Combining enzyme and metabolic engineering for microbial supply of therapeutic phytochemicals.

The history of pharmacology is deeply intertwined with plant-derived compounds, which continue to be crucial in drug development. However, their complex structures and limited availability in plants challenge drug discovery, optimization, development, and industrial production via chemical synthesis or natural extraction. This review delves into the integration of metabolic and enzyme engineering to leverage micro-organisms as platforms for the sustainable and reliable production of therapeutic phytochemicals. We argue that engineered microbes can serve a triple role in this paradigm: facilitating pathway discovery, acting as cell factories for scalable manufacturing, and functioning as platforms for chemical derivatization. Analyzing recent progress and outlining future directions, the review highlights microbial biotechnology's transformative potential in expanding plant-derived human therapeutics' discovery and supply chains.

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.

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.

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.

Protein engineering using mutability landscapes: Controlling site-selectivity of P450-catalyzed steroid hydroxylation.

Directed evolution and rational design have been used widely in engineering enzymes for their application in synthetic organic chemistry and biotechnology. With stereoselectivity playing a crucial role in catalysis for the synthesis of valuable chemical and pharmaceutical compounds, rational design has not achieved such wide success in this specific area compared to directed evolution. Nevertheless, one bottleneck of directed evolution is the laborious screening efforts and the observed trade-offs in catalytic profiles. This has motivated researchers to develop more efficient protein engineering methods. As a prime approach, mutability landscaping avoids such trade-offs by providing more information of sequence-function relationships. Here, we describe an application of this efficient protein engineering method to improve the regio-/stereoselectivity and activity of P450BM3 for steroid hydroxylation, while keeping the mutagenesis libraries small so that they will require only minimal screening.

Research for Cytomegalovirus Mutations Associated With Resistance to Antivirals in Kidney Transplant Receptors.

Cytomegalovirus (CMV) mutations associated with antiviral resistance have become a major problem related to high mortality in kidney transplant patients. The aim of the study was to investigate mutations in the CMV genes UL97 and UL54 associated with antiviral resistance. A retrospective observational cohort study was carried out at Hospital Ophir Loyola (HOL), a reference in Kidney Transplantation. A total of 81 patients who underwent kidney transplantation were followed up between 2016 and 2018 were monitored for CMV viral load by performing qPCR. Sanger sequencing was performed on 66 patients. All CMV-positive kidney transplant recipients were included. Mutations were observed in 15 samples (22.72%) from patients. Most cases involved UL97 mutations. Mutation in UL54 without mutation in UL97 was detected in only 2 cases. Resistance mutations in UL97 were identified, such as M460V, L595S, H520Q, two co-mutations D465R + Del524 and A594P + D413A and a 3 codon deletion (del598-601). The search for mutations in the CMV genes identified mutations that confer resistance to conventional antivirals, such as ganciclovir and cidofovir, used in the treatment of these patients. Confirmation of the association with increased CMV viral load in transplanted patients, due to mutation in resistance genes, requires phenotypic analysis for confirmation purposes. These were the first findings in patients in northern Brazil that we know of.

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

Radon prevalence in domestic water in the Ría de Vigo coastal basin (NW Iberian Peninsula).

The Ría de Vigo catchment is situated in the largest radon-prone area of the Iberian Peninsula. High local indoor radon (222Rn) levels are the preeminent source of radiation exposure, with negative effects on health. Nevertheless, information on radon levels of natural waters and the potential human exposure risks associated with their domestic use is very sparse. To elucidate the environmental factors increasing human exposure risk to radon during domestic water use, we undertook a survey of local water sources, including springs, rivers, wells, and boreholes, over different temporal scales. Continental waters were highly enriched in 222Rn: activities ranged from 1.2 to 20.2 Bq L-1 in rivers and levels one to two orders of magnitude higher were found in groundwaters (from 8.0 to 2737 Bq L-1; median 121.1 Bq L-1). The geology and hydrogeology of local crystalline aquifers support one order of magnitude higher 222Rn activities in groundwater stored in deeper fractured rock compared to that contained within the highly weathered regolith at the surface. During the mean dry season, 222Rn activities nearly doubled in most sampled waters in comparison to the wet period (from 94.9 during the dry season to 187.3 Bq L-1 during wet period; n = 37). Seasonal water use and recharge cycles and thermal convection are postulated to explain this variation in radon activities. The high 222Rn activities cause the total effective dose of radiation received from domestic use of untreated groundwaters to exceed the recommended 0.1 mSv y-1. Since more than 70% of this dose comes from indoor water degassing and subsequent 222Rn inhalation, preventative health policy in the form of 222Rn remediation and mitigation measures should be implemented prior to pumping untreated groundwater into dwellings, particularly during the dry period.

Six new species of Elpidium Müller, 1880 (Podocopida: Limnocytheridae) from Eastern Brazil.

