and empirical basis for biologically informed conservation strategies and actions, in order to minimize the potential extinction of populations due to climate change.

Lizard assemblages on sandy coastal plains in southeastern Brazil: An analysis of occurrence and composition, and the role of habitat structure.

Data on the structure of lizard communities in the different biomes and ecosystems of Brazil are still limited. In this study, we related the species richness, abundance and the spatial occurrence of lizards to the structure of the vegetation found on the coast of the state of Espírito Santo, southeastern Brazil, to determine whether habitat influences the structure of lizard communities. We conducted fieldwork in 2012, 2013 and 2014, collecting data in standardized samples. We analyze whether variables of vegetation structure influenced species richness and abundance, using Generalized Linear Models (GLMs). We recorded 12 lizard species from eight families. In general, species richness and abundance were similar among sites. Locally, we recorded the highest species richness in shrubby vegetation, open Clusia vegetation, and the restinga forest zone. Bromeliads explained the occurrence of teiids, although there was no systematic relationship between species richness and vegetation structure. Our results provide important insights into the characteristics of the lizard communities found on sandy coastal plains and contribute to the conservation of these species in these ecosystems.

In vitro assessment of cytotoxic, genotoxic and mutagenic effects of antimalarial drugs artemisinin and artemether in human lymphocytes.

Artemisinin is a substance extracted from the Chinese plant Artemisia annua L. widely used in natural medicine for the treatment of various diseases. Artemether is a substance synthesized from artemisinin, and both drugs are commonly administered in the treatment of malaria. Although considered effective antimalarial drugs, very little is known about the genotoxic, cytotoxic and mutagenic effects of these drugs. Therefore, in the present study, we evaluated the genotoxic, mutagenic and cytotoxic effects of artemisinin (12.5, 25 and 50 µg/mL) and artemether (7.46; 14.92 and 29.84 µg/mL) in cultured human lymphocytes using the comet assay, the micronucleus test and the cytotoxicity assay for detection of necrosis and apoptosis by acridine orange/ethidium bromide staining. Our results showed a significant increase (p < 0.05) in the rate of DNA damage measured by comet assay and in the micronucleus frequency after treatment with both drugs. It was also observed that only artemisinin induced a statistically significant increase (p < 0.05) in the number of lymphocytes with death by necrosis 48 h after treatment. The results demonstrated that these two drugs induce mutagenic, genotoxic and cytotoxic effects in cultured human lymphocytes. Our data indicate the need for caution in the use of such drugs, since genotoxic/mutagenic effects may increase the risk of carcinogenesis.

Site-Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater.

Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site-selective injection of vibrational energy into a protein, that is, localized heating, is required for their investigation. Here, we solved this problem by the site-specific incorporation of the non-canonical amino acid ß-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kasha's rule, AzAla undergoes ultrafast internal conversion and heating after S1 excitation while upon S2 excitation, it serves as a fluorescent label. We equipped PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. We indeed observed VET from the incorporation sites in the protein to a bound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in a wide range of proteins.