Design and Analytical Evaluation of a Rapid Plasma Screening Assay for Circulating Human Papillomavirus DNA via Thermostable Enzyme Chemistries.

Infection with oncogenic strains of human papillomavirus (HPV), such as HPV-16 and HPV-18, can lead to malignant progression and tumorigenesis. As an adjunct to traditional invasive tissue sampling methods, the use of modern thermostable enzyme chemistries can aid in the development of innovative assay workflows to extract and detect circulating HPV DNA (cHPV-DNA) in liquid biopsies. In this work, we first successfully generated a model system to replicate fragmented cHPV-DNA in human plasma. Using this model system, we designed a novel thermostable enzyme chemistry-based cHPV-DNA assay for rapid clinical HPV screening and robustly evaluated its analytical assay performance. Our findings demonstrated that the use of thermostable enzymes provided faster cHPV-DNA extraction and amplification, leading to an overall three-fold improvement in overall assay time as compared to the current standard assay workflow and achieving clinically relevant levels of analytical specificity, sensitivity, and precision for accurate cHPV-DNA detection with excellent 100% sensitivity and specificity in contrived human plasma specimens. In summary, we have devised a rapid laboratory workflow to facilitate the emerging use of liquid biopsies for minimally invasive, rapid, and scalable HPV DNA testing. With facile assay modifications, our thermostable enzyme-based cHPV-DNA assay can be utilized for the detection of other clinically high-risk HPV genotypes.

Residues at the interface between zinc binding and winged helix domains of human RECQ1 play a significant role in DNA strand annealing activity.

RECQ1 is the shortest among the five human RecQ helicases comprising of two RecA like domains, a zinc-binding domain and a RecQ C-terminal domain containing the winged-helix (WH). Mutations or deletions on the tip of a ß-hairpin located in the WH domain are known to abolish the unwinding activity. Interestingly, the same mutations on the ß-hairpin of annealing incompetent RECQ1 mutant (RECQ1T1) have been reported to restore its annealing activity. In an attempt to unravel the strand annealing mechanism, we have crystallized a fragment of RECQ1 encompassing D2-Zn-WH domains harbouring mutations on the ß-hairpin. From our crystal structure data and interface analysis, we have demonstrated that an α-helix located in zinc-binding domain potentially interacts with residues of WH domain, which plays a significant role in strand annealing activity. We have shown that deletion of the α-helix or mutation of specific residues on it restores strand annealing activity of annealing deficient constructs of RECQ1. Our results also demonstrate that mutations on the α-helix induce conformational changes and affects DNA stimulated ATP hydrolysis and unwinding activity of RECQ1. Our study, for the first time, provides insight into the conformational requirements of the WH domain for efficient strand annealing by human RECQ1.

Human RecQ helicases in transcription-associated stress management: bridging the gap between DNA and RNA metabolism.

RecQ helicases are a highly conserved class of DNA helicases that play crucial role in almost all DNA metabolic processes including replication, repair and recombination. They are able to unwind a wide variety of complex intermediate DNA structures that may result from cellular DNA transactions and hence assist in maintaining genome integrity. Interestingly, a huge number of recent reports suggest that many of the RecQ family helicases are directly or indirectly involved in regulating transcription and gene expression. On one hand, they can remove complex structures like R-loops, G-quadruplexes or RNA:DNA hybrids formed at the intersection of transcription and replication. On the other hand, emerging evidence suggests that they can also regulate transcription by directly interacting with RNA polymerase or recruiting other protein factors that may regulate transcription. This review summarizes the up to date knowledge on the involvement of three human RecQ family proteins BLM, WRN and RECQL5 in transcription regulation and management of transcription associated stress.

Functionalisation of Polyvinylpyrrolidone on Gold Nanoparticles Enhances Its Anti-Amyloidogenic Propensity towards Hen Egg White Lysozyme.

Protein amyloids are characterized by aggregates that usually consist of fibres containing misfolded proteins and having a cross ß-sheet conformation. These aggregates can eventually lead to several degenerative diseases like Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease and Parkinson's disease. The present study describes the effect of chemically synthesized polyvinylpyrrolidone (PVP)-conjugated gold nanoparticles (PVP-AuNps) on hen egg white lysozyme (HEWL) amyloids. The synthesized nanoparticles have been characterized using various biophysical techniques like Ultraviolet-Visible (UV-Vis) Spectroscopy, Transmission electron microscopy (TEM), X-ray diffraction (XRD) analysis, dynamic light scattering (DLS), zeta-potential measurement and Fourier transform infrared spectroscopy (FTIR). The aggregation studies showed that PVP acts as a partial inhibitor of HEWL amyloidogenesis. However, when conjugated to gold nanoparticle surface, it leads to complete inhibition of amyloid formation. Apart from inhibition, PVP-conjugated gold nanoparticles also exhibited a significant disaggregation effect on mature amyloids and hence can be exploited as an effective therapeutic agent against hereditary systemic amyloidosis.

