...skilled research manager in the domain of heritable attributes of human health and precision medicine.
Skills and Expertise
Jan 2006 - Jun 2007
Henley Business School
Field of study
Jan 1994 - Dec 1997
Novartis Research Institute and Brunel University
Field of study
- Bioengineering, immunology, virology
Research Items (55)
Africa is experiencing a rapid increase in adult obesity and associated cardiometabolic diseases (CMDs). The H3Africa AWI-Gen Collaborative Centre was established to examine genomic and environmental factors that influence body composition, body fat distribution and CMD risk, with the aim to provide insights towards effective treatment and intervention strategies. It provides a research platform of over 10 500 participants, 40–60 years old, from Burkina Faso, Ghana, Kenya and South Africa. Following a process that involved community engagement, training of project staff and participant informed consent, participants were administered detailed questionnaires, anthropometric measurements were taken and biospecimens collected. This generated a wealth of demographic, health history, environmental, behavioural and biomarker data. The H3Africa SNP array will be used for genome-wide association studies. AWI-Gen is building capacity to perform large epidemiological, genomic and epigenomic studies across seve
Introduction: The use of antiretroviral therapy has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless, gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) gene editing system. Areas covered: CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease-deficient Cas9 fused to transcription activation domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus. Expert opinion: The diversity of the CRISPR/Cas9 technologies offers great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV.
One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein, such as human serum transferrin. To examine whether this is feasible, a peptide sequence cleavable by the human immunodeficiency virus type 1 protease (VSQNYPIVL) was inserted into various regions of human serum transferrin, and the resultant proteins were tested for function. Experimentally, molecular modeling was used to identify five candidate insertion sites in surface exposed loops of human serum transferrin that were distant from biologically active domains. These insertions were cloned using polymerase chain reaction mutagenesis, and the proteins were expressed using a baculovirus expression vector system. Analysis of the mutant proteins provided a number of important findings: (a) they retained native human serum transferrin function, (b) the inserted peptide sequence was surface exposed, and most importantly, (c) two of these mutants could be cleaved by human immunodeficiency virus-1 protease. In conclusion, this investigation has validated the use of human serum transferrin as a carrier protein for functional peptide domains introduced into its structure using protein engineering. These findings will be useful for developing a novel class of therapeutic agents for a broad spectrum of diseases.
Semiquantitative polymerase chain reaction (PCR) is a powerful tool for determining DNA and RNA copy num-bers in biological samples (4,5,8). Essentially, the technique involves ampli-fication of the target with specific primers and comparison of the signal intensity with that of a standard that was amplified within the same tube af-ter separation of the products by gel electrophoresis. Signal intensity is pro-portional to target copy number in semiquantitative PCR. The standard can either be an endogenous sequence (e.g. housekeeping genes such as glyc-eraldehyde-3-phosphate dehydrogen-ase [GAPDH]; see References 16 and 17) or be added prior to amplification at a specific copy number (14). An optimal competitor sequence should be amplified at a similar effi-ciency as the target gene. Therefore, it should use the same primers as the tar-get DNA and have similar size, nu-cleotide composition and secondary structure. Hence, optimal competitior 'mimics' tend to be constructed from the cloned target gene. Several strate-gies exist to do this. Restriction en-zymes can be used to create a deletion within the target gene or permit inser-tion of a stuffer fragment (9). However, in the absence of suitable restriction sites, this approach is not feasible. A re-cent publication describes the applica-tion of inverse polymerase chain reac-tion (IPCR) to generate PCR mimics without the requirement of restriction enyzmes (18). This method is rapid and simple, but does have one major draw-back. If the plasmid containing the tar-get gene is large, then amplification of the entire plasmid can be difficult un-less the target gene is first subcloned into a smaller vector. We present an application of PCR-ligation-PCR (PLP) mutagenesis (2,3) for the production of an ideal mimic se-quence for any semiquantitative PCR application. Like the IPCR method, this method is rapid and does not require the use of restriction sites at the point of the deletion. However, unlike IPCR, there is no requirement for
[This corrects the article DOI: 10.1371/journal.pone.0206326.].
Question - Is there any harm done by mixing plasmids the night BEFORE transfecting, without buffer or reagent?
Some tubes are sticky and adsorb DNA - which can be an issue if you have low concentrations of DNA. Try using Eppendoref Low Bind tubes and freeze the samples at -20C. Defrost on ice and vortex well to ensure resuspension.
