Development of a selection to recover improved DNA ligase enzymes during directed evolution

DNA ligases are essential enzymes used in many molecular biology applications. Of particular note, they are important enzymes in next generation sequencing (NGS) technologies. The improved speed, efficiency, and affordability of NGS over Sanger sequencing has greatly expanded the applications of DNA sequencing. In most NGS technologies ligase enzymes play a crucial role, for instance in ligating adaptors onto sequence fragments during sample preparation. This key step requires a blunt-ended ligation reaction, with highly efficient ligases required in order to create a sample library of high quality. The current go-to enzyme is T4 DNA ligase, which has not evolved in Nature to perform blunt ended ligations, and as such has relatively poor levels of activity when compared to other substrates. There is therefore potential to improve upon this enzyme and engineer a ligase that is more efficient with blunt-ended substrates. We have developed a novel function-based directed evolution selection to evolve blunt-ended ligases that have greater catalytic efficiency. The basis for this approach is the over-expression of a ligase enzyme variant which is then incubated with a linearised plasmid encoding for that same ligase variant. More efficient ligases will ligate the plasmid encoding for their own gene variant more efficiently (in a blunt-ended ligation), and so greater numbers of the circularised plasmid will be produced. Through successive rounds of transformation, amplification and ligation the most improved enzyme variants are enriched. This selection approach is being used to evaluate a panel of ligase variants in order to identify the best ligases for blunt-ended ligation applications. Please click Additional Files below to see the full abstract

Trajectories of temperament from late childhood through adolescence and associations with anxiety and depression in young adulthood

Anxiety and depression are pervasive and pernicious mental health problems for young adults. Developmental trajectories of adolescent temperament (Effortful Control, Negative Emotionality, and Positive Emotionality) may help us predict who will experience anxiety/depression during young adulthood. The present study used longitudinal data from a large, community sample of Mexican-origin youth ( N = 674) to examine how temperament develops across adolescence (age 10–16) and whether the developmental trajectories of temperament are associated with anxiety/depression during young adulthood (ages 19 and 21). Results indicate that Effortful Control, Negatively Emotionality, and the Affiliation facet of Positive Emotionality tend to decrease across adolescence, whereas Surgency tends to increase. Smaller decreases in Effortful Control and greater increases in Positive Emotionality across adolescence were associated with fewer anxiety/depression symptoms during young adulthood, whereas smaller decreases in Negative Emotionality were associated with more anxiety/depression symptoms later on. Thus, temperament development serves as both a protective factor (Effortful Control, Positive Emotionality) and a risk factor (Negative Emotionality) for later anxiety/depression in Mexican-origin youth

Gene editing enables rapid engineering of complex antibiotic assembly lines

From Springer Nature via Jisc Publications RouterHistory: received 2021-09-09, accepted 2021-11-02, registration 2021-11-08, pub-electronic 2021-11-25, online 2021-11-25, collection 2021-12Publication status: PublishedFunder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268; Grant(s): BB/L013754/1, BB/N023536/1Abstract: Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized

Genetic and Clinical Predictors of Age of ESKD in Individuals With Autosomal Dominant Tubulointerstitial Kidney Disease Due to UMOD Mutations

n/

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells

Doubly Robust Regression Analysis for Data Fusion

10.5705/ss.202018.0334Statistica Sinic

Escherichia coli NemA is an efficient chromate reductase that can be biologically immobilized to provide a cell free system for remediation of hexavalent chromium.

Hexavalent chromium is a serious and widespread environmental pollutant. Although many bacteria have been identified that can transform highly water-soluble and toxic Cr(VI) to insoluble and relatively non-toxic Cr(III), bacterial bioremediation of Cr(VI) pollution is limited by a number of issues, in particular chromium toxicity to the remediating cells. To address this we sought to develop an immobilized enzymatic system for Cr(VI) remediation. To identify novel Cr(VI) reductase enzymes we first screened cell extracts from an Escherichia coli library of soluble oxidoreductases derived from a range of bacteria, but found that a number of these enzymes can reduce Cr(VI) indirectly, via redox intermediates present in the crude extracts. Instead, activity assays for 15 candidate enzymes purified as His6-tagged proteins identified E. coli NemA as a highly efficient Cr(VI) reductase (k(cat)/K(M)= 1.1×10(5) M(-1) s(-1) with NADH as cofactor). Fusion of nemA to the polyhydroxyalkanoate synthase gene phaC from Ralstonia eutropha enabled high-level biosynthesis of functionalized polyhydroxyalkanoate granules displaying stable and active NemA on their surface. When these granules were combined with either Bacillus subtilis glucose dehydrogenase or Candida boidinii formate dehydrogenase as a cofactor regenerating partner, high levels of chromate transformation were observed with only low initial concentrations of expensive NADH cofactor being required, the overall reaction being powered by consumption of the cheap sacrificial substrates glucose or formic acid, respectively. This system therefore offers promise as an economic solution for ex situ Cr(VI) remediation

The use of 3D geological models in the development of the conceptual groundwater model

The conceptual groundwater model is heavily dependent on the geological framework which is used to defi ne the aquifer being studied. In the past, two-dimensional datasets such as geological maps and cross-sections were used in coordination with site-specifi c point data to build a conceptual understanding at the site or catchment scale. This is then simplified and it is this simplifi ed version which is used to build the framework for the numerical groundwater fl ow model. Due to the way the geological framework model and the conceptual groundwater model were generated they could not be viewed together; this inevitably led to a signifi cant loss of information and understanding. With the current rapid developments in 3D modelling software and the increasing availability of digital geological data it is now possible to produce detailed 3D geological models of complex aquifer sequences. In this paper we will use two case studies (Chalk aquifer of the London Basin and the Jurassic limestone aquifer of the Cotswolds) to demonstrate that by developing a detailed 3D geological model signifi cant benefi ts are gained in the understanding and development of the conceptual groundwater model

The role of temperament in the onset of suicidal ideation and behaviors across adolescence: Findings from a 10-year longitudinal study of Mexican-origin youth.

Suicide among young people is an increasingly prevalent and devastating public health crisis around the world. To reduce the rate of suicide, it is important to identify factors that can help us better predict suicidal ideation and behaviors. Adolescent temperament (effortful control, negative emotionality, positive emotionality) may be a source of risk and resilience for the onset of suicidal ideation, plans, and attempts. The present study uses longitudinal data from a large, community sample of Mexican-origin youth (N = 674), assessed annually from age 12 to 21, to examine how temperament is associated with the onset of suicidal ideation and behaviors during adolescence and young adulthood. Results indicate that higher levels of effortful control (activation control, inhibitory control, attention) are associated with decreased probability of experiencing the onset of suicidal ideation, plans, and attempts, whereas higher levels of negative emotionality (particularly aggression, frustration, and depressed mood) are associated with increased probability of experiencing the onset of suicidal ideation and behaviors. Positive emotionality (surgency, affiliation) was not associated with the onset of suicidal ideation and behaviors. Supplemental analyses showed conceptually similar findings for the Big Five, with Conscientiousness associated with decreased risk, Neuroticism associated with increased risk, and the other three dimensions showing largely null results. The findings did not vary significantly for boys and girls or for youth born in the U.S. versus Mexico. Overall, these findings suggest that adolescent temperament serves as both a protective factor (via effortful control/Conscientiousness) and a risk factor (via negative emotionality/Neuroticism) for suicidal ideation and behaviors in Mexican-origin youth. (PsycInfo Database Record (c) 2022 APA, all rights reserved)