Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. Isolation, separation and purification of various types of proteins and peptides, as well as of other specific molecules, is used in almost all branches of biosciences and biotechnologies. Separation science and technology is thus very important area necessary for further developments in bio-oriented research and technology. New separation techniques, capable of treating dilute solutions or solutions containing only minute amounts of target molecules in the presence of vast amounts of accompanying compounds in both small and large-scale processes, even in the presence of particulate matter, are necessary.In the area of biosciences and biotechnology the isolation of proteins and peptides is usually performed using variety of chromatography, electrophoretic, ultrafiltration, precipitation and other procedures, affinity chromatography being one of the most important techniques. Affinity ligand techniques represent currently the most powerful tool available to the downstream processing both in term of their selectivity and recovery. The strength of column affinity chromatography has been shown in thousands of successful applications, especially in the laboratory scale. However, the disadvantage of all standard column liquid chromatography procedures is the impossibility of the standard column systems to cope with the samples containing particulate material so they are not suitable for work in early stages of the isolation/purification process where suspended solid and fouling components are present in the sample. In this case magnetic affinity, ion-exchange, hydrophobic or adsorption batch separation processes, applications of magnetically stabilized fluidized beds or magnetically modified two-phase systems have shown their usefulness.The basic principle of batch magnetic separation is very simple. Magnetic carriers bearing an immobilized affinity or hydrophobic ligand or ion-exchange groups, or magnetic biopolymer particles having affinity to the isolated structure, are mixed with a sample containing target compound(s). Samples may be crude cell lysates, whole blood, plasma, ascites fluid, milk, whey, urine, cultivation media, wastes from food and fermentation industry and many others. Following an incubation period when the target compound(s) bind to the magnetic particles the whole magnetic complex is easily and rapidly removed from the sample using an appropriate magnetic separator. After washing out the contaminants, the isolated target compound(s) can be eluted and used for further work.Magnetic separation techniques have several advantages in comparison with standard separation procedures. This process is usually very simple, with only a few handling steps. All the steps of the purification procedure can take place in one single test tube or another vessel. There is no need for expensive liquid chromatography systems, centrifuges, filters or other equipment. The separation process can be performed directly in crude samples containing suspended solid material. In some cases  it is even possible to integrate the disintegration and separation steps and thus shorten the total separation time . Due to Magnetic separation is usually very gentle to the target proteins or peptides. Even large protein complexes that tend to be broken up by traditional column chromatography techniques may remain intact when using the very gentle magnetic separation procedure . Both thSeparation of target proteins using standard chromatography techniques often leads to the large volume of diluted protein solution. In this case appropriate magnetic particles can be used for their concentration instead of ultrafiltration, precipitation etc. .The purpose of this review is to summarize various methodologies and strategies which can be employed for the isolation and purification of target proteins and peptides with the help of magnetic materials. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. All these information will help the scientists to select the optimal magnetic material and the purification procedure.The basic equipment for laboratory experiments is very simple. Magnetic carriers with immobilized affinity or hydrophobic ligands, magnetic particles prepared from a biopolymer exhibiting affinity for the target compound(s) or magnetic ion-exchangers are usually used to perform the isolation procedure. Magnetic separators of different types can be used for magnetic separations, but many times cheap strong permanent magnets are equally efficient, especially in preliminary experiments.Magnetic carriers and adsorbents can be either prepared in the laboratory, or commercially available ones can be used. Such carriers are usually available in the form of magnetic particles prepared from various synthetic polymers, biopolymers or porous glass, or magnetic particles based on the inorganic magnetic materials such as surface modified magnetite can be used. Many of the particles behave like superparamagnetic ones responding to an external magnetic field, but not interacting themselves in the absence of magnetic field. This is important due to the fact that magnetic particles can be easily resuspended and remain in suspension for a long time. In most cases, the diameter of the particles differs from ca 50 nm to approx. 10 μm. However, also larger magnetic affinity particles, with the diameters up to millimetre range, have been successfully used [lly used . MagnetiCommercially available magnetic particles can be obtained from a variety of companies. In most cases polystyrene is used as a polymer matrix, but carriers based on cellulose, agarose, silica, porous glass or silanized magnetic particles are also available. Examples of magnetic particles used (or usable) for proteins and peptides separation can be found elsewhere -7.Particles with immobilised affinity ligands are available for magnetic affinity adsorption. Streptavidin, antibodies, protein A and Protein G are used most often in the course of protein and peptides isolation. Magnetic particles with above mentioned immobilised ligands can also serve as generic solid phases to which native or modified affinity ligands can be immobilised .2 are used for immobilisation, in some cases magnetic particles are