Diversity and Evolution of Methane-Related Pathways in Archaea.

Methane is one of the most important greenhouse gases on Earth and holds an important place in the global carbon cycle. Archaea are the only organisms that use methanogenesis to produce energy and rely on the methyl-coenzyme M reductase complex (Mcr). Over the last decade, new results have significantly reshaped our view of the diversity of methane-related pathways in the Archaea. Many new lineages that synthesize or use methane have been identified across the whole archaeal tree, leading to a greatly expanded diversity of substrates and mechanisms. In this review, we present the state of the art of these advances and how they challenge established scenarios of the origin and evolution of methanogenesis, and we discuss the potential trajectories that may have led to this strikingly wide range of metabolisms.

Components of iron-Sulfur cluster assembly machineries are robust phylogenetic markers to trace the origin of mitochondria and plastids.

Establishing the origin of mitochondria and plastids is key to understand 2 founding events in the origin and early evolution of eukaryotes. Recent advances in the exploration of microbial diversity and in phylogenomics approaches have indicated a deep origin of mitochondria and plastids during the diversification of Alphaproteobacteria and Cyanobacteria, respectively. Here, we strongly support these placements by analyzing the machineries for assembly of iron-sulfur ([Fe-S]) clusters, an essential function in eukaryotic cells that is carried out in mitochondria by the ISC machinery and in plastids by the SUF machinery. We assessed the taxonomic distribution of ISC and SUF in representatives of major eukaryotic supergroups and analyzed the phylogenetic relationships with their prokaryotic homologues. Concatenation datasets of core ISC proteins show an early branching of mitochondria within Alphaproteobacteria, right after the emergence of Magnetococcales. Similar analyses with the SUF machinery place primary plastids as sister to Gloeomargarita within Cyanobacteria. Our results add to the growing evidence of an early emergence of primary organelles and show that the analysis of essential machineries of endosymbiotic origin provide a robust signal to resolve ancient and fundamental steps in eukaryotic evolution.

Neurodegeneration and neuroinflammation are linked, but independent of alpha-synuclein inclusions, in a seeding/spreading mouse model of Parkinson's disease.

A key pathological process in Parkinson's disease (PD) is the transneuronal spreading of α-synuclein. Alpha-synuclein (α-syn) is a presynaptic protein that, in PD, forms pathological inclusions. Other hallmarks of PD include neurodegeneration and microgliosis in susceptible brain regions. Whether it is primarily transneuronal spreading of α-syn particles, inclusion formation, or other mechanisms, such as inflammation, that cause neurodegeneration in PD is unclear. We used a model of spreading of α-syn induced by striatal injection of α-syn preformed fibrils into the mouse striatum to address this question. We performed quantitative analysis for α-syn inclusions, neurodegeneration, and microgliosis in different brain regions, and generated gene expression profiles of the ventral midbrain, at two different timepoints after disease induction. We observed significant neurodegeneration and microgliosis in brain regions not only with, but also without α-syn inclusions. We also observed prominent microgliosis in injured brain regions that did not correlate with neurodegeneration nor with inclusion load. Using longitudinal gene expression profiling, we observed early gene expression changes, linked to neuroinflammation, that preceded neurodegeneration, indicating an active role of microglia in this process. Altered gene pathways overlapped with those typical of PD. Our observations indicate that α-syn inclusion formation is not the major driver in the early phases of PD-like neurodegeneration, but that microglia, activated by diffusible, oligomeric α-syn, may play a key role in this process. Our findings uncover new features of α-syn induced pathologies, in particular microgliosis, and point to the necessity for a broader view of the process of α-syn spreading.

A Comprehensive Evolutionary Scenario of Cell Division and Associated Processes in the Firmicutes.

The cell cycle is a fundamental process that has been extensively studied in bacteria. However, many of its components and their interactions with machineries involved in other cellular processes are poorly understood. Furthermore, most knowledge relies on the study of a few models, but the real diversity of the cell division apparatus and its evolution are largely unknown. Here, we present a massive in-silico analysis of cell division and associated processes in around 1,000 genomes of the Firmicutes, a major bacterial phylum encompassing models (i.e. Bacillus subtilis, Streptococcus pneumoniae, and Staphylococcus aureus), as well as many important pathogens. We analyzed over 160 proteins by using an original approach combining phylogenetic reconciliation, phylogenetic profiles, and gene cluster survey. Our results reveal the presence of substantial differences among clades and pinpoints a number of evolutionary hotspots. In particular, the emergence of Bacilli coincides with an expansion of the gene repertoires involved in cell wall synthesis and remodeling. We also highlight major genomic rearrangements at the emergence of Streptococcaceae. We establish a functional network in Firmicutes that allows identifying new functional links inside one same process such as between FtsW (peptidoglycan polymerase) and a previously undescribed Penicilin-Binding Protein or between different processes, such as replication and cell wall synthesis. Finally, we identify new candidates involved in sporulation and cell wall synthesis. Our results provide a previously undescribed view on the diversity of the bacterial cell cycle, testable hypotheses for further experimental studies, and a methodological framework for the analysis of any other biological system.

