Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Acceptability and perceived feasibility of the KaziKidz health promotion intervention among educators and caregivers in schools from South Africa: a qualitative descriptive study.

BACKGROUND: Despite the uncontested benefits of physical activity, its promotion lags behind in the public health agenda of low- and middle-income countries (LMICs). School-based interventions are promising strategies to foster health during childhood, but evidence of their effectiveness is limited and inconclusive for LMICs. Thus, further investigation is needed on contextual factors associated with intervention implementation in low-resource settings. We studied the acceptability and feasibility of the KaziKidz health promotion intervention and its implementation and make recommendations to improve future adoption and sustainability. METHODS: KaziKidz was implemented in four primary schools from low-income communities in South Africa in 2019. Semi-structured interviews with four school principals, three focus group interviews with 16 educators, and another three with 16 caregivers were conducted between October and November 2021. Participants were purposively recruited. Interview transcripts were analyzed via thematic analysis using a deductive and reflexive approach. RESULTS: Three main themes influencing intervention implementation and adoption were identified: (1) prioritizing teachers' needs (2), integrating the program into the school structure, and (3) creating opportunities in the community. Supporting recommendations included: (theme 1) adopting intervention approaches that are inclusive of educators' health and providing them with capacity development and external support; (theme 2) fostering a feeling of ownership and belonging among school stakeholders to adapt interventions to specific resources and needs; and (theme 3) raising community awareness to encourage individuals to claim power over and actively engage with the program. CONCLUSIONS: Comprehensive interventions comprising health support, adequate training, and ongoing assistance for educators combined with school-wide and community outreach actions seeking to strengthen program ownership, accountability, and engagement can enhance uptake of school-based interventions and long-term maintenance. TRIAL REGISTRATION: ISRCTN15648510, registered on 17/04/2020.

An intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly and the recruitment of Pol-α to Mcm2-7.

Mcm10 is an essential eukaryotic factor required for DNA replication. The replication fork helicase is composed of Cdc45, Mcm2-7 and GINS (CMG). DDK is an S-phase-specific kinase required for replication initiation, and the DNA primase-polymerase in eukaryotes is pol α. Mcm10 forms oligomers in vitro, mediated by the coiled-coil domain at the N-terminal region of the protein. We characterized an Mcm10 mutant at the N-terminal Domain (NTD), Mcm10-4A, defective for self-interaction. We found that the Mcm10-4A mutant was defective for stimulating DDK phosphorylation of Mcm2, binding to eighty-nucleotide ssDNA, and recruiting pol α to Mcm2-7 in vitro. Expression of wild-type levels of mcm10-4A resulted in severe growth and DNA replication defects in budding yeast cells, with diminished DDK phosphorylation of Mcm2. We then expressed the mcm10-4A in mcm5-bob1 mutant cells to bypass the defects mediated by diminished stimulation of DDK phosphorylation of Mcm2. Expression of wild-type levels of mcm10-4A in mcm5-bob1 mutant cells resulted in severe growth and DNA replication defects, along with diminished RPA signal at replication origins. We also detected diminished GINS and pol-α recruitment to the Mcm2-7 complex. We conclude that an intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly, and the recruitment of pol α to Mcm2-7.

Mcm10 coordinates the timely assembly and activation of the replication fork helicase.

Mcm10 is an essential replication factor that is required for DNA replication in eukaryotes. Two key steps in the initiation of DNA replication are the assembly and activation of Cdc45-Mcm2-7-GINS (CMG) replicative helicase. However, it is not known what coordinates helicase assembly with helicase activation. We show in this manuscript, using purified proteins from budding yeast, that Mcm10 directly interacts with the Mcm2-7 complex and Cdc45. In fact, Mcm10 recruits Cdc45 to Mcm2-7 complex in vitro. To study the role of Mcm10 in more detail in vivo we used an auxin inducible degron in which Mcm10 is degraded upon addition of auxin. We show in this manuscript that Mcm10 is required for the timely recruitment of Cdc45 and GINS recruitment to the Mcm2-7 complex in vivo during early S phase. We also found that Mcm10 stimulates Mcm2 phosphorylation by DDK in vivo and in vitro. These findings indicate that Mcm10 plays a critical role in coupling replicative helicase assembly with helicase activation. Mcm10 is first involved in the recruitment of Cdc45 to the Mcm2-7 complex. After Cdc45-Mcm2-7 complex assembly, Mcm10 promotes origin melting by stimulating DDK phosphorylation of Mcm2, which thereby leads to GINS attachment to Mcm2-7.

Role of the LEXE motif of protein-primed DNA polymerases in the interaction with the incoming nucleotide.

The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding ϕ29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg(2+) ions, a reaction highly improved when Mn(2+) was used as metal activator. These results, together with previous crystallographic resolution of ϕ29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg(2+). Recovery of both reactions with Mn(2+) supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP.

Development and characterization of a non-human primate model of disseminated synucleinopathy.