available already in the activated form .Also some other affinity ligands  are already immobilised to commercially available carriers. To immobilise other ligands of interest to both commercial and laboratory made magnetic particles standard procedures used in affinity chromatography can be employed. Usually functional groups available on the surface of magnetic particles such as -COOH, -OH or -NHIn the laboratory magnetite  particles can be surface modified by silanization. This process modifies the surface of the inorganic particles so that appropriate functional groups become available, which enable easy immobilisation of affinity ligands . In exceBiopolymers such as agarose, chitosan, kappa carrageenan and alginate can be easily prepared in a magnetic form. In the simplest way the biopolymer solution is mixed with magnetic particles and after bulk gel formation the magnetic gel formed is mechanically broken into fine particles . AlternaBasically the same procedures can be used to prepare magnetic particles from synthetic polymers such as polyacrylamide, poly and many others .In another approach used standard affinity or ion-exchange chromatography material was post-magnetised by interaction of the sorbent with water-based ferrofluid. Magnetic particles accumulated within the pores of chromatography adsorbent thus modifying this material into magnetic form ,13. AlteMagnetoliposomes (magnetic derivatives of standard liposomes), either in the original form or after immobilization of specific proteins, have the potential for the separation of antiphospholipid antibodies , IgG antRecently also non-spherical magnetic structures, such as magnetic nanorods have been tested as possible adsorbent material for specific separation of target proteins .Magnetic separators are necessary to separate the magnetic particles from the system. In the simplest approach, a small permanent magnet can be used, but various magnetic separators employing strong rare-earth magnets can be obtained at reasonable prices. Commercial laboratory scale batch magnetic separators are usually made from magnets embedded in disinfectant-proof material. The racks are constructed for separations in Eppendorf micro-tubes, standard test tubes or centrifugation cuvettes, some of them have a removable magnetic plate to facilitate easy washing of separated magnetic particles. Other types of separators enable separations from the wells of microtitration plates and the flat magnetic separators are useful for separation from larger volumes of suspensions (up to approx. 500 – 1000 ml). Examples of typical batch magnetic separators are shown in Fig. Flow-through magnetic separators are usually more expensive, and high gradient magnetic separators (HGMS) are the typical examples. Laboratory scale HGMS is composed from a column packed with fine magnetic grade stainless steel wool or small steel balls which is placed between the poles of an appropriate magnet. The suspension is pumped through the column, and magnetic particles are retained within the matrix. After removal the column from the magnetic field, the particles are retrieved by flow and usually by gentle vibration of the column.For work in dense suspensions, open gradient magnetic separators may be useful. A very simple experimental set-up for the separation of magnetic affinity adsorbents from litre volumes of suspensions was described .Currently many projects require the analysis of a high number of individual proteins or variants. Therefore, methods are required that allows multiparallel processing of different proteins. There are several multiple systems for high throughput nucleic acid and proteins preparation commercially available. The most often used approach for proteins isolation is based on the isolation and assay of 6xHis-tagged recombinant proteins using magnetic beads with Ni-nitriloacetic acid ligand . The comMagnetic separations of proteins and peptides are usually convenient and rapid. Nevertheless, several hints may be helpful to obtain good results.Proteins and peptides in the free form can be directly isolated from different sources. Membrane bound proteins have to be usually solubilized using appropriate detergents. When nuclei are broken during sample preparation, DNA released into the lysate make the sample very viscous. This DNA may be sheared by repeated passage up and down through a 21 gauge hypodermic syringe needle before isolation of a target protein. Alternatively, DNase can be added to enzymatically digest the DNA.Magnetic beads in many cases exhibit low non-specific binding of non-target molecules present in different samples. Certain samples may still require preclearing to remove molecules which have high non-specific binding activity. If preclearing is needed, the sample can be mixed with magnetic beads not coated with the affinity ligand. In the case of immunomagnetic separation, magnetic beads coated with secondary antibody or with irrelevant antibodies have been used. The non-specific binding can also be minimised by adding a non-ionic detergent both in the sample and in the washing buffers after isolation of the target.In general, magnetic affinity separations can be performed in two different modes. In the direct method, an appropriate affinity ligand is directly coupled to the magnetic particles or biopolymer exhibiting the affinity towards target compound(s) is used in the course of preparation of magnetic affinity particles. These particles are added to the sample and target compounds then bind to them. In the indirect method the free affinity ligand (in most cases an appropriate antibody) is added to the solution or suspension to enable the interaction with the target compound. The resulting complex is then captured by appropriate magnetic particles. In case antibodies are used as free affinity ligands, magnetic particles with immobilised secondary antibodies, protein A or protein G are used for capturing of the complex. Alternatively the free affinity ligands can be biotinylated and magnetic particles with immobilised streptavidin or avidin are used to capture the complexes formed. In both methods, magnetic particles with isolated target compound(s) are magnetically separated and then a series of washing steps is performed to remove