Question - NEB 10-beta competent E.coli cells. Good trasformation but why the colonies don't grow?
To follow through with Hanna, make sure that the Amp is fresh, and that the agar is not too hot (less than 60C) when you mix. if The recombinant plasmid is toxic you will probably notice that clones are smaller - to do this make sure you have a pUC control. For suspension cultures you can drop the temp room right down to 27C and extend shaking from overnight to overnight plus a day.
Question - How do you sequence the cap gene of an AAV?
Hi Laurence. As you have probably noted, there is so much secondary structure in the ITR that it is very difficult to sequence. In my lab, if there is a question about sequence, we have instead resorted to detailed restriction analysis, and/or sub-cloned the region between the ITRs back into a parental vector for which we can be sure has the correct sequence.
Background: African populations are characterised by diversity at many levels including: demographic history, genetic ancestry, language, wealth, socio-political landscape, culture and behaviour. Several of these have a profound impact on body fat mass. Obesity, a key risk factor for cardiovascular and metabolic diseases, in the wake of the epidemiological and health transitions across the continent, requires detailed analysis together with other major risk factors. Objective: To compare regional and sex-specific body mass index (BMI) distributions, using a cross-sectional study design, in adults aged 40-60 years across six study sites in four sub-Saharan African (SSA) countries and to compare the determinants of BMI at each. Methods: Anthropometric measurements were standardised across sites and BMI calculated. Median BMI and prevalence of underweight, lean, overweight and obesity were compared between the sexes and across sites. Data from multivariable linear regression models for the principal determinants of BMI were summarised from the site-specific studies. Results: BMI was calculated in 10,702 participants (55% female) and was significantly higher in women than men at nearly all sites. The highest prevalence of obesity was observed at the three South African sites (42.3-66.6% in women and 2.81-17.5% in men) and the lowest in West Africa (1.25-4.22% in women and 1.19-2.20% in men). Across sites, higher socio-economic status and educational level were associated with higher BMI. Being married and increased dietary intake were associated with higher BMI in some communities, whilst smoking and alcohol intake were associated with lower BMI, as was HIV infection in the regions where it was prevalent. Conclusion: In SSA there is a marked variation in the prevalence of obesity both regionally and between men and women. Our data suggest that the drive for social upliftment within Africa will be associated with rising levels of obesity, which will require the initiation of targeted sex-specific intervention programmes across specific African communities.
Project - PCR Mutagenesis
I have attached a copy of the book chapter discussing in detail the PCR-Ligation-PCR mutagenesis, which was one of the original PCR mutagenesis techniques.
Dyslipidaemia is a primary risk factor for cardiometabolic disease, causing over 17 million deaths globally in 2015. However, the burden of dyslipidaemia and factors associated with lipid levels remain unknown in many rural African populations. Therefore, this study evaluated the association of socio-demographic, anthropometric and behavioural factors with lipid levels in rural Ghana. The prevalence of hypercholesterolaemia, hypertriglyceridaemia and elevated LDL-C in the total population of 1839 (846 men and 993 women) was 4.02%, 2.12%, and 5.55% respectively and did not differ between genders. The prevalence of low HDL-C levels was 60.30% and differed (p = 0.005) between men (56.86%) and women (63.24%). Subcutaneous abdominal fat was associated with TC (β = 0.067, p = 0.015) and TG (β = 0.137, p
Question - Could this Cas9 anti-HIV strategy possibly work?
I would like to suggest review of the following paper that presents a review of, and methods to, address treatment of HIV using existing and novel approaches usind CRISP/Cas.
Question - Incorrect band size: Q5 Site-Directed Mutagenesis?
Question - Could you suggest a Mutagenesis kit?
Question - How to do in vitro mutagenesis of a particular gene?
Question - Which is the best commercial kit for site directed mutagenesis?
Question - What is the cheapest option for amutagenesis kit?
Coming from old school, looking to basic science instead of kits, I would like to revommend the technique or PCR-Ligation-PCR mutagenesis that can be used to generate a whole range of mutations. This is reference in ResearchGate here:
Question - How to fuse two independent domain?
Question - Ponceau S Stain prior to probing the membrane with antibody?
Once you have the Ponceau S stained membrane, remember to image it (so that you have a reference for where the protein bands are), then make sure the membrane is washed fully to remove the dye before adding primary antibody,
Question - Title for my proposal, i would like to know which one to use?
Try to frame the title of your proposal as a question. In this way you will provide a direct link to the hypothesis you will be addressing in your work.