Phosphorylation-dependent activation of the cell wall synthase PBP2a in Streptococcus pneumoniae by MacP.

Most bacterial cells are surrounded by an essential cell wall composed of the net-like heteropolymer peptidoglycan (PG). Growth and division of bacteria are intimately linked to the expansion of the PG meshwork and the construction of a cell wall septum that separates the nascent daughter cells. Class A penicillin-binding proteins (aPBPs) are a major family of PG synthases that build the wall matrix. Given their central role in cell wall assembly and importance as drug targets, surprisingly little is known about how the activity of aPBPs is controlled to properly coordinate cell growth and division. Here, we report the identification of MacP (SPD_0876) as a membrane-anchored cofactor of PBP2a, an aPBP synthase of the Gram-positive pathogen Streptococcus pneumoniae We show that MacP localizes to the division site of S. pneumoniae, forms a complex with PBP2a, and is required for the in vivo activity of the synthase. Importantly, MacP was also found to be a substrate for the kinase StkP, a global cell cycle regulator. Although StkP has been implicated in controlling the balance between the elongation and septation modes of cell wall synthesis, none of its substrates are known to modulate PG synthetic activity. Here we show that a phosphoablative substitution in MacP that blocks StkP-mediated phosphorylation prevents PBP2a activity without affecting the MacP-PBP2a interaction. Our results thus reveal a direct connection between PG synthase function and the control of cell morphogenesis by the StkP regulatory network.

GeneSpy, a user-friendly and flexible genomic context visualizer.

Summary: The exploration and comparison of genome organization is routinely used in the frame of genomic and phylogenomic analyses. As a consequence, in the past few years, various tools allowing visualizing genomic contexts have been developed. However, their use is often hampered by a lack of flexibility, particularly concerning associated databases input formats and figure customization. Here we present GeneSpy, a graphical user interface that allows the visualization and dynamic exploration of eukaryotic and prokaryotic annotated genomes. GeneSpy relies on user-friendly manageable local databases and allows the easy customization and production of figures in a multitude of formats. Availability and implementation: GeneSpy is freely available at https://lbbe.univ-lyon1.fr/GeneSpy/ for Linux, Mac OS and Windows under CeCILL license (http://www.cecill.info/licences/). It is written in Python 2.7 and depends on Matplotlib, Tkinter and Sqlite libraries. Supplementary information: Supplementary data are available at Bioinformatics online.

The mouse brain metabolome: region-specific signatures and response to excitotoxic neuronal injury.

Neurodegeneration is a multistep process characterized by a multitude of molecular entities and their interactions. Systems analyses, or omics approaches, have become an important tool in characterizing this process. Although RNA and protein profiling made their entry into this field a couple of decades ago, metabolite profiling is a more recent addition. The metabolome represents a large part or all metabolites in a tissue, and gives a snapshot of its physiology. By using gas chromatography coupled to mass spectrometry, we analyzed the metabolic profile of brain regions of the mouse, and found that each region is characterized by its own metabolic signature. We then analyzed the metabolic profile of the mouse brain after excitotoxic injury, a mechanism of neurodegeneration implicated in numerous neurological diseases. More important, we validated our findings by measuring, histologically and molecularly, actual neurodegeneration and glial response. We found that a specific global metabolic signature, best revealed by machine learning algorithms, rather than individual metabolites, was the most robust correlate of neuronal injury and the accompanying gliosis, and this signature could serve as a global biomarker for neurodegeneration. We also observed that brain lesioning induced several metabolites with neuroprotective properties. Our results deepen the understanding of metabolic changes accompanying neurodegeneration in disease models, and could help rapidly evaluate these changes in preclinical drug studies.

When iron and sulfur met on an anoxic planet and eventually made clusters essential for life.