majority of contaminating compounds and particles. The target compounds are then usually eluted, but for specific applications  they can be used still attached to the particles, such as in the case of polymerase chain reaction, magnetic ELISA etc.The two methods perform equally well, but, in general, the direct technique is more controllable. The indirect procedure may perform better if affinity ligands have poor affinity for the target compound.In most cases, magnetic batch adsorption is used to perform the separation step. This approach represents the simplest procedure available, enabling to perform the whole separation in one test-tube or flask. If larger magnetic particles (with diameters above ca 1 μm) are used, simple magnetic separators can be employed. In case magnetic colloids (diameters ranging between tens and hundreds of nanometres) are used as affinity adsorbents, high-gradient magnetic separators have usually to be used to remove the magnetic particles from the system.Alternatively magnetically stabilised fluidised beds (MSFB), which enable a continuous separation process, can be used. The use of MSFB is an alternative to conventional column operation, such as packed-bed or fluidised bed, especially for large-scale purification of biological products. Magnetic stabilisation enables the expansion of a packed bed without mixing of solid particles. High column efficiency, low pressure drop and elimination of clogging can be reached ,22.Also non-magnetic chromatographic adsorbents can be stabilized in magnetically stabilized fluidized beds if sufficient amount of magnetically susceptible particles is also present. The minimum amount of magnetic particles necessary to stabilize the bed is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations .Biocompatible two phase systems, composed for example from dextran and polyethylene glycol, are often used for isolation of biologically active compounds, subcellular organelles and cells. One of the disadvantages of this system is the slow separation of the phases when large amounts of proteins and cellular components are present. The separation of the phases can be accelerated by the addition of fine magnetic particles or ferrofluids to the system followed by the application of a magnetic field. This method seems to be useful when the two phases have very similar densities, the volumetric ratio between the phases is very high or low, or the systems are viscous. Magnetically enhanced phase separation usually increases the speed of phase separation by a factor of about 10 in well-behaved systems, but it may increase by a factor of many thousands in difficult systems. The addition of ferrofluids and/or iron oxide particles was shown to have usually no influence on enzyme partioning or enzyme activity ,25.Proteins and peptides isolated using magnetic techniques have to be usually eluted from the magnetic separation materials. In most cases bound proteins and peptides can be submitted to standard elution methods such as the change of pH, change of ionic strength, use of polarity reducing agents  or the use of deforming eluents containing chaotropic salts. Affinity elution  may be both a very efficient and gentle procedure.Magnetic affinity and ion-exchange separations have been successfully used in various areas, such as molecular biology, biochemistry, immunochemistry, enzymology, analytical chemistry, environmental chemistry etc -29. TablIn the case of proteins and peptides purifications, no simple strategy for magnetic affinity separations exists. Various affinity ligands have been immobilised on magnetic particles, or magnetic particles have been prepared from biopolymers exhibiting the affinity for target enzymes or lectins. Immunomagnetic particles, i.e. magnetic particles with immobilised specific antibodies against the target structures, have been used for the isolation of various antigens, both molecules and cells  and can Magnetic separation procedures can be employed in several ways. Preparative isolation of the target protein or peptide is usually necessary if further detailed study is intended. In other cases, however, the magnetic separation can be directly followed (after elution with an appropriate buffer) with SDS electrophoresis. Magnetically separated proteins and peptides can also be used for further mass spectroscopy characterization ,31. The Enzyme isolation is usually performed using immobilised inhibitors, cofactors, dyes or other suitable ligands, or magnetic beads prepared from affinity biopolymers can be used  are often short-lived and in low abundance. A rapid and sensitive method, based on the immobilization of biotinylated DNA/RNA fragments containing the specific binding sequence to the magnetic streptavidin particles, can be used. The bound DNA/RNA binding proteins are usually eluted with high salt buffer or change of pH .Sc from its conformer which is a cellular protein called PrPC. In fact, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes [Other types of proteins were isolated using specific affinity-based procedures. For example, plasminogen immobilized on magnetic particles was used to separate scrapie and bovine spongiform encephalopathy associated prion protein PrPpurposes ,40.Magnetic separation was also successfully used for the recovery of proteins expressed in the form of inclusion bodies, involving at first chemical extraction from the host cells, then adsorptive capture of the target protein onto small magnetic adsorbents, followed by rapid collection of the product-loaded supports with the aid of high gradient magnetic fields .A new approach for analytical ion-exchange separation of native proteins and proteins enzymatic digest products has been described recently . MagnetiMagnetic solid phase extraction (MSPE) enables to preconcentrate target analytes from larger volumes of solutions or suspensions using relatively small amount of magnetic specific adsorbent. Up to now this procedure was used for preconcentration of low-molecular weight xenobiotics ,43 but uXenopus egg extracts for analyses of the cell mitosis mechanisms [Sometimes the removal of certain proteins will reveal functions involving the depleted proteins or will help in the course of subsequent protein isolation. As an example, