Hydrogen Exchange Mass Spectrometry for Studying Protein-Ligand Interactions

Hydrogen deuterium exchange (HDX) coupled with mass spectrometry is widely used for probing protein structure and dynamics. Protein-ligand interactions usually induce a reduction in the measured HDX rates an effect that may be ascribed to stabilization of the protein structure. This work aims to improve the general understanding of the changes in HDX patterns associated with ligand binding. We initially applied HDX for studying differences between oxy-hemoglobin (Oxy-Hb) and aquomet-hemoglobin (Chapter 2). The results show that the α and β subunits respond differently to the oxy to aquomet transition with the heme binding pocket being destabilized in both cases. The results suggest that enhanced structural dynamics in the heme binding pocket may have adverse effects on heme-protein interactions. Chapter 3 focuses on the different scenarios that can be encountered in an HDX experiment upon ligand binding. Myoglobin and hemoglobin were used as model systems, focusing on the oxy and deoxy states of both proteins. Our results demonstrate that ligand binding can be stabilizing or destabilizing, leading to decreased or increased HDX rates respectively. In Chapters 4 HDX was used to probe the changes in structural dynamics of caseinolytic protease P (ClpP), an antibiotic drug target, after binding ADEP antibiotics. The mechanism of ADEP binding and the N-terminal structure of ClpP is not well understood with conflicting x-ray structures reported in literature. Our findings demonstrate that the N-terminus of ClpP remains quite unstructured after ADEP binding, while belt region undergoes tightening. Pin 1, a peptidyl prolyl isomerase, binding to a cyclic peptide inhibitor was studied in Chapter 5. Characterization of Pin1-CRYPEVEIC interactions by other techniques has been iv difficult. This study demonstrates that binding of the inhibitor triggers an overall stabilization of Pin 1. We identify a loop that interacts with basic sites of the ligand and that becomes destabilized upon ligand binding. This destabilization is ascribed to steric clashes between the peptide inhibitor and the protei

THE DETECTION OF HEPATITIS C VIRAL NS3-4A PROTEASE AND PROSTATE SPECIFIC ANTIGEN USING ELECTROCHEMICAL TECHNIQUES

This work attempts to detect hepatitis C viral (Hep CV) NS3-4A protease and prostate specific antigen (PSA) proteins using electrochemical methods. The sensing probes used in detecting these biological markers are their inhibitory peptides. The inhibitory peptide for the hepatitis C viral NS3-4A protease is Asp-Glu-Ile-Val-Pro-Nva with an IC50of 7.5 pM and the inhibitory peptide for prostate specific antigen is the peptide Ser-Lys-Ser- Lys-Ser-Lys-Ser-Cys-Val-Phe-Ala-His-Asn-Tyr-Asp-Tyr-Leu-Val-Cys with a K, of 0.8 pM. The steps involved in the fabrication of the sensor surface include the formation of a self assembled monolayer (SAM) using a thioctic acid modified ferrocene compound. The use of a thioctic acid labelled compound facilitates the chemisorption of the ferrocene probe to the electrode surface. The SAM is then diluted using an ethanolic solution of hexanethiol. This will help backfill the films and remove physisorbed molecules from the electrode surface. The sensing probes are then covalently attached to the surface and are used for subsequent studies with either NS3-4A protease or PSA. Our studies show that the Hep CV NS3-4A protease can be detected using the biosensor approach at very low concentrations and this sensor approach is also very specific in the presence of high concentrations of human serum albumin (HSA). The changes in sensor surface can be directly correlated to the concentration o f the protease. Studies using this approach with PSA also show similar result

The prevalence of occult hepatitis B virus (hbv) infection in a large multi-ethnic haemodialysis cohort.

Haemodialysis patients are at increased risk of exposure to blood borne viruses. To reduce transmission in the UK, all haemodialysis patients are regularly screened, and if susceptible to Hepatitis B virus (HBV) infection, vaccinated

Patient satisfaction with care provided by a district dental clinic

Magister Scientiae Dentium - MSc(Dent)Patient satisfaction is critical for the growth of oral health service and practice. The present study was a descriptive study on patient satisfaction with oral health care provided by a district dental clinic. The aim of the study was to determine whether patients attending the dental clinic of the Lagos State University hospital were satisfied with the care they received.South Afric

Prevalence and Socio-behavioral Influence of Early Childhood Caries, ECC, and Feeding Habits among 6-36 Months Old Children in Uganda and Tanzania.