[FeS] clusters are co-factors that are essential for life and are synthesized by dedicated multiprotein cellular machineries. In this review, we present the current scenario for the emergence and the diversification of the [FeS] cluster biosynthesis machineries. In addition to well-known NIF, ISC and SUF machineries, two alternative minimal systems, SMS, and MIS, were recently identified. Taxonomic distribution and phylogeny analyses indicate that SMS and MIS were present in the Last Universal Common Ancestor (LUCA), well before the increase of oxygen on Earth. ISC, SUF and NIF systems emerged later in the history of life. The possible reasons for the emergence and diversification of these machineries are discussed.

A molecular comparison of [Fe-S] cluster-based homeostasis in Escherichia coli and Pseudomonas aeruginosa.

Iron-sulfur [Fe-S] clusters are essential protein cofactors allowing bacteria to perceive environmental redox modification and to adapt to iron limitation. Escherichia coli, which served as a bacterial model, contains two [Fe-S] cluster biogenesis systems, ISC and SUF, which ensure [Fe-S] cluster synthesis under balanced and stress conditions, respectively. However, our recent phylogenomic analyses revealed that most bacteria possess only one [Fe-S] cluster biogenesis system, most often SUF. The opportunist human pathogen Pseudomonas aeruginosa is atypical as it harbors only ISC. Here, we confirmed the essentiality of ISC in P. aeruginosa under both normal and stress conditions. Moreover, P. aeruginosa ISC restored viability, under balanced growth conditions, to an E. coli strain lacking both ISC and SUF. Reciprocally, the E. coli SUF system sustained growth and [Fe-S] cluster-dependent enzyme activities of ISC-deficient P. aeruginosa. Surprisingly, an ISC-deficient P. aeruginosa strain expressing E. coli SUF showed defects in resistance to H2O2 stress and paraquat, a superoxide generator. Similarly, the P. aeruginosa ISC system did not confer stress resistance to a SUF-deficient E. coli mutant. A survey of 120 Pseudomonadales genomes confirmed that all but five species have selected ISC over SUF. While highlighting the great versatility of bacterial [Fe-S] cluster biogenesis systems, this study emphasizes that their contribution to cellular homeostasis must be assessed in the context of each species and its own repertoire of stress adaptation functions. As a matter of fact, despite having only one ISC system, P. aeruginosa shows higher fitness in the face of ROS and iron limitation than E. coli. IMPORTANCE: ISC and SUF molecular systems build and transfer Fe-S cluster to cellular apo protein clients. The model Escherichia coli has both ISC and SUF and study of the interplay between the two systems established that the ISC system is the house-keeping one and SUF the stress-responding one. Unexpectedly, our recent phylogenomic analysis revealed that in contrast to E. coli (and related enterobacteria such as Salmonella), most bacteria have only one system, and, in most cases, it is SUF. Pseudomonas aeruginosa fits the general rule of having only one system but stands against the rule by having ISC. This study aims at engineering P. aeruginosa harboring E. coli systems and vice versa. Comparison of the recombinants allowed to assess the functional versatility of each system while appreciating their contribution to cellular homeostasis in different species context.

Single cell transcriptome analysis of the THY-Tau22 mouse model of Alzheimer's disease reveals sex-dependent dysregulations.

Alzheimer's disease (AD) progression and pathology show pronounced sex differences, but the factors driving these remain poorly understood. To gain insights into early AD-associated molecular changes and their sex dependency for tau pathology in the cortex, we performed single-cell RNA-seq in the THY-Tau22 AD mouse model. By examining cell type-specific and cell type-agnostic AD-related gene activity changes and their sex-dimorphism for individual genes, pathways and cellular sub-networks, we identified both statistically significant alterations and interpreted the upstream mechanisms controlling them. Our results confirm several significant sex-dependent alterations in gene activity in the THY-Tau22 model mice compared to controls, with more pronounced alterations in females. Both changes shared across multiple cell types and cell type-specific changes were observed. The differential genes showed significant over-representation of known AD-relevant processes, such as pathways associated with neuronal differentiation, programmed cell death and inflammatory responses. Regulatory network analysis of these genes revealed upstream regulators that modulate many of the downstream targets with sex-dependent changes. Most key regulators have been previously implicated in AD, such as Egr1, Klf4, Chchd2, complement system genes, and myelin-associated glycoproteins. Comparing with similar data from the Tg2576 AD mouse model and human AD patients, we identified multiple genes with consistent, cell type-specific and sex-dependent alterations across all three datasets. These shared changes were particularly evident in the expression of myelin-associated genes such as Mbp and Plp1 in oligodendrocytes. In summary, we observed significant cell type-specific transcriptomic changes in the THY-Tau22 mouse model, with a strong over-representation of known AD-associated genes and processes. These include both sex-neutral and sex-specific patterns, characterized by consistent shifts in upstream master regulators and downstream target genes. Collectively, these findings provide insights into mechanisms influencing sex-specific susceptibility to AD and reveal key regulatory proteins that could be targeted for developing treatments addressing sex-dependent AD pathology.

Cognitive impairment and dementia after intracerebral hemorrhage: a cross-sectional study of a hospital-based series.

BACKGROUND: Frequencies of cognitive impairment and dementia have not been assessed in spontaneous intracerebral hemorrhage (ICH). The objective of this study was to determine the frequencies and patterns of cognitive impairment and dementia in a cross-sectional study of consecutive patients hospitalized in a single university medical center. METHODS: Of 183 consecutive patients hospitalized between 2002 and 2006, 80 survivors were contacted and 78 were included (mean time since stroke 40 months). Thirty patients were scored with the Informant Questionnaire on Cognitive Decline in the Elderly and Instrumental Activities of Daily Living in a telephone interview, and 48 underwent a comprehensive clinical and neuropsychological assessment. RESULTS: Dementia was observed in 18 of 78 patients (23%; 95% confidence interval [CI] 13-32%) and cognitive impairment without dementia was seen in 37 of 48 patients (77%; 95% CI 65-89%). The cognitive disorders mainly concerned episodic memory (52%), psychomotor speed (44%), and executive function (37%), followed by language and visuoconstructive abilities. In a logistic regression analysis, Rankin score >1 at discharge and hemorrhage volume were the initial factors to be selected as a predictor of long-term dementia. CONCLUSIONS: This single-center, cross-sectional study revealed that the prevalence of dementia and cognitive impairment without dementia after ICH are high and are similar to those observed in cerebral infarct. Further longitudinal, prospective studies are required to assess accurately the prevalence, mechanisms and predictors of post-ICH dementia.

A Diverged Transcriptional Network for Usage of Two Fe-S Cluster Biogenesis Machineries in the Delta-Proteobacterium Myxococcus xanthus.

Myxococcus xanthus possesses two Fe-S cluster biogenesis machineries, ISC (iron-sulfur cluster) and SUF (sulfur mobilization). Here, we show that in comparison to the phylogenetically distant Enterobacteria, which also have both machineries, M. xanthus evolved an independent transcriptional scheme to coordinately regulate the expression of these machineries. This transcriptional response is directed by RisR, which we show to belong to a phylogenetically distant and biochemically distinct subgroup of the Rrf2 transcription factor family, in comparison to IscR that regulates the isc and suf operons in Enterobacteria. We report that RisR harbors an Fe-S cluster and that holo-RisR acts as a repressor of both the isc and suf operons, in contrast to Escherichia coli, where holo-IscR represses the isc operon whereas apo-IscR activates the suf operon. In addition, we establish that the nature of the cluster and the DNA binding sites of RisR, in the isc and suf operons, diverge from those of IscR. We further show that in M. xanthus, the two machineries appear to be fully interchangeable in maintaining housekeeping levels of Fe-S cluster biogenesis and in synthesizing the Fe-S cluster for their common regulator, RisR. We also demonstrate that in response to oxidative stress and iron limitation, transcriptional upregulation of the M. xanthus isc and suf operons was mediated solely by RisR and that the contribution of the SUF machinery was greater than the ISC machinery. Altogether, these findings shed light on the diversity of homeostatic mechanisms exploited by bacteria to coordinately use two Fe-S cluster biogenesis machineries. IMPORTANCE Fe-S proteins are ubiquitous and control a wide variety of key biological processes; therefore, maintaining Fe-S cluster homeostasis is an essential task for all organisms. Here, we provide the first example of how a bacterium from the Deltaproteobacteria branch coordinates expression of two Fe-S cluster biogenesis machineries. The results revealed a new model of coordination, highlighting the unique and common features that have independently emerged in phylogenetically distant bacteria to maintain Fe-S cluster homeostasis in response to environmental changes. Regulation is orchestrated by a previously uncharacterized transcriptional regulator, RisR, belonging to the Rrf2 superfamily, whose members are known to sense diverse environmental stresses frequently encountered by bacteria. Understanding how M. xanthus maintains Fe-S cluster homeostasis via RisR regulation revealed a strategy reflective of the aerobic lifestyle of this organsim. This new knowledge also paves the way to improve production of Fe-S-dependent secondary metabolites using M. xanthus as a chassis.

