Community child psychiatry: evolution and direction.
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Black or African American↗
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Results for “directed evolution”
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[Selection by structured traits and the direction of evolution].
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Animals↗
[Nikolai L'vovich Gerbil'skii and ecologo-physiologic directions in evolution morphology].
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Anatomy↗
Disruption of efflux activity reduces biofilm formation through multiple pathways.
Free-swimming bacteria must undergo large-scale changes in gene expression to form structured, aggregated biofilm communities. These regulatory changes are susceptible to environmental stimuli such as exposure to antimicrobials, which can affect adhesion, biofilm matrix production, pathogenicity and multidrug susceptibility. Previously, we found that genetic or chemical inactivation of efflux activity in Escherichia coli and Salmonella Typhimurium disrupts biofilm formation with a wide range of pathways sensitive to efflux inhibition, including reduced expression of csgD, a major regulator of biofilm matrix production. How the regulatory networks controlling efflux activity and biofilm formation overlap and how perturbing efflux impacts biofilm formation is still unclear. To address this, we used a combination of directed evolution experiments and large-scale functional genomics screens (TraDIS-Xpress) to identify the genes and pathways affecting efflux activity and biofilm formation in Salmonella enterica serovar Typhimurium and E. coli. This work describes the landscape of pathways linking efflux activity and biofilm formation. Whilst no singular gene or pathway was found to control the link between the two phenotypes, we propose changes in membrane potential following efflux inactivation are sensed through multiple response regulators that each in turn contribute to repression of biofilm development. These include the two-component signal transduction system EnvZ-OmpR and AraC/XylS family transcriptional regulators, RamA and MarA, which have extensive overlapping regulons and demonstrate high degrees of functional redundancy. This work deepens our understanding of the regulatory networks governing efflux activity and biofilm formation in Enterobacteriaceae and highlights the level of overlapping regulation and functional redundancy between them.
Salmonella typhimurium↗
[Anthropophysiologic basis of the formation of arterial hypertension in primates].
Comparative physiological analysis suggested evolutional direction of typical organization of the blood orthostatic regulation and its dependence upon the posture status in pronograde animals (rats, rabbits, cats, dogs) as well as in primates (humans, monkeys). Data on the evidence of functional adaptation of cardiovascular system under the conditions of experimental bipedal posture in monkeys are presented and the meaning of blood regulation according to the gravity factor in the development of arterial hypertension is discussed.
Adaptation, Physiological↗
Studies on the mechanism of Tris-induced inactivation of oxygen evolution.
A study was made of the inactivation by Tris of O2 evolution in chloroplasts and the subsequent reactivation of O2 evolution. We conclude: 1. At concentrations of Tris sufficient to inhibit O2 evolution directly, a slow rate (t 1/2 approximately 20--25 min) of inactivation occurs; 2. Inactivation is accelerated (t 1/2 approximately 2 min) by weak light absorbed by system II and is rate limited by a dark step with a half-time of about 200 s; 3. Minimally one quantum event within System II is sufficient to inactive 50--70% of the O2 evolving centers; 4. This process is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive but is inhibited by reduced dichlorophenol indophenol and phenazine methosulfate, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, 2-(3-chloro-4-trifluoromethyl)-anilino-3,5-dinitrothiophene and tetraphenylboron; 5. Partial reactivation of inactive O2 evolving centers is affected by the use of the same reagents inhibiting the light induced inactivations; 6. The life-time (t 1/2 approximately 1 to 3 h) of the activable state is correlated with diffusion across thylakoids of the larger manganese pool released from binding sites and remaining in thylakoids following inactivation of O2 evolution.
Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗
Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids.
More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina.
Animals↗
Decoding the distribution, structure-function-redox potential relationship and recent advances in fungal laccases: a systematic approach.
Laccases, categorized as multicopper oxidases, are recognized for their multifaceted roles in ecosystems and their utility in diverse industrial applications. Laccases from higher fungi, specifically Ascomycota and Basidiomycota, have garnered significant research interest due to their elevated redox potentials and their capacity to degrade lignin in decaying wood, alongside other industrial uses. Here, we have conducted a comprehensive and systematic analysis on fungal laccases using Web of Science, Scopus, PubMed, and ScienceDirect. The genomic distribution, phylogenetic affiliation, and structural organization of laccase-encoding genes in higher fungal species were investigated, as were the catalytic mechanisms of the corresponding enzymes. Additionally, the study explores the correlation between structural domains and redox potential, as well as the impact of post-translational modifications like glycosylation on enzyme activity. Furthermore, the recent advancements in laccase engineering, employing strategies such as rational design, directed evolution, and heterologous expression are discussed. The review also explores the scope of "artificial intelligence and machine learning" in deducing the structure-function relationships, optimizing codon usage, predicting signal peptides, enhancing enzymatic performance, and developing host-specific genetic engineering techniques is also discussed for tailoring fungal laccases to meet the demands of industrial biocatalysis for improved activity and stability.
