M. Sohail (Khan)

The design and exogenous delivery of siRNA for post-transcriptional gene silencing.

RNA interference (RNAi) is a natural cellular process that effects post-transcriptional gene silencing in eukaryotic systems. Small interfering RNA (siRNA) molecules are the key intermediaries in this process which when exogenously administered can inhibit or “silence” the expression of any given target gene. Thus, siRNA molecules hold great promise as biological tools and as potential therapeutic agents for targeted inhibition of disease-causing genes. However, key challenges to the effective and widespread use of these polyanionic, macromolecular duplexes of RNA are their appropriate design and efficient delivery to cells in vitro and in vivo. This review highlights the current strategies used in the design of effective siRNA molecules and also summarises the main strategies being considered for the exogenous delivery of siRNA for both in vitro and in vivo applications.

Gene Silencing Nucleic Acids Designed by Scanning Arrays: Anti-EGFR Activity of siRNA, Ribozyme and DNA Enzymes Targeting a Single Hybridization-accessible Region using the Same Delivery System

Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC50 values of around 750 nM. In contrast, siRNA was significantly more potent with an IC50 of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.

A conserved serine of heterogeneous nuclear ribonucleoprotein L (hnRNP L) mediates depolarization-regulated alternative splicing of potassium channels

Background: Excitable cells show activity-dependent alternative splicing of ion channels. Results: CaMKIV phosphorylates hnRNP L at Ser-513, which is essential for depolarization-repression of a Slo1 potassium channel exon and splicing factor U2AF65. Conclusion: Depolarization controls alternative splicing of Slo1 channels through Ser-513 phosphorylation and inhibition of U2AF65. Significance: This provides the first direct link between depolarization/CaMKIV and the constitutive spliceosome. Molecular mechanisms of gene regulation underlying the activity-dependent long term changes of cellular electrical properties, such as those during memory, are largely unknown. We have shown that alternative splicing can be dynamically regulated in response to membrane depolarization and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) activation, through special CaM kinase responsive RNA elements. However, proteins that mediate this regulation and how they are affected by CaMKIV are not known. Here we show that the regulation of the stress axis-regulated exon of the Slo1 potassium channel transcripts by membrane depolarization requires a highly conserved CaMKIV target serine (Ser-513) of the heterogeneous ribonucleoprotein L. Ser-513 phosphorylation within the RNA recognition motif 4 enhanced heterogeneous ribonucleoprotein L interaction with the CaMKIV-responsive RNA element 1 of stress axis-regulated exon and inhibited binding of the large subunit of the U2 auxiliary factor U2AF65. Both of these activities were abolished by a S513A mutation. Thus, through Ser-513, membrane depolarization/calcium signaling controls a critical spliceosomal assembly step to regulate the variant subunit composition of potassium channels.

A locus for hereditary hypotrichosis localized to human chromosome 18q21.1.

Hereditary hypotrichosis is a rare autosomal recessive condition characterized clinically by alopecia. Three consanguineous kindreds with multiple affected individuals were ascertained from different regions of Pakistan. A novel hypotrichosis locus was mapped to a 5.5 cM region on chromosome 18q21.1. A maximum two-point LOD score of 5.25 was obtained at marker D18S36 (θ=0.0). Three genes each for desmoglein and desmocollin proteins are located in this region. The expression in epidermal desmosomes and their connection to the keratin intermediate filaments make these genes excellent candidates for recessive hypotrichosis.

Performance indicators for 'micro-projects' in developing countries

The majority of public works contracts undertaken in developing countries have a total cost of less than US

Strengthening accountability for urban services

15 000. They include the provision of enhanced water and sanitation, access ways and pavements, small community buildings and solid‐waste related construction. These are termed ‘micro‐projects’ and there is a general lack of information for such projects. This paper describes the development and testing of 67 performance indicators for use on micro‐projects. They include not only general performance indicators but also indicators for inter‐organizational and socio‐economic issues. These indicators are based on data from over 800 micro‐projects undertaken in developing countries. For each indicator, a statement of why the indicator was selected, the key sources of information and how to determine the indicator is provided. Examples of the use of the indicators are also presented.

Towards improved labour standards for construction of minor works in low income countries

This article explores participatory governance in the context of participatory mechanisms of accountability for urban services. In the past few years, accountability has become a buzzword internationally and, as is the case with many such terms, there is confusion as to what exactly is meant. The people and organizations promoting accountability view it as critical to solving problems with urban services in an increasingly fragmented context of service provision. This article seeks to explore the growing interest in accountability and to assess the potential of participatory governance in improving the provision of urban services. Other objectives are to: • consider contemporary innovations in the way urban services are delivered – the context of accountability; • define accountability using the existing literature and present current models for accountability; • discuss how the concept of participatory governance can be operationalized in the context of urban services; • present initial findings from case studies undertaken in South Africa, Bangladesh, South Korea and the UK; these case studies are used to illustrate different functions of accountability; and • examine the potential of accountability arrangements demonstrated in these case studies to improve the quality of local services and the responsiveness of service providers.

A Participatory Approach to Urban Transport Planning in Developing Countries

Purpose – The construction industry is one of the largest employment providers in the developing world. It is also one of the least safe industries, with a high frequency of accidents resulting in financial losses, injuries, disabilities and deaths. Decent working conditions and resulting improved worker satisfaction are key to sustainable productivity in the industry. International standards safeguarding construction workers are abundant and ratified by most low‐income countries. This paper aims to examine if these standards are adequately reflected in contracts for construction works; and if they not, how contract clauses can be improved and put into operation.Design/methodology/approach – Based on research undertaken in Ghana, India and Zambia from 2000 to 2003, this paper explores the aforementioned points. The paper comprises analysis of contract clauses from the International Federation of Consulting Engineers (FIDIC) and developing country contracts, along with case study findings.Findings – This p...

Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.

Abstract Traditionally transport planning and policy-making has used quantitative surveys to predict future demand for public transport. However, this article argues that a more participatory approach is required in order to better understand household activity patterns and the effects and implications of travel on livelihoods. Such an understanding will enable transport planning and policy to support the needs of those on low income and achieve broader poverty alleviation objectives. This article draws on case studies undertaken in Harare (Zimbabwe), Accra (Ghana) and Colombo (Sri Lanka) as part of a broader study carried out for the UK Department for International Development (DfID) Knowledge and Research Programme. The article examines the effect of public transport on certain dimensions of poverty, considers the links between urban transport and other sectors (health, education and employment) and summarizes key methods of enquiry that might be adopted in effecting a more participatory approach to transport planning.

Alternate Water Supply Arrangements in Peri-Urban Localities: Awami (people’s) Tanks in Orangi Township, Karachi

Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.