Question - GFP tagged protein screening?
Depending on the components of the ligation reaction you may find a reduction in transformation frequency, so you might want to try in parallel transforming a 1:10 dilution of the ligation mix.
Question - Is it possible to make more cDNA out of cDNA made directly from RT of RNA?
You could create more copies using PCR, but use as few cycles of amplification as possible, as you risk introducing mutations. Rather repeat the cDNA reaction for a longer period or use a reverse transcriptase with higher processivity.
There is an alarming tide of cardiovascular and metabolic disease (CMD) sweeping across Africa. This may be a result of an increasingly urbanized lifestyle characterized by the growing consumption of processed and calorie-dense food, combined with physical inactivity and more sedentary behaviour. While the link between lifestyle and public health has been extensively studied in Caucasian and African American populations, few studies have been conducted in Africa. This paper describes the detailed methods for Phase 1 of the AWI-Gen study that were used to capture phenotype data and assess the associated risk factors and end points for CMD in persons over the age of 40 years in sub-Saharan Africa (SSA). We developed a population-based cross-sectional study of disease burden and phenotype in Africans, across six centres in SSA. These centres are in West Africa (Nanoro, Burkina Faso, and Navrongo, Ghana), in East Africa (Nairobi, Kenya) and in South Africa (Agincourt, Dikgale and Soweto). A total of 10,702 individuals between the ages of 40 and 60 years were recruited into the study across the six centres, plus an additional 1021 participants over the age of 60 years from the Agincourt centre. We collected socio-demographic, anthropometric, medical history, diet, physical activity, fat distribution and alcohol/tobacco consumption data from participants. Blood samples were collected for disease-related biomarker assays, and genomic DNA extraction for genome-wide association studies. Urine samples were collected to assess kidney function. The study provides base-line data for the development of a series of cohorts with a second wave of data collection in Phase 2 of the study. These data will provide valuable insights into the genetic and environmental influences on CMD on the African continent.
Background There is a high prevalence of hypertension and related cardiovascular diseases in sub-Saharan Africa, yet few large studies exploring hypertension in Africa are available. The actual burden of disease is poorly understood and awareness and treatment to control it is often suboptimal. Objectives The study sought to report the prevalence of measured hypertension and to assess awareness and control of blood pressure among older adults in rural and urban settings in 6 sites located in West, East, and Southern Africa. In addition, we examined regional, sex, and age differences related to hypertension. Methods A population-based cross-sectional study was performed at 6 sites in 4 African countries: Burkina Faso (Nanoro), Ghana (Navrongo), Kenya (Nairobi), and South Africa (Agincourt, Dikgale, Soweto). Blood pressure measurements were taken using standardized procedures on 10,696 adults 40 to 60 years of age. Hypertension was defined as systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg or taking antihypertensive medication. Results The mean prevalence of hypertension ranged from 15.1% in Nanoro to 54.1% in Soweto. All 3 of the South African sites had a mean prevalence of hypertension of over 40.0%, significantly higher than in Nairobi (25.6%) and Navrongo (24.5%). Prevalence increased with age in both sexes and at all sites. A significantly higher prevalence of hypertension was observed in women in Agincourt, Dikgale, and Nairobi, whereas in Nanoro this trend was reversed. Within the hypertensive group the average proportion of participants who were aware of their blood pressure status was only 39.4% for men and 53.8% for women, and varied widely across sites.
