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Autism spectrum disorder (ASD) is among the most common developmental disorders of great social significance. Adequate intervention can increase socialization of autistic individuals, both increasing their quality of life and decreasing financial burden. Initiatives to introduce modern standards of autism treatment and education in Russia were launched several years ago; unfortunately, there is little information on the perceived problems and barriers, which may hinder further development of autism support system. This paper presents the results of a stakeholder survey including both service receivers (parents of children with ASD) and service providers (professionals working with ASD children) from different regions of Russia. The study reveals that the quality of specialized services related to autism treatment is assessed much higher than the quality of inclusive education and accessibility of general education and other general services available to children and adults with ASD, highlighting the importance of implementing an interdepartmental system providing continuous support to people with ASD and facilitating their inclusion and social integration. The results also show that general attitude to distant diagnostics and education, on par to the estimates of quality of services related to distant education, are lower in non-capital regions, where the need for such services is higher due to lower accessibility of person-to person services. The present study may be of use both for professionals in autism treatment and education, and for the specialists who may encounter persons with autism once in a while, as it brings a detailed estimation of several aspects of autism support both through the eyes of support receivers and providers.

The biosynthesis of butyric acid from glucose through inverted fatty acid β-oxidation by recombinant Escherichia coli strains was optimized. An increased yield of the target compound was achieved resulting from the plasmid expression of the atoB, fadB, and fadE/fabI genes in the core strain MG∆4 PL-tesB ΔyciA (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, PL-SDϕ10-tesB, ∆yciA). The positive effect of enforced ATP hydrolysis on microaerobic conversion of the carbohydrate substrate to the final product by the recombinants was demonstrated. Activation of the futile cycle of pyruvate–phosphoenolpyruvate–pyruvate, due to the increased expression of the ppsA gene, ensured a marked increase in glucose consumption by the recombinants and led to an increase in the molar yield of butyric acid up to 39.5%. When the components of the H+-ATP synthase complex were uncoupled resulting from the deletion of atpFH genes, the molar yield of butyric acid from glucose demonstrated by the strain forming butyryl-CoA by the action of enoyl-ACP reductase FabI reached 46%. [ABSTRACT FROM AUTHOR]

Inverted fatty acid β-oxidation represents a versatile biochemical platform for biosynthesis by the engineered microbial strains of numerous value-added chemicals from convenient and abundant renewable carbon sources, including biomass-derived sugars. Although, in recent years, significant progress has been made in the production through this pathway of n-alcohols, 1,3-diols, and carboxylic acids and its 2,3-unsaturated derivatives, the potential of the pathway for the biosynthesis of 3-hydroxycarboxylic acids remained almost undisclosed. In this study, we demonstrate the microaerobic production of even-chain-length C4–C8 3-hydroxycarboxylic acids from glucose through the inverted fatty acid β-oxidation by engineered E. coli strains. The notable accumulation of target compounds was achieved upon the strong constitutive expression of the genes atoB, fadA, fadB, fadE/fabI, and tesB, which code for the key enzymes catalysing reactions of aerobic fatty acid β-oxidation and thioesterase II, in strains devoid of mixed-acid fermentation pathways and lacking nonspecific thioesterase YciA. The best performing recombinants were able to synthesise up to 14.5 mM of 3-hydroxycarboxylic acids from glucose with a total yield of 0.34 mol/mol and a C4/C6/C8 ratio averaging approximately 63/28/9. The results provide a framework for the development of highly efficient strains and processes for the bio-based production of valuable 3-hydroxycarboxylates from renewable raw materials. [ABSTRACT FROM AUTHOR]

The effect of anaplerotic pathways activation on CO2-dependent anaerobic glucose utilization by Escherichia coli strains deficient in the main fermentation pathways and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO2 generation in the strains was ensured resulting from oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Sodium bicarbonate dissolved in the medium was used as an external source of CO2. The genes of heterologous pyruvate carboxylase and native NADH-dependent malic enzyme were overexpressed in the strains to allow anaplerotic carboxylation of pyruvic acid to oxaloacetic or malic acid. The ability of the strains to reoxidize NADH utilizing carboxylation products was additionally increased due to enhanced expression of malate dehydrogenase gene. In the case of endogenous CO2 formation, the activation of anaplerotic pathways did not cause a notable increase in the anaerobic glucose consumption by the constructed strains. At the same time, the expression of pyruvate carboxylase led to a pronounced decrease in the secretion of pyruvic acid with the concomitant increase in the yield of four-carbon metabolites. Further enhancement of NADH-dependent malic enzyme expression provoked activation of a pyruvate - oxaloacetate - malate - pyruvate futile cycle in the strains. The availability in the medium of the external CO2 source sharply increased the anaerobic utilization of glucose by strains expressing pyruvate carboxylase. The activity of the futile cycle has raised with the increased malic enzyme expression and dropped upon enhancement of malate dehydrogenase expression. As a result, the efficiency of CO2-dependent anaerobic glucose utilization coupled to the formation of four-carbon carboxylation products increased in the studied strains resulting from the primary anaplerotic conversion of pyruvic acid into oxaloacetic acid followed by the involvement of the precursor formed in NADH-consuming biosynthetic reactions dominating over the reactions of the revealed futile cycle. [ABSTRACT FROM AUTHOR]

Background: The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmidless marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on in vitro and in vivo recombination processes have been developed. Results: Here, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant E. coli strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial ψ80-attB sites into desired points of the chromosome followed by two site-specific recombination processes: first, the ψ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with ψ80-attP-site, and second, the λ system is used for excision of inserted vector part, including the plasmid ori-replication and the marker, flanked by ψ-attL/R-sites. Conclusion: The developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry. [ABSTRACT FROM AUTHOR]

Background: RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet. Results: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing PlacUV5→lad. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range. Conclusion: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production. [ABSTRACT FROM AUTHOR]

Escherichia coli open reading frames ydiO and ydiQRST were identified as genes encoding components of the acyl-CoA dehydrogenase complex of anaerobic fatty acid β-oxidation. Individual or concomitant inactivation of fadE gene, encoding known aerobic acyl-CoA dehydrogenase, and ydiO and/or ydiQRST genes did not affect cellular growth on glucose as a sole carbon source. Aerobic growth on sodium oleate was observed only for the cells with intact fadE gene. With an alternative electron acceptor, the cells possessing intact fadE gene demonstrated anaerobic growth on sodium oleate irrespective of the presence or absence of ydiO and ydiQRST genes. For the fadE-deficient mutants, anaerobic growth on sodium oleate was observed only for cells with intact ydiO and ydiQRST genes, while the fadE/ydiO and fadE/ydiQRST mutants failed to grow under the similar conditions.