Early childhood caries (ECC) is a serious problem that has remained unexplored in sub-Saharan Africa. This study aimed to identify possible socio-behavioral correlates of ECC focusing 6-36 months old children and their caretakers.\ud Cross sectional studies were conducted in a high fluoride rural area, Manyara, Tanzania and a low fluoride urban area, Kampala, Uganda. Totals of 1221 and 816 child - caretaker pairs attending health care facilities for growth monitoring were recruited in Manyara and Kampala, respectively. All caretakers completed face to face interviews at the health care facility. Children underwent oral clinical examination whereby ECC and Enamel hypoplasia were recorded using the dmft (WHO 1997) and the DDE index (FDI 1992). The prevalence of ECC was 3.7% in Manyara and 17.6% in Kampala. According to multiple logistic regression analyses, received oral health information from health worker was the strongest determinant of ECC in Manyara, adjusted OR 0.3, 95% CI 0.09 - 0.93. In Kampala, visible plaque, high sugar intake and presence of enamel hypoplasia associated with ECC, adjusted ORs 2.8 (95% CI 1.61- 4.95), 3.0 (95% CI 1.39 - 6.34) and 2.3 (95% CI 1.36 - 3.95). Oral health education aimed at caretakers of 6-36 months, including health care workers' information regarding the detrimental consequences for oral health of frequent sugar consumption and poor oral hygiene is important for prevention of ECC in Tanzania and Uganda

Pin1 and neurodegeneration: a new player for prion disorders?

Pin1 is a peptidyl-prolyl isomerase that catalyzes the cis/trans conversion of phosphorylated proteins at serine or threonine residues which precede a proline. The peptidyl-prolyl isomerization induces a conformational change of the proteins involved in cell signaling process. Pin1 dysregulation has been associated with some neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. Proline-directed phosphorylation is a common regulator of these pathologies and a recent work showed that it is also involved in prion disorders. In fact, prion protein phosphorylation at the Ser-43-Pro motif induces prion protein conversion into a disease-associated form. Furthermore, phosphorylation at Ser-43-Pro has been observed to increase in the cerebral spinal fluid of sporadic Creutzfeldt-Jakob Disease patients. These findings provide new insights into the pathogenesis of prion disorders, suggesting Pin1 as a potential new player in the disease. In this paper, we review the mechanisms underlying Pin1 involvement in the aforementioned neurodegenerative pathologies focusing on the potential role of Pin1 in prion disorders

Single-dose administration and the influence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of four randomised trials.

BACKGROUND: The ChAdOx1 nCoV-19 (AZD1222) vaccine has been approved for emergency use by the UK regulatory authority, Medicines and Healthcare products Regulatory Agency, with a regimen of two standard doses given with an interval of 4-12 weeks. The planned roll-out in the UK will involve vaccinating people in high-risk categories with their first dose immediately, and delivering the second dose 12 weeks later. Here, we provide both a further prespecified pooled analysis of trials of ChAdOx1 nCoV-19 and exploratory analyses of the impact on immunogenicity and efficacy of extending the interval between priming and booster doses. In addition, we show the immunogenicity and protection afforded by the first dose, before a booster dose has been offered. METHODS: We present data from three single-blind randomised controlled trials-one phase 1/2 study in the UK (COV001), one phase 2/3 study in the UK (COV002), and a phase 3 study in Brazil (COV003)-and one double-blind phase 1/2 study in South Africa (COV005). As previously described, individuals 18 years and older were randomly assigned 1:1 to receive two standard doses of ChAdOx1 nCoV-19 (5 × 1010 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 1010 viral particles) of the ChAdOx1 nCoV-19 for the first dose. The primary outcome was virologically confirmed symptomatic COVID-19 disease, defined as a nucleic acid amplification test (NAAT)-positive swab combined with at least one qualifying symptom (fever ≥37·8°C, cough, shortness of breath, or anosmia or ageusia) more than 14 days after the second dose. Secondary efficacy analyses included cases occuring at least 22 days after the first dose. Antibody responses measured by immunoassay and by pseudovirus neutralisation were exploratory outcomes. All cases of COVID-19 with a NAAT-positive swab were adjudicated for inclusion in the analysis by a masked independent endpoint review committee. The primary analysis included all participants who were SARS-CoV-2 N protein seronegative at baseline, had had at least 14 days of follow-up after the second dose, and had no evidence of previous SARS-CoV-2 infection from NAAT swabs. Safety was assessed in all participants who received at least one dose. The four trials are registered at ISRCTN89951424 (COV003) and ClinicalTrials.gov, NCT04324606 (COV001), NCT04400838 (COV002), and NCT04444674 (COV005). FINDINGS: Between April 23 and Dec 6, 2020, 24 422 participants were recruited and vaccinated across the four studies, of whom 17 178 were included in the primary analysis (8597 receiving ChAdOx1 nCoV-19 and 8581 receiving control vaccine). The data cutoff for these analyses was Dec 7, 2020. 332 NAAT-positive infections met the primary endpoint of symptomatic infection more than 14 days after the second dose. Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4-74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group. There were no hospital admissions for COVID-19 in the ChAdOx1 nCoV-19 group after the initial 21-day exclusion period, and 15 in the control group. 108 (0·9%) of 12 282 participants in the ChAdOx1 nCoV-19 group and 127 (1·1%) of 11 962 participants in the control group had serious adverse events. There were seven deaths considered unrelated to vaccination (two in the ChAdOx1 nCov-19 group and five in the control group), including one COVID-19-related death in one participant in the control group. Exploratory analyses showed that vaccine efficacy after a single standard dose of vaccine from day 22 to day 90 after vaccination was 76·0% (59·3-85·9). Our modelling analysis indicated that protection did not wane during this initial 3-month period. Similarly, antibody levels were maintained during this period with minimal waning by day 90 (geometric mean ratio [GMR] 0·66 [95% CI 0·59-0·74]). In the participants who received two standard doses, after the second dose, efficacy was higher in those with a longer prime-boost interval (vaccine efficacy 81·3% [95% CI 60·3-91·2] at ≥12 weeks) than in those with a short interval (vaccine efficacy 55·1% [33·0-69·9] at <6 weeks). These observations are supported by immunogenicity data that showed binding antibody responses more than two-fold higher after an interval of 12 or more weeks compared with an interval of less than 6 weeks in those who were aged 18-55 years (GMR 2·32 [2·01-2·68]). INTERPRETATION: The results of this primary analysis of two doses of ChAdOx1 nCoV-19 were consistent with those seen in the interim analysis of the trials and confirm that the vaccine is efficacious, with results varying by dose interval in exploratory analyses. A 3-month dose interval might have advantages over a programme with a short dose interval for roll-out of a pandemic vaccine to protect the largest number of individuals in the population as early as possible when supplies are scarce, while also improving protection after receiving a second dose. FUNDING: UK Research and Innovation, National Institutes of Health Research (NIHR), The Coalition for Epidemic Preparedness Innovations, the Bill & Melinda Gates Foundation, the Lemann Foundation, Rede D'Or, the Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Radiological Assessment of Primordial Radionuclides in Crab Species from Igbokoda River in Southwest of Nigeria

Communication in Physical Sciences, 2021, 7(1): 1-7 Authors: Olusegun&nbsp; Sowole* and&nbsp; Adesoji A. R. Adebambo Received 30 January 2021/Accepted 11 February 2021 Primordial radionuclides are natural radionuclides of specific half-lives that are capable of disintegrating with the release of ionizing radiations along with huge amount of energy which can be so harmful to living organisms. Activity concentrations and radiological indices of 40K, 226Ra and 228Ra in crab species from Igbokoda River in the coastal area of South Western Nigeria, have been determined using gamma spectrometry method. Twenty (20) samples of three different species (Callinectes latimanus, Callinectes amnicola and Cadiosoma armatum) of crabs were collected from the River. The results obtained indicated that the highest dose rates of 40K, 226Ra and 228Ra were associated with Callinectes latimanus while the highest annual committed effective dose of 40K to man was 0.0026mSvyr-1 and was associated with Callinectes latimanus for 226Ra (0.0068mSvyr-1) and 228Ra (0.0208mSvyr-1). The highest excess lifetime cancer risk associated with 40K, 226Ra and 228Ra 0.0237 x 10-3 0.0728 x 10-3. All the values obtained were within the limits recommended globally; indicating that there was no significant radiological health implication to the aquatic animals and man the consume