Introduction: The presence of a widespread cortical synucleinopathy is the main neuropathological hallmark underlying clinical entities such as Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB). There currently is a pressing need for the development of non-human primate (NHPs) models of PDD and DLB to further overcome existing limitations in drug discovery. Methods: Here we took advantage of a retrogradely-spreading adeno-associated viral vector serotype 9 coding for the alpha-synuclein A53T mutated gene (AAV9-SynA53T) to induce a widespread synucleinopathy of cortical and subcortical territories innervating the putamen. Four weeks post-AAV deliveries animals were sacrificed and a comprehensive biodistribution study was conducted, comprising the quantification of neurons expressing alpha-synuclein, rostrocaudal distribution and their specific location. Results: Intraputaminal deliveries of AAV9-SynA53T lead to a disseminated synucleinopathy throughout ipsi- and contralateral cerebral cortices, together with transduced neurons located in the ipsilateral caudal intralaminar nuclei and in the substantia nigra pars compacta (leading to thalamostriatal and nigrostriatal projections, respectively). Cortical afferent systems were found to be the main contributors to putaminal afferents (superior frontal and precentral gyri in particular). Discussion: Obtained data extends current models of synucleinopathies in NHPs, providing a reproducible platform enabling the adequate implementation of end-stage preclinical screening of new drugs targeting alpha-synuclein.

Intervention effects and long-term changes in physical activity and cardiometabolic outcomes among children at risk of noncommunicable diseases in South Africa: a cluster-randomized controlled trial and follow-up analysis.

Introduction: Risk factors for noncommunicable diseases such as insufficient physical activity (PA), overweight or hypertension are becoming increasingly predominant among children globally. While school-based interventions are promising preventive strategies, evidence of their long-term effectiveness, especially among vulnerable populations, is scarce. We aim to assess the short-term effects of the physical and health KaziKidz intervention on cardiometabolic risk factors and the long-term, pre-and post-COVID-19 pandemic changes thereof in high-risk children from marginalized communities. Methods: The intervention was tested in a cluster-randomized controlled trial between January and October 2019 in eight primary schools near Gqeberha, South Africa. Children with overweight, elevated blood pressure, pre-diabetes, and/or borderline dyslipidemia were identified and re-assessed 2 years post-intervention. Study outcomes included accelerometry-measured PA (MVPA), body mass index (BMI), mean arterial pressure (MAP), glucose (HbA1c), and lipid levels (TC to HDL ratio). We conducted mixed regression analyses to assess intervention effects by cardiometabolic risk profile, and Wilcoxon signed-rank tests to evaluate longitudinal changes in the high-risk subpopulation. Results: We found a significant intervention effect on MVPA during school hours for physically inactive children, and among active as well as inactive girls. In contrast, the intervention lowered HbA1c and TC to HDL ratio only in children with glucose or lipid values within the norm, respectively. At follow-up, the intervention effects were not maintained in at-risk children, who showed a decline in MVPA, and an increase in BMI-for-age, MAP, HbA1c and TC to HDL ratio. Conclusion: We conclude that schools are key settings in which to promote PA and improve health; however, structural changes are necessary to ensure that effective interventions reach marginalized school populations and achieve sustainable impact.

Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome.

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. KEY MESSAGES: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.

Relationship between Body Mass Index and Physical Activity among Children from Low-Income Communities in Gqeberha, South Africa: A Cross-Sectional Study.

This study aimed to establish the prevalence of underweight, overweight and obesity, the level of moderate-to-vigorous physical activity (MVPA) and the association thereof among vulnerable children from low-income communities in South Africa. Cross-sectional data were collected from 916 children (467 boys and 449 girls) aged 8-13 years (x̄ = 10.4 ± 1.2 years) attending eight low-income schools in Gqeberha, South Africa. Measured outcomes included accelerometery-measured physical activity (PA), weight, height and body mass index (BMI). Analysis of variance was used to determine the mean difference of total MVPA stratified by sex and BMI classification. Overall, 13% of the cohort were underweight, 19% were overweight/obese and 64% engaged in 60 min of MVPA per day. Girls presented nearly twice the odds of being overweight or obese than boys (95% CI: 1.40-2.77). Underweight to normal-weight children (boys: OR = 3.89, 95% CI: 2.18-6.93; girls: OR = 1.78, 95% CI: 1.13-2.80) were more likely to engage in 60 min/day of MVPA than overweight to obese children. There is an inverse association between BMI categories and theduration of MVPA achieved per day. Special attention should be aimed at increasing awareness of healthy nutrition and promoting a variety of PA, especially among girls and children with excess weight.

Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes.

DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium Thermus thermophilus strain HB27 encodes a Ppol protein among the genes present in mobile element ICETh2, absent in other T. thermophilus strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every ppol mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the ppol mutants in combination with addAB mutants. The results show that knockout addAB mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas ppol mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the ppol and/or addAB mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains. All these data support that Ppol appears to keep in balance the activity of AddAB as a part of the DNA housekeeping maintenance in T. thermophilus HB27, thus, playing a key role in its genome stability.