Fiber deprivation and microbiome-borne curli shift gut bacterial populations and accelerate disease in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurological disorder characterized by motor dysfunction, dopaminergic neuron loss, and alpha-synuclein (αSyn) inclusions. Many PD risk factors are known, but those affecting disease progression are not. Lifestyle and microbial dysbiosis are candidates in this context. Diet-driven gut dysbiosis and reduced barrier function may increase exposure of enteric neurons to toxins. Here, we study whether fiber deprivation and exposure to bacterial curli, a protein cross-seeding with αSyn, individually or together, exacerbate disease in the enteric and central nervous systems of a transgenic PD mouse model. We analyze the gut microbiome, motor behavior, and gastrointestinal and brain pathologies. We find that diet and bacterial curli alter the microbiome and exacerbate motor performance, as well as intestinal and brain pathologies, but to different extents. Our results shed important insights on how diet and microbiome-borne insults modulate PD progression via the gut-brain axis and have implications for lifestyle management of PD.

Synthesis of aminopyridines and aminopyridones by cobalt-catalyzed [2+2+2] cycloadditions involving yne-ynamides: scope, limitations, and mechanistic insights.

An in-depth study of the cobalt-catalyzed [2+2+2] cycloaddition between yne-ynamides and nitriles to afford aminopyridines has been carried out. About 30 nitriles exhibiting a broad range of steric demand and electronic properties have been evaluated, some of which open new perspectives in metal-catalyzed arene formation. In particular, the use of [CpCo(CO)(dmfu)] (dmfu=dimethyl fumarate) as a precatalyst made possible the incorporation of electron-deficient nitriles into the pyridine core. Modification of the substitution pattern at the yne-ynamide allows the regioselectivity to be switched toward 3- or 4-aminopyridines. Application of this synthetic methodology to the construction of the aminopyridone framework using a yne-ynamide and an isocyanate was also briefly examined. DFT computations suggest that 3-aminopyridines are formed by formal [4+2] cycloaddition between the nitrile and the intermediate cobaltacyclopentadiene, whereas 4-aminopyridines arise from an insertion pathway.

Single-Cell Transcriptional Profiling and Gene Regulatory Network Modeling in Tg2576 Mice Reveal Gender-Dependent Molecular Features Preceding Alzheimer-Like Pathologies.

Alzheimer's disease (AD) onset and progression is influenced by a complex interplay of several environmental and genetic factors, one of them gender. Pronounced gender differences have been observed both in the relative risk of developing AD and in clinical disease manifestations. A molecular level understanding of these gender disparities is still missing, but could provide important clues on cellular mechanisms modulating the disease and reveal new targets for gender-oriented disease-modifying precision therapies. We therefore present here a comprehensive single-cell analysis of disease-associated molecular gender differences in transcriptomics data from the neocortex, one of the brain regions most susceptible to AD, in one of the most widely used AD mouse models, the Tg2576 model. Cortical areas are also most commonly used in studies of post-mortem AD brains. To identify disease-linked molecular processes that occur before the onset of detectable neuropathology, we focused our analyses on an age with no detectable plaques and microgliosis. Cell-type specific alterations were investigated at the level of individual genes, pathways, and gene regulatory networks. The number of differentially expressed genes (DEGs) was not large enough to build context-specific gene regulatory networks for each individual cell type, and thus, we focused on the study of cell types with dominant changes and included analyses of changes across the combination of cell types. We observed significant disease-associated gender differences in cellular processes related to synapse organization and reactive oxygen species metabolism, and identified a limited set of transcription factors, including Egr1 and Klf6, as key regulators of many of the disease-associated and gender-dependent gene expression changes in the model. Overall, our analyses revealed significant cell-type specific gene expression changes in individual genes, pathways and sub-networks, including gender-specific and gender-dimorphic changes in both upstream transcription factors and their downstream targets, in the Tg2576 AD model before the onset of overt disease. This opens a window into molecular events that could determine gender-susceptibility to AD, and uncovers tractable target candidates for potential gender-specific precision medicine for AD.