Elpidium is the most common ostracod genus occurring in phytotelmata in the Neotropical region, with distributions ranging from Florida, USA in the north to Rio Grande do Sul, Brazil in the south. However, the genus remains poorly known both in terms of diversity and of the distributional pattern of its species. Here, we describe six new species of Elpidium, E. oxumae n. sp., E. cordiforme n. sp., E. picinguabaense n. sp., E. eriocaularum n. sp., E. higutiae n. sp., E. purium n. sp., all from phytotelm environments in the Brazilian Atlantic rain forest. In addition, we discuss the distributional pattern and endemicity levels of Elpidium species in the light of these new taxonomic results and argue about possible misunderstandings on the distribution of the type species E. bromeliarum.

Qualitative assessment of hand hygiene knowledge, attitudes and practices among healthcare workers prior to the implementation of the WHO Hand Hygiene Improvement Strategy at Faranah Regional Hospital, Guinea.

Healthcare-associated infections are a serious burden globally. Few qualitative studies have explored healthcare workers' knowledge, attitudes and practices of hand hygiene. Prior to the implementation of the World Health Organization's Hand Hygiene Improvement Strategy at Faranah Regional Hospital in the Upper Region of Guinea in December 2018, we conducted a qualitative baseline assessment of knowledge, attitudes and practices of hand hygiene among healthcare workers to guide future hand hygiene interventions. The qualitative study consisted of direct observations, In-Depth Interviews (IDIs) and Focus Group Discussions (FGDs). We found that the 2013-16 Ebola outbreak had had a pivotal impact on healthcare workers' knowledge, attitudes and practices. The severity of the disease and the training provided for infection control were responsible for their knowledge acquisition and adoption of good attitudes and practices. However, negligence, resulting in poor hand hygiene practices, rose after the outbreak, once the "cue of fear" that had motivated workers for their own self-protection had waned. Our results suggest that local capacity building through training and availability of hand hygiene materials would be a sustainable approach to enhance hand hygiene culture at the hospital. Our study suggests that there is a need for a high and long-term commitment of authorities and healthcare workers at all levels for a sustainable hand hygiene culture.

Communicating risk during early phases of COVID-19: Comparing governing structures for emergency risk communication across four contexts.

Background: Emergency risk communication (ERC) is key to achieving compliance with public health measures during pandemics. Yet, the factors that facilitated ERC during COVID-19 have not been analyzed. We compare ERC in the early stages of the pandemic across four socio-economic settings to identify how risk communication can be improved in public health emergencies (PHE). Methods: To map and assess the content, process, actors, and context of ERC in Germany, Guinea, Nigeria, and Singapore, we performed a qualitative document review, and thematically analyzed semi-structured key informant interviews with 155 stakeholders involved in ERC at national and sub-national levels. We applied Walt and Gilson's health policy triangle as a framework to structure the results. Results: We identified distinct ERC strategies in each of the four countries. Various actors, including governmental leads, experts, and organizations with close contact to the public, collaborated closely to implement ERC strategies. Early integration of ERC into preparedness and response plans, lessons from previous experiences, existing structures and networks, and clear leadership were identified as crucial for ensuring message clarity, consistency, relevance, and an efficient use of resources. Areas of improvement primarily included two-way communication, community engagement, and monitoring and evaluation. Countries with recurrent experiences of pandemics appeared to be more prepared and equipped to implement ERC strategies. Conclusion: We found that considerable potential exists for countries to improve communication during public health emergencies, particularly in the areas of bilateral communication and community engagement as well as monitoring and evaluation. Building adaptive structures and maintaining long-term relationships with at-risk communities reportedly facilitated suitable communication. The findings suggest considerable potential and transferable learning opportunities exist between countries in the global north and countries in the global south with experience of managing outbreaks.

The Antidepressant Sertraline Affects Cell Signaling and Metabolism in Trichophyton rubrum.

The dermatophyte Trichophyton rubrum is responsible for most human cutaneous infections. Its treatment is complex, mainly because there are only a few structural classes of fungal inhibitors. Therefore, new strategies addressing these problems are essential. The development of new drugs is time-consuming and expensive. The repositioning of drugs already used in medical practice has emerged as an alternative to discovering new drugs. The antidepressant sertraline (SRT) kills several important fungal pathogens. Accordingly, we investigated the inhibitory mechanism of SRT in T. rubrum to broaden the knowledge of its impact on eukaryotic microorganisms and to assess its potential for future use in dermatophytosis treatments. We performed next-generation sequencing (RNA-seq) to identify the genes responding to SRT at the transcript level. We identified that a major effect of SRT was to alter expression for genes involved in maintaining fungal cell wall and plasma membrane stability, including ergosterol biosynthetic genes. SRT also altered the expression of genes encoding enzymes related to fungal energy metabolism, cellular detoxification, and defense against oxidative stress. Our findings provide insights into a specific molecular network interaction that maintains metabolic stability and is perturbed by SRT, showing potential targets for its strategic use in dermatophytosis.