- Oct 2015
- Postdoctoral Forum, Faculty of Health Sciences, University of the Witwatersrand
Background: The only HIV vaccine clinical trial to show efficacy was RV144 conducted in Thailand in 2009. The study reported an efficacy of 31.2%, highly correlated with the presence of antibodies targeting the variable 2 (V2) region of the HIV envelope. Given the importance of the V2 region as a broadly neutralizing antibody (bnAb) target, there is a need to better understand both the neutralizing and effectors functions, including antibody dependent cell-mediated cytotoxicity (ADCC) of such antibodies. To this end, monoclonal antibodies (mAbs) isolated from B cells of two subjects in the CAPRISA cohort (CAP228 and CAP256) were selected based on V2-directed function and binding. Heavy and light chains of each mAb were combined into single-plasmid expression cassettes. Subsequent packaging into adeno-associated virus (AAV) vectors will allow delivery into humanized transgenic murine models for HIV. Through these studies, we aim to probe the neutralizing and ADCC potential of mAbs through gene therapy using vectored immunoprophylaxis (VIP). Methods: The variable regions of the heavy and light chains of CAP228 (19F) and CAP256 (VRC26.08, VRC26.09, VRC26.16, VRC26.21) were cloned from individual plasmids, using Gibson Assembly, into an immunoglobulin G (IgG) plasmid backbone to create single-plasmid expression vectors. All cloned regions were verified by Sanger sequencing. The mAbs were expressed by transient transfection of HEK 293T cells and quantified using an IgG-specific ELISA. The CAP228 mAb was characterized using a V2-specific ELISA and the neutralizing antibody titers of CAP256 mAbs were determined using a TZM-bl reporter-gene assay. Results: CAP228 (19F) and CAP256 (VRC26.08, VRC26.09, VRC26.16, VRC26.21) mAbs were successful cloned into a single-plasmid expression vectors. Transfections of HEK 293T cells yielded 1-2 µg mAb per 3 ml transfection supernatant. CAP228-19F functioned comparably to a purified CAP228-19F control when tested in V2 ELISA for differential binding to sorting antigens CAP45 and CAP45 K169E. In preliminary neutralizing antibody assays, the CAP256 antibodies yielded inhibitory concentrations similar to expected values when tested against viruses CAP256 SU, CAP256 SU K169E, DU422, and Q23.17. Conclusions: The heavy and light chains of CAP228 and CAP256 mAbs were successfully cloned into single plasmid expression vectors, which produced functional mAbs that retained V2 binding specificity of the HIV envelope, and differentially neutralized various HIV-1 viruses. The novel application of the Gibson Assembly using a generic IgG backbone, facilitates rapid cloning of mAb transgenes, and paves the way for delivery using AAV. Testing of these AAV-vectored mAbs in humanized mouse models for HIV will further our understanding of V2-based immunogenic responses and aid the development of vectored immunoprophylaxis to create a preventative vaccine.
Question - Reusable chambers for the automated cell viability assay?
The Countess has a reusable slide option.
Question - How much DNA template (genomic or plasmid DNA) is used for a general PCR?
...Remembering that if the PCR product is going to be used directly for cloning then you want to avoid too much contaminating plasmid vector. Of course there is always the option of removing excess vector with a DpnI digest.
Question - How much DNA template (genomic or plasmid DNA) is used for a general PCR?
I usually start with around 10 ng of a typical plasmid template (assuming 3-5kb), or an equivalent molar quantity of template if not.
One needs to strike a balance between the amount of template, and the degree of amplification that is required to generate an adequate amount of product. Too little template will require more cycles of amplification, and therefore increase the possibility of introducing errors. On the other hand, too much template could result a "dirty" PCR with low yields, and a lot of non-specific amplification. Of course, always make sure that the clone is sequenced.
Question - How do I purify intracellular protein from transfected cells without it losing it's function?
Few things to consider:
1. Assuming that you have fused the protein to a secretory signal, you might want to check that this sequence is still attached, and has not been cleaved off; remembering that some proteins get processed at their N-terminal during maturation.
2. If the protein contains any native intracellular targeting domains, then these might limit the extent to which it is secreted.
3. Perhaps consider whether the protein might be susceptible to proteolysis in the supernatant - especially after 7 days. In which case, sample earlier, and add a protease inhibitor to the culture medium.
Good luck! Stuart
Question - Can anyone help with PCR "Splicing by Overlap Extension" trouble?
You could also try PCR-ligation-PCR as an alternative to SOE PCR. We developed it back in the 90's as a universal method for joining PCR fragments and generating mutations. See BioTechniques 18(5)746-50, or my ResearchGate profile.
Question - ligation of PCR Products?
Some good answers above. You might want to try increasing the concentration of inserts in order to "force" the three-way ligation. Alternatively, you could also try a modification of PCR-ligation-PCR cloning that we invented back in 1995 (see Biotechniques 18(5) 746-50). Here, you would ligate your XbaI digested fragments, then PCR amplify using the upstream primer of the first fragment, and downstream primer of the second. BamHI/KpnI digestion would then generate the single insert you require.
Good luck! Stuart
Question - Has anybody used PCR product from agarose gel band for cloning DNA?
If you have a well separated, strong, PCR band, you could also try in-gel ligation. It may save the worry about purification. Just need to make sure that you are good at estimating the relative molar concentrations of the vector/insert, and keep time on the UV box to minimum.
Question - What is the difference in method and protocol of extracting RNA from whole blood vs. serum?
