Development of Hsmar1 transposon tools for research and biotechnology

DNA transposons are mobile genetic elements that relocate between genomic loci. This relocation is termed transposition and is catalysed by an element-encoded enzyme called transposase. Hsmar1 is a DNA transposon that invaded the primate genome ~50 Mya and was active for ~13 My. Over the last decade, the molecular mechanisms of Hsmar1 transposition and autoregulation have been covered extensively. I took advantage of this mechanistic understanding to develop transposon technologies using Hsmar1 as a proof-of-concept platform. In research and biotechnology, transposons are used for insertional mutagenesis, transgenesis, gene therapy and next-generation sequencing. To improve existing transposon tools, I sought to establish increased control over the transposition reaction. Most transposases integrate their transposons semi-randomly throughout the genome. To control where transposons are integrated, I fused the Hsmar1 transposase to a catalytically inactive Cas9 variant, dCas9. The aim was to bias transposon insertions into the vicinity of a pre-determined target site bound by a guide RNA-dCas9-transposase ribonucleoprotein complex. I detected a 14.8-fold enrichment of transposon insertions into a 600 bp target site in an in vitro plasmid-to-plasmid assay compared to unfused transposase. Additionally, I show that 100 % of targeted insertions occur within 22 bp to one side of the guide RNA binding site. Next, I developed a light-inducible transposase for non-invasive, spatio-temporal control over the onset of transposition. Light-activated transposase reached up to 50 % of the activity of wild-type. To find hyperactive transposases, I constructed a library of 2 x 105 transposase mutants. I isolated three hyperactive variants and found that hyperactivity occurs due to faster transposon-end synapsis. Lastly, I developed an Hsmar1 transpososome, which is a pre-assembled synaptic complex in vitro. I show that this complex can be chemically transformed into E. coli for one-step insertional mutagenesis. Furthermore, the transpososome can also be lipofected into human cells, where the level of transposition exceeds that of the traditionally used transposase-helper plasmids

Targeted DNA transposition in vitro using a dCas9-transposase fusion protein

Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1. Integrations were unidirectional and tightly constrained to one side of the sgRNA binding site. Further analysis of the nucleoprotein intermediates demonstrated that the transposase and Cas9 moieties can bind their respective substrates independently or in concert. Kinetic analysis of the reaction in the presence of the Cas9 target–DNA revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli. Specific integration into the lacZ gene in E. coli was obscured by a high background of random integrations. Nevertheless, Cas9 is an attractive candidate for transposon-targeting because it has a high affinity and long dwell-time at its target site. This will facilitate a future optogenetic strategy for the temporal control of integration, which will increase the ratio of targeted to untargeted events

Development of Hsmar1 transposon tools for research and biotechnology

DNA transposons are mobile genetic elements that relocate between genomic loci. This relocation is termed transposition and is catalysed by an element-encoded enzyme called transposase. Hsmar1 is a DNA transposon that invaded the primate genome ~50 Mya and was active for ~13 My. Over the last decade, the molecular mechanisms of Hsmar1 transposition and autoregulation have been covered extensively. I took advantage of this mechanistic understanding to develop transposon technologies using Hsmar1 as a proof-of-concept platform. In research and biotechnology, transposons are used for insertional mutagenesis, transgenesis, gene therapy and next-generation sequencing. To improve existing transposon tools, I sought to establish increased control over the transposition reaction. Most transposases integrate their transposons semi-randomly throughout the genome. To control where transposons are integrated, I fused the Hsmar1 transposase to a catalytically inactive Cas9 variant, dCas9. The aim was to bias transposon insertions into the vicinity of a pre-determined target site bound by a guide RNA-dCas9-transposase ribonucleoprotein complex. I detected a 14.8-fold enrichment of transposon insertions into a 600 bp target site in an in vitro plasmid-to-plasmid assay compared to unfused transposase. Additionally, I show that 100 % of targeted insertions occur within 22 bp to one side of the guide RNA binding site. Next, I developed a light-inducible transposase for non-invasive, spatio-temporal control over the onset of transposition. Light-activated transposase reached up to 50 % of the activity of wild-type. To find hyperactive transposases, I constructed a library of 2 x 105 transposase mutants. I isolated three hyperactive variants and found that hyperactivity occurs due to faster transposon-end synapsis. Lastly, I developed an Hsmar1 transpososome, which is a pre-assembled synaptic complex in vitro. I show that this complex can be chemically transformed into E. coli for one-step insertional mutagenesis. Furthermore, the transpososome can also be lipofected into human cells, where the level of transposition exceeds that of the traditionally used transposase-helper plasmids

Depth-Wise Distribution of Soil Chemical Properties, Micro-nutrients Status, and Bacterial Population under Quercus leucotrichophora and Shorea robusta Forest of Chakrata and Thano Region of Uttarakhand

