Restriction Inhibition Assay: A Qualitative and Quantitative Method to Screen Sequence Specific DNA Binder from Herbal Plants

Purpose: To employ restriction inhibition assay (RIA) to screen phytochemical-rich fractions (PRFs) with high affinity for EcoRI and HindIII restriction sequences and correlate their interaction to an anticancer activity.Methods: pBR322 linear plasmid DNA was used as a template to screen the sequence-selective inhibition of aqueous extracts of Cinnamomum zeylanicum and Picrorhiza kurroa, respectively. The template was further incubated with different concentrations of PRFs prior to digestion with restriction endonucleases HindIII and EcoRI. The Expressed Sequence Tags (ESTs) and Sequence Tag Sites (STS) of oncogenes were screened for the presence of EcoRI and HindIII restriction sequences to associate an anticancer property to PRF.Results: The inhibitory concentrations of Cinnamomum zeylanicum aqueous extract against HindIII and EcoRI endonucleases were approximately 2.5 and 5 μg/μl, respectively. No binding was observed for Picrorhiza kurroa at both HindIII and EcoRI restriction sites. The saponin-rich fractions of Cinnamomum zeylanicum showed significant (p < 0.001) inhibition as compared to control at concentrations of 0.28±1.45 μg/μl for EcoRI and 0.11±2.68 μg/μl for HindIII endonucleases. Both EcoRI and HindIII restriction sites were found repeatedly in the STS and ESTs of BRCA2, the early onset oncogene.Conclusion: The inhibition of endonucleases by phytochemical-rich fractions provides direct evidence of the use of RIA for screening as well as demonstrating the binding specificity of these PRFs. The presence of 5’-AAGCTT-3’ & 5’-GAATTC-3’ in the ESTs of BRAC2 provides an insight into the use of screened components as leads in the search for novel anticancer compounds.Keywords: Restriction endonucleases, Restriction sites, Phytochemicals, Restriction inhibition assay (RIA), Binding specificity, Oncogenes, Sequence tag sites, Expressed sequence tag, Anticancer

Beneficiation Studies on Cobalt Bearing Ore from Africa

Cobalt bearing ore is used in Africa for hydro-metallurg-ical extraction of Co. The party desired to know whether it is possible to pre-concentrate this ore in order to reduce the material handled by metallurgical operations. This cobalt bearing sample (5% Co) was beneficiated in the Modern Mineral Processing Laboratory and Pilot Plant of Indian Bureau of Mines at Nagpur. The cobalt was ident-ified by EPMA to be present as WAD where manganese is replaced by cobalt. The as-received sample was dominated by fines. Screening the as received sample on 10 mesh followed by desliming of -10 mesh fraction yielded a Co concentrate assaying 7.2% Co with 94.6% cobalt recovery. Although the concentrate assays only 7.2% Co, this simple process developed offers the advantage that around 33% of the slimes (<20 microns in size assaying around I% Co) can bypass the metallurgical operations to follow thus drast-ically reducing the handling, settling and filtration problems in the extraction of Co by hydrometallurgical route with minimum Co losses in the tailings

Tuberculosis (TB) Aftermath: study protocol for a hybrid type I effectiveness-implementation non-inferiority randomized trial in India comparing two active case finding (ACF) strategies among individuals treated for TB and their household contacts

Background : Approximately 7% of all reported tuberculosis (TB) cases each year are recurrent, occurring among people who have had TB in the recent or distant past. TB recurrence is particularly common in India, which has the largest TB burden worldwide. Although patients recently treated for TB are at high risk of developing TB again, evidence around effective active case finding (ACF) strategies in this population is scarce. We will conduct a hybrid type I effectiveness-implementation non-inferiority randomized trial to compare the effectiveness, cost-effectiveness, and feasibility of two ACF strategies among individuals who have completed TB treatment and their household contacts (HHCs). Methods : We will enroll 1076 adults (≥ 18 years) who have completed TB treatment at a public TB unit (TU) in Pune, India, along with their HHCs (averaging two per patient, n = 2152). Participants will undergo symptom-based ACF by existing healthcare workers (HCWs) at 6-month intervals and will be randomized to either home-based ACF (HACF) or telephonic ACF (TACF). Symptomatic participants will undergo microbiologic testing through the program. Asymptomatic HHCs will be referred for TB preventive treatment (TPT) per national guidelines. The primary outcome is rate per 100 person-years of people diagnosed with new or recurrent TB by study arm, within 12 months following treatment completion. The secondary outcome is proportion of HHCs < 6 years, by study arm, initiated on TPT after ruling out TB disease. Study staff will collect socio-demographic and clinical data to identify risk factors for TB recurrence and will measure post-TB lung impairment. In both arms, an 18-month “mop-up” visit will be conducted to ascertain outcomes. We will use the RE-AIM framework to characterize implementation processes and explore acceptability through in-depth interviews with index patients, HHCs and HCWs (n = 100). Cost-effectiveness will be assessed by calculating the incremental cost per TB case detected within 12 months and projected for disability-adjusted life years averted based on modeled estimates of morbidity, mortality, and time with infectious TB. Discussion : This novel trial will guide India’s scale-up of post-treatment ACF and provide an evidence base for designing strategies to detect recurrent and new TB in other high burden settings. Trial registration : NCT04333485, registered April 3, 2020. CTRI/2020/05/025059 [Clinical Trials Registry of India], registered May 6 2020.Research reported in this manuscript was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health(NIH) under award number R01AI143748. See funding documentation in Additional file3. The funding body does not have a role in the collection, analysis, and interpretation of data for TB Aftermath nor were they involved in writing this manuscript

Interaction of daunomycin with deoxydinucleotide d-CpG by two-dimensional proton magnetic resonance techniques

Interaction of daunomycin with d-CpG has been studied by taking 500-MHz proton NMR spectra of a mixture of 5.5 mM daunomycin with 11.0 mM d-CpG in the temperature range 277-320 K. The 2D COSY and NOESY spectra of the complex are investigated at 297 K. The nonexchangeable base protons of d-CpG and the ring protons of drug chromophore shift upfield up to ~0.27 ppm on interaction. Changes in chemical shift decrease with temperature and are attributed to stacking of drug chromophore between CG and GC base pairs. The sugar is predominantly in the S conformational state for both cytosine and guanine residues in the right-handed helix of the d-CpG complex. The glycosyl angles are about -120 &#177; 20&#176; and -90 &#177; 20&#176; for C and G residues, respectively. In daunomycin, the spin-spin couplings-J(1'H-2'eqH), J(1'H-2'axH), J(4'H-5'CH3), J(7H-8axH), and J(7H-8eqH)-are altered on complexation with d-CpG. Further, the interproton distances-1'H-2'axH, 7H-8axH, 7H-8eqH, 5'H-8axH, and 5'H-8eqH-are altered significantly on complexation. The observed intermolecular NOEs-CH6-2H, 1H; CH6, CH5-4OCH3; CH1'-2'axH; and GH4'-9COCH3-demonstrate the existence of specific conformation of the complex which reflects the anti-cancerous action of the drug