The guanidinium-acid-phenol methods work well - but I am a bit old-school (!) You just need to keep a check on the release of RNAses when working with whole blood. Keep samples on ice, spin lysates in a cooled centrifuge, work quickly, and use an RNAse inhibitor. Good comments made in the earlier answers.
Question - Can you give me some information about synthetic biology?
Googling is a great start - but the number of hits grow with a doubling time faster than E. coli (!) Useful starting points are igem.org (see above)) and the BioBricks foundation (biobricks.org). You might also want to check out the group at MIT (synbio.mit.edu), who are doing some bleeding edge work on metabolic engineering.
Of course, many people look to the "guru" J. Craig Venter. His research centre (jcvi.org) produced the first synthetic self-replicating bacterial cell, which sparked a huge number of public debates (youtube search...!) concerning ethics and future capabilities.
Good luck, and have fun!
Question - Is climate change location specific?
Have enjoyed following the discussion.
It may be interesting to consider the additional dimension of any (un)measured "potential" for change.
Take, for example, global warming. Some propose that our predictions are way off...the increases in atmospheric temperature seem to be half that predicted. Others argue to the contrary, claiming that the oceans have provided a heat sink to store that extra energy.
So, let's consider the dimension of "potential" for change. If the ocean is a buffer against changes in atmospheric temperature, it can also be assumed that, as a buffer, its potential to mediate change over time is a function of both its present capacity (to buffer), and the changing environment in which it acts. And these changes may, or may not be, local.
Be it weather, sociology or innovation, the notion of a changing buffering capacity in any system brings a whole different level of complexity and intellectual challenge.
One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein such as human serum transferrin (HST). However, a potential drawback is that the HST may increase the immunoreactivity of the peptide, in the same way that carrier proteins can be used to generate highly immunogenic peptide hapten conjugates. In this study we have generated a recombinant HST carrier protein that contains a peptide substrate of HIV-1 protease (VSQNYPIVL). The protein retained native HST function, and the peptide was surface exposed since it was immunoreactive in native dot blots, and was cleaved by HIV-1 protease. Immunisation of rabbits with the recombinant protein elicited only a very poor anti-peptide immune response. In contrast, strong anti-peptide immune responses were raised against both the peptide alone, and a chemical conjugate of the peptide with HST. These data demonstrate that it is possible to attenuate the immune response normally directed against an immunogenic peptide sequence by engineering into a surface exposed loop of HST. These findings may have an important impact on the future design of peptide delivery systems.
We have developed a baculovirus expression system for the rapid and efficient production of large quantities (>5 mg/10(8) cells) of ICP8. The recombinant ICPS is fully functional and binds to single-stranded DNA. Secondary structure calculations from circular dichroism measurements indicate a content of 34.5% or-helix and 15.4% beta-sheet. This is the first structural report for ICP8 using CD analysis, which will be very useful for high-throughput assay development and mechanistic studies, (C) 1999 Academic Press.
We have developed a baculovirus expression system for the rapid and efficient production of large quantities (>10 mg/l) of VP16. The recombinant VP16 binds to a complex of host cell transcription factors and TAAT-GARAT motif. Secondary structure calculations from circular dichroism measurements indicate a content of 32.0 % alpha-helix and 17.5 % beta-sheet. This is the first structural CD analysis of VP16 which will be very useful for high-throughput assay development and mechanistic studies. (C) 1998 Academic Press.
Here we describe a novel PCR method for site-specific mutagenesis. Unlike splice overlap extension (SOE) PCR, this technique does not require primers with overlapping complementary sequences, but still maintains sequence specificity at the recombination junction. Like the PCR process itself, the principle is only inherently obvious after it has been explained. Remarkably, this simple technique can be applied universally to generate gene fusions, deletions, insertions and point mutations. The method is practical, cost effective and reproducible, and it therefore offers many advantages over existing techniques and commercially available mutagenesis kits.
Recently, there has been much interest in expressing recombinant human serum transferrin (HST) and mutants thereof for structural and functional studies. We have developed a baculovirus expression system for the rapid and efficient production of large quantities of HST (> 20 mg/l). Like native HST, the recombinant protein can bind two ferric ions in the presence of bicarbonate, and is actively taken up by receptor-mediated endocytosis. Secondary structure calculations from CD measurements indicate a content of 42% alpha-helix and 28% beta-sheet. This is the first reported use of a non-mammalian expression system to produce functional HST, and will provide a practical tool to allow expression of a wide range of HST variants for mutagenesis studies.