Forest trees have an appreciable demand for nutrients as they have longer rotation than crops and this is easily replenished by the constant release of nutrients resulting from weathering under favorable conditions in the soils developed from parent material rich in nutrient-bearing minerals. A study was undertaken to assess the chemical properties, micro-nutrient status, and bacterial population; two sites were taken for the collection of soil samples: the oak forest of Chakrata and the Sal forest of Thano. Soils were drawn at three depths viz. 0-30 cm, 30-60 cm, 60-90 cm. All soil parameters examined at three depths viz. 0-30 cm, 30-60 cm, 60-90 cm. Oak (Quercus Leucotrichophora) forests have higher microbial activity than Sal forests. The soil in both the studied areas was rich in nutrients, where, the maximum mean standard deviation values of pH (6.85), soil organic carbon (5.9 %), available nitrogen (0.04 %) reported in the Oak forest of Chakrata, and available phosphorus were almost similar in both forest regions. Oak forests have higher organic carbon, water-holding capacity, and nutrient availability compared to Sal forests. However, it is concluded that, for better conditions of these forests for soil and carbon-storing potential, the forest needs good management practices, especially in community forest areas to avoid illegal felling, impact of fire, and over-exploitation of fuel and fodder. Good management practices would help mitigate the impact of climate change and sustainable outcomes of the resources for the community's benefit

Electric Field Driven Reversible Spinodal Dewetting of Thin Liquid Films on Slippery Surfaces

Abstract The stability of thin liquid films on a surface depends on the excess free energy of the system involving various short‐range and long‐range interactions. When forced to wet an unfavorable surface, thin liquid films dewet into multiple small‐sized droplets via spinodal, homogeneous, or heterogeneous nucleation process. However, if the total excess free energy of the system can be manipulated using external stimuli, for example, electric field, temperature, or light, one can control the stability of thin liquid films on demand. Here the electric‐field induced reversible dewetting and rewetting of thin liquid films underneath aqueous drops on slippery surfaces is studied. Upon applying voltage, hundreds of nanometer thick stable liquid films dewet in a manner identical to the spinodal dewetting of few nanometer‐thick liquid films following the linear stability analysis. Upon removing the applied voltage, the dewetted droplets spread, coalesce with neighboring ones, and form a uniform film again, albeit taking significantly longer times. The characteristic features defined via spatial and temporal evolution of the dewetting and rewetting processes are present over multiple cycles suggesting the complete reversibility of the process

Vermistabilization of mango tree pruning waste with five earthworm species: A biochemical and heavy metal assessment

Mango tree pruning results in high biomass output, which is a serious agricultural and environmental problem. Vermicomposting is a potential, fast and sustainable tool to address these challenges. For sixty days, the experiment was carried out in six vermireactors containing five earthworm species by Eudrilus eugeniae, Eisenia fetida, Aporrectodea rosea, Lumbricus rubellus, and Lampito mauritii, as well as composting (without earthworm) using mango tree pruning waste biomass along with cattle dung as an instant preferred feeding material for earthworms. The pH, TOC, C/N and C/P ratios of the waste were substantially reduced by the earthworm activity. However, after vermicomposting, the levels of macronutrients (N, P, K, Ca, Mg, S) and micronutrients (Fe, Mn, Zn, and Cu) and microbial count substantially increased. The TOC content of waste was reduced by 42–55%, and the C/N of vermicompost ranged from 5.58 to 11.38. The results showed that earthworm fecundity was highest in vermireactors containing Eudrilus eugeniae and Eisenia fetida. The current study was ultimately determine that vermicomposting using Eudrilus eugeniae or Eisenia fetida is an effective strategy for utilising mango tree pruning waste, ensuring environmental sustainability and improving farmer revenue

Complementary and alternative medicine/therapy for tobacco cessation in India: A secondary analysis of GATS-1 and 2

Background: India has nearly 267 million adult tobacco users, with a slowly improving quitting rate. Among the many approaches to quitting the habit, such as counseling, nicotine replacement therapy, nicotine patch or gum, and prescribed allopathic medicines. Complementary and alternative medicine/therapy (CAM), a thousand-year-old practice in India, may also prove to be a potential method in tobacco cessation; however, there is scarce literature on the extent of use of CAM among tobacco users who attempt to quit the habit. Therefore, this study attempts to examine the potential of CAM as a strategy for tobacco control in India. Material and Methods: We undertook a secondary analysis of the data from both rounds of the Global Adult Tobacco Survey (GATS 2009 and 2016). The dependent variable included in the analysis was the use of traditional medicine as a method for quitting tobacco in three types of users—smokers, smokeless tobacco users, and dual users. The prevalence of CAM use was reported, and Chi-square test was applied to find the factors significantly associated with the use of CAM among tobacco users considering a P value of 0.05 to be statistically significant. Results: The overall prevalence of traditional medicine use for GATS-1 was observed to be more among dual users (4%), while for GATS-2, it was highest among smokers (3%). For both rounds of the GATS survey, the use of traditional medicine was found to be higher among males, rural residents, users with no education or less than primary education, and the eastern region. Conclusions: CAM has a promising potential for supporting tobacco cessation provided a concerted effort is undertaken to standardize pharmacopeia and establish robust clinical evidence. In addition, there is a need to create awareness, build the capacity of healthcare providers, and foster academic-industrial research in indigenous Ayurveda, Yoga, Naturopathy, Unani, Siddha, and Homeopathy (AYUSH) systems