Laccase↗
Oral granular cell tumors: a clinicopathologic and immunocytochemical study.
To investigate the histogenesis of the granular cell, a large series of granular cell tumors was studied for clinical and histopathologic features with emphasis on immunocytochemical markers. The nongingival granular cell tumors (NGGCT) were found to be more prevalent among females than males by a ratio of 2:1 and arose on the tongue (67%), the buccal mucosa (13%), the lips (8%), the soft palate (6%), and other sites (6%). With the use of the avidin-biotin-peroxidase method, polyclonal rabbit antisera were employed. The antisera were directed to the following antigens: S-100 protein, myoglobin, myosin, actin, desmin, alpha-1-antitrypsin, and muramidase. Results indicated that granular cell tumors are not homogenous for immunocytochemical markers. Nongingival granular cell tumors were universally positive for S-100 protein and failed to exhibit immunoreactivity for myogenous or histiocytic markers. Alternatively, the gingival granular cell tumor of infancy was negative for all markers, whereas rhabdomyoma was reactive with myogenous markers and a subpopulation of tumor cells displayed S-100 protein immunoreactivity. The granular cell ameloblastoma was reactive only with antiserum to alpha-1-antitrypsin. Ultrastructurally, granular cells from one of two NGGCT showed a direct evolution from skeletal muscle fibers. It is concluded that the oral NGGCT is a tumor positive for S-100 protein that may arise from muscle or nerve sheath.
Adolescent↗
Ultrahigh-throughput screening assay for PET-degrading enzymes.
In recent years, several PET-degrading enzymes have been identified from both known microorganisms and metagenomic sources in response to the growing environmental issue of polyethylene terephthalate (PET) accumulation. Despite this progress, there is a limited number of (ultra)high-throughput screening methods for assessing PET-hydrolyzing activity without relying on surrogate substrates. This method utilizes the coupled activity of ketoreductases (KREDs) and diaphorase to produce a fluorescent compound (resorufin) in the presence of PET degradation products, offering a more direct and efficient screening approach. A metagenomic KRED was coupled with the diaphorase from Clostridium kluyveri to enable the detection of the hydrolysis of PET degradation products catalyzed by the Bacillus subtilis BS2 esterase. The coupled reaction was established in water-in-oil microdroplets, encapsulating a single E. coli cell per droplet, demonstrating its potential for use in the ultrahigh-throughput screening of metagenomic libraries or randomized libraries for directed evolution campaigns.
High-Throughput Screening Assays↗
A base editor facilitates simultaneous purine and pyrimidine substitutions for ex vivo and in vivo mutagenesis screens.
Genetic mutations are closely linked to human diseases, yet the relationship between many mutations and their corresponding phenotypes remains poorly understood. Furthermore, tools to study the connection between nucleotide variations and phenotypes are limited. To address this issue, we developed ACGBEmax by fusing the dual-functional deaminase, engineered N-methylpurine DNA glycosylase, and evolved SOS response associated peptidase domain with nCas9(D10A). ACGBEmax enables the precise conversion of A, C, and G to other bases in mammalian cells, thereby generating an extensive range of base mutations types. We used ACGBEmax to generate HPRT variants, identifying mutations conferring resistance to 6-thioguanine. Additionally, we performed in situ mutagenesis of Ctnnb1 in mouse liver, identifying both known and potential oncogenic mutations. Our results prove that ACGBEmax is a powerful tool for generating a wide spectrum of mutation types at specific gene loci, highlighting its significant potential for applications in functional screening and the directed evolution of protein variants.
Animals↗
Novel human liver-tropic AAV variants define transferable domains that markedly enhance the human tropism of AAV7 and AAV8.
Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. Here, we report the bioengineering of a set of next-generation AAV vectors, named AAV-SYDs (where "SYD" stands for Sydney, Australia), with increased human hepato-tropism in a liver xenograft mouse model repopulated with primary human hepatocytes. We followed a two-step process that staggered directed evolution and domain-swapping approaches. Using DNA-family shuffling, we first mapped key AAV capsid regions responsible for efficient human hepatocyte transduction in vivo. Focusing on these regions, we next applied domain-swapping strategies to identify and study key capsid residues that enhance primary human hepatocyte uptake and transgene expression. Our findings underscore the potential of AAV-SYDs as liver gene therapy vectors and provide insights into the mechanism responsible for their enhanced transduction profile.
AAV↗
Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism.
No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.
Animals↗
Programmable enzymes for targeted gene insertion.
Genome editing technologies have advanced from nuclease-based reagents that generate programmed DNA double-strand breaks, which can cause deleterious effects, to next-generation reagents that perform controlled DNA modification through double-strand break-independent mechanisms, such as base editing and prime editing. Although these approaches enable precise small-scale sequence changes, methods for programmable insertion of large DNA cargos have been limited. The ability to write entire genes or large regions into the genome could transform the treatment of genetically heterogeneous disorders, for which numerous pathogenic variants underlie a common disease and mutation-specific editing strategies are impractical. Recent advances in computational genome mining have accelerated the discovery of naturally occurring enzymes with novel biochemical and functional properties, including recombinases and transposases capable of large-scale modifications. Moreover, directed evolution, rational engineering and expanded homologue discovery are enabling the repurposing and optimization of these systems for genome engineering. Here we review recent technology development efforts that harness diverse enzymes for kilobase-scale genome engineering, with a particular focus on CRISPR-associated transposase systems.
Journal Article↗
Binary vector copy number engineering improves Agrobacterium-mediated transformation.
The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.
Genetic Vectors↗
Fitness effects of a deletion mutation increasing transcription of the 6-phosphogluconate dehydrogenase gene in Escherichia coli.
Directed evolution in microbial organisms provides an experimental approach to molecular evolution in which selective forces can be controlled and favorable mutations analyzed at the molecular level. Here we present an analysis of a mutation selected in Escherichia coli in response to growth in a chemostat in which the limiting nutrient was gluconate. The selectively favored mutation, designated gnd+ (862), occurred in the gene gnd coding for 6-phosphogluconate dehydrogenase, used in gluconate metabolism. Although the allele is strongly favored in chemostats in which the limiting nutrient is gluconate, the selective effects of gnd+ (862) are highly dependent on growth conditions. In chemostats in which growth is limited by a mixture of gluconate and either ribose, glucose, or succinate, the gnd+ (862) allele is favored, disfavored, or neutral according to the relative concentrations of the substrates. The gnd+ (862) allele results from a deletion of 385 nucleotide pairs in the region 5' to the promoter of gnd, and one endpoint of the deletion is contiguous with the terminus of an IS5 insertion sequence located near gnd in E. coli K12. The gnd+ (862) allele shows a marked increase in transcription that accounts for most or all of the increased enzyme activity.
Alleles↗
Histopathologic and immunopathologic study of pyoderma gangrenosum.
Sixty-three patients with pyoderma gangrenosum were seen and studied at the Mayo Clinic from 1971 to 1980. Biopsies from the erythematous border or necrotic edge of the pyoderma gangrenosum lesions usually demonstrated a characteristic pathogenic morphologic evolution. The early lesions revealed mild to moderate perivascular lymphocytic infiltrate associated with endothelial swelling. The fully developed lesions demonstrated necrosis in addition to a dense lymphocytic infiltration surrounding as well as involving the blood vessels. Extravasation of erythrocytes and thrombosis sometimes were seen. Ulceration, infarction, and abscess formation were found in the later stages of evolution. Direct immunofluorescence results were positive in the blood vessels of 36 of 65 (55%) specimens. IgM, C3, and fibrin were found in the papillary and reticular dermal vessels. IgG and IgA were only occasionally present. Pyoderma gangrenosum appears to be a reactive process that is manifested as a vasculitis. Biopsy material from the advancing active erythematous border has early characteristic dermatopathologic findings of lymphocytic vasculitis. Cutaneous vascular immune deposits suggest an immune pathogenesis of either an immune complex disease or lymphocytotoxic reaction.
Biopsy↗
AAV's anatomy: roadmap for optimizing vectors for translational success.
Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success.
Cystic Fibrosis↗