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.

Impact Of The COVID-19 Pandemic on Hospital Admissions and In-Hospital Lethality From Cardiovascular Diseases in Brazil: An Ecological and Time Series Study.

Since the onset of the coronavirus disease 2019 (COVID-19) pandemic in Brazil, several government policies have been taken. Herein, we aimed to assess the impact of the COVID-19 pandemic on hospital admissions and in-hospital lethality for cardiovascular diseases (CVD) in Brazil in 2020. An ecological and time-series study on hospitalizations and deaths from CVD in Brazil was conducted from January 2018 to December 2020.The hospital admission rate for CVD reduced by 17.1%, with a significant decreasing trend between January and May 2020 (Annual Percent Change: -8,7%; P-value < 0.001). The in-hospital lethality rate increased from 8.2% in 2018 to 9.3% in 2020. During this period, Brazil totaled 21.8 million days of hospital stay. Indicators of hospital admissions and lethality from CVD in Brazil were impacted by the emergence of the COVID-19 pandemic in different ways in the regions and depending on the nature of the indicator.

We're building it up to burn it down: fire occurrence and fire-related climatic patterns in Brazilian biomes.

Background: Terrestrial biomes in South America are likely to experience a persistent increase in environmental temperature, possibly combined with moisture reduction due to climate change. In addition, natural fire ignition sources, such as lightning, can become more frequent under climate change scenarios since favourable environmental conditions are likely to occur more often. In this sense, changes in the frequency and magnitude of natural fires can impose novel stressors on different ecosystems according to their adaptation to fires. By focusing on Brazilian biomes, we use an innovative combination of techniques to quantify fire persistence and occurrence patterns over time and evaluate climate risk by considering key fire-related climatic characteristics. Then, we tested four major hypotheses considering the overall characteristics of fire-dependent, fire-independent, and fire-sensitive biomes concerning (1) fire persistence over time; (2) the relationship between climate and fire occurrence; (3) future predictions of climate change and its potential impacts on fire occurrence; and (4) climate risk faced by biomes. Methods: We performed a Detrended Fluctuation Analysis to test whether fires in Brazilian biomes are persistent over time. We considered four bioclimatic variables whose links to fire frequency and intensity are well-established to assess the relationship between climate and fire occurrence by confronting these climate predictors with a fire occurrence dataset through correlative models. To assess climate risk, we calculated the climate hazard, sensitivity, resilience, and vulnerability of Brazilian biomes, and then we multiplied the Biomes' vulnerability index by the hazards. Results: Our results indicate a persistent behaviour of fires in all Brazilian biomes at almost the same rates, which could represent human-induced patterns of fire persistence. We also corroborated our second hypothesis by showing that most fire-dependent biomes presented high thermal suitability to fire, while the fire-independent biome presented intermediate suitability and fire-sensitive biomes are the least suitable for fire occurrence. The third hypothesis was partially corroborated since fire-dependent and independent biomes are likely to increase their thermal suitability to fire, while fire-sensitive biomes are likely to present stable-to-decreasing thermal suitability in the future. Finally, our fourth hypothesis was partially corroborated since most fire-dependent biomes presented low climate risk, while the fire-independent biome presented a high risk and the fire-sensitive biomes presented opposite trends. In summary, while the patterns of fire persistence and fire occurrence over time are more likely to be related to human-induced fires, key drivers of burned areas are likely to be intensified across Brazilian biomes in the future, potentially increasing the magnitude of the fires and harming the biomes' integrity.

A New Species of Elpidium (Crustacea: Ostracoda: Limnocytheridae) from Brazil and a Morphological Phylogenetic Proposal for the Genus.

Elpidium species exclusively inhabiting confined and temporary environments, such as those of tank-bromeliads, are a source of interesting and diverse studies on taxonomy, evolution and ecology, to name a few. However, despite its great diversity of species or potential for study, this genus (and other phytotelm members) has been poorly studied. In the last years, however, description of Elpidium species increased from six before 2013 to 11 today. This study is an effort to keep uncovering its great diversity and to go further in order to deeply understand the genus Elpidium. To this end, this study describes another species in the genus, Elpidium litoreum sp. nov., and proposes a phylogenetic reconstruction of it based on morphological characters. Our results point to the monophyly of Elpidium and puts Intrepidocythere ibipora as its sister-group. Although the phylogeny revealed some interesting relations, it also exposed some incongruities that ultimately demonstrate how superficial the current knowledge about the genus is. All these questions are discussed in detail. We see this work as at the same time an effort to better understand Elpidium and a stimulus to other researches to turn their attention to the historically neglected phytotelmata community.

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.