An early origin of iron-sulfur cluster biosynthesis machineries before Earth oxygenation.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential for life. It is largely thought that the emergence of oxygenic photosynthesis and progressive oxygenation of the atmosphere led to the origin of multiprotein machineries (ISC, NIF and SUF) assisting Fe-S cluster synthesis in the presence of oxidative stress and shortage of bioavailable iron. However, previous analyses have left unclear the origin and evolution of these systems. Here, we combine exhaustive homology searches with genomic context analysis and phylogeny to precisely identify Fe-S cluster biogenesis systems in over 10,000 archaeal and bacterial genomes. We highlight the existence of two additional and clearly distinct 'minimal' Fe-S cluster assembly machineries, MIS (minimal iron-sulfur) and SMS (SUF-like minimal system), which we infer in the last universal common ancestor (LUCA) and we experimentally validate SMS as a bona fide Fe-S cluster biogenesis system. These ancestral systems were kept in archaea whereas they went through stepwise complexification in bacteria to incorporate additional functions for higher Fe-S cluster synthesis efficiency leading to SUF, ISC and NIF. Horizontal gene transfers and losses then shaped the current distribution of these systems, driving ecological adaptations such as the emergence of aerobic lifestyles in archaea. Our results show that dedicated machineries were in place early in evolution to assist Fe-S cluster biogenesis and that their origin is not directly linked to Earth oxygenation.

An Optimized Workflow for the Discovery of New Antimicrobial Compounds Targeting Bacterial RNA Polymerase Complex Formation.

Bacterial resistance represents a major health problem worldwide and there is an urgent need to develop first-in-class compounds directed against new therapeutic targets. We previously developed a drug-discovery platform to identify new antimicrobials able to disrupt the protein-protein interaction between the ß' subunit and the σ70 initiation factor of bacterial RNA polymerase, which is essential for transcription. As a follow-up to such work, we have improved the discovery strategy to make it less time-consuming and more cost-effective. This involves three sequential assays, easily scalable to a high-throughput format, and a subsequent in-depth characterization only limited to hits that passed the three tests. This optimized workflow, applied to the screening of 5360 small molecules from three synthetic and natural compound libraries, led to the identification of six compounds interfering with the ß'-σ70 interaction, and thus was capable of inhibiting promoter-specific RNA transcription and bacterial growth. Upon supplementation with a permeability adjuvant, the two most potent transcription-inhibiting compounds displayed a strong antibacterial activity against Escherichia coli with minimum inhibitory concentration (MIC) values among the lowest (0.87-1.56 µM) thus far reported for ß'-σ PPI inhibitors. The newly identified hit compounds share structural feature similarities with those of a pharmacophore model previously developed from known inhibitors.

Correction to "Discovery of Novel α-Carboline Inhibitors of the Anaplastic Lymphoma Kinase".

[This corrects the article DOI: 10.1021/acsomega.2c00507.].

Cellular assays identify barriers impeding iron-sulfur enzyme activity in a non-native prokaryotic host.

Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.

Ciliary neurotrophic factor cell-based delivery prevents synaptic impairment and improves memory in mouse models of Alzheimer's disease.

The development of novel therapeutic strategies for Alzheimer's disease (AD) represents one of the biggest unmet medical needs today. Application of neurotrophic factors able to modulate neuronal survival and synaptic connectivity is a promising therapeutic approach for AD. We aimed to determine whether the loco-regional delivery of ciliary neurotrophic factor (CNTF) could prevent amyloid-beta (Abeta) oligomer-induced synaptic damages and associated cognitive impairments that typify AD. To ensure long-term administration of CNTF in the brain, we used recombinant cells secreting CNTF encapsulated in alginate polymers. The implantation of these bioreactors in the brain of Abeta oligomer-infused mice led to a continuous secretion of recombinant CNTF and was associated with the robust improvement of cognitive performances. Most importantly, CNTF led to full recovery of cognitive functions associated with the stabilization of synaptic protein levels in the Tg2576 AD mouse model. In vitro as well as in vivo, CNTF activated a Janus kinase/signal transducer and activator of transcription-mediated survival pathway that prevented synaptic and neuronal degeneration. These preclinical studies suggest that CNTF and/or CNTF receptor-associated pathways may have AD-modifying activity through protection against progressive Abeta-related memory deficits. Our data also encourage additional exploration of ex vivo gene transfer for the prevention and/or treatment of AD.