Isolation and partial structural evaluation of a cardiotoxic factor from Indian common murrel (Channa striatus L.) skin extract.

It was earlier reported from this laboratory that, Channa striatus, L a common edible fish, whose skin extract (CSSE) was pharmacologicaclly potent and contains several bioactive compounds. In the present communication a cardiotoxic factor was isolated and purified by thin layer chromatography followed by silica gel and neutral alumina column chromatography. Spectroscopic studies (UV, IR, 1H and 13C NMR, FAB-MS) indicated that the lethal cardiotoxic factor (CSS-CTF II) was an aromatic alkaloid compound with -NH, > C = C < and -OH functional groups. The molecular weight was found to be 413 dalton. LD50 of CSS-CTF II was found to be 42.5 mg/kg (i.v) in Swiss albino male mice. Pharmacological studies showed that CSS-CTF II possesses hypotensive and cardiotoxic activities and produced death through apnoea in experimental animals but had no effect on nerve muscle preparations. The haematological and biochemical data also indicated the toxic nature of CSS-CTF II, through significant fall in haemoglobin, total RBC, WBC, platelet count and increased cardiac marker enzyme CPK and CPK-MB value in experimental animals. The present investigation thus established the toxic nature of CSS-CTF II isolated from edible fish C. striatus skin extract. Further work is needed to identify CSS-CTF II's mechanism of action and its antagonism for therapeutic purpose.

Help-seeking patterns for children with epilepsy in rural India: implications for service delivery.

PURPOSE: Most people in the world with epilepsy are untreated with antiepileptic drugs (AEDs). In some developing countries, this is because treatment facilities are unavailable or difficult to access. It has even been suggested that indigenous health systems threaten the prospect of the global control of epilepsy with AEDs. We have investigated patterns and costs of help seeking for children with epilepsy in a region of rural India where only 12% of children with epilepsy were in treatment. Our objective was to find out (a) whom families had consulted; (b) if nonconsulting families differed in demographic or child medical factors; (c) if indigenous treatment was taken, exclusive of allopathic treatment; and (d) the direct and indirect cost of various providers. METHODS: We conducted a cross-sectional interview study in a community-based program for childhood epilepsy in rural West Bengal, India. We interviewed parents of 85 children aged 2 to 18 years with untreated epilepsy who had entered a clinical trial of AEDs during 1995 through 1996. RESULTS: Eighty percent of families had sought some help in the past: 62% with an allopathic practitioner, 44% with traditional practitioners. Primary health centres (PHCs) and quacks were not popular. Twenty-four percent of families never sought help of any kind, and this was unassociated with sex, income, maternal literacy, or medical variables. There was evidence of both exclusivity and pluralism: 42% of families first consulting allopathic practitioners also visited traditional practitioners, whereas 30% of families first consulting traditional practitioners also went to allopathic practitioners. One visit to a physician cost a median of 9-13% of monthly income and 5-12 person-hours; the cost of visiting indigenous providers was negligible. CONCLUSIONS: Most families sought some form of help and were motivated to spend large amounts of money and time for allopathic treatments from qualified practitioners. The typical cost of allopathic treatment was unsustainable in the long term. Medical pluralism is common and does not adversely influence use of allopathic treatment. The phenomenon of nonconsulting merits further study. Traditional practitioners play a complementary role and might become involved in community treatment programs. Low-cost, local treatment is essential to the public health control of epilepsy.

Social integration of children with epilepsy in rural India.

Only a small fraction of people with epilepsy in developing countries has access to medical facilities. Even with effective treatment, their psychosocial needs are often overlooked in the absence of obvious disability. In rural areas, community-based rehabilitation programmes assist in the integration of people with disabilities into employment and the community. However, the functional impairment associated with epilepsy is not well recognised in intervention programmes in developing countries. We report, for the first time, the social activities of children with epilepsy and their peers in rural India. We employed a cross-sectional design using a new age and sex-specific social activity questionnaire. Population screening in the context of a community-based rehabilitation programme identified 88 children with epilepsy and 250 randomly selected controls. A trained interviewer administered the questionnaire to mothers in Bengali. Girls' activities were principally domestic, whilst boys' were mostly outdoors and involved peers. All groups of children with epilepsy had significant social deficits, equally for boys and girls in the age range from 2 to 18 years (p < 0.05). Boys with epilepsy had limited peer group activities, and parents conferred fewer responsibilities to school age and adolescent children compared to controls. The nature and degree of deficits were beyond the constraints imposed by neurological impairment. Our findings in pre-schoolers were consistent with parental attitudes of overprotection found in previous research. We conclude that social integration needs active and early promotion among children with epilepsy. The assessment of remediable risk and protective factors in the family and community is an important practical area for research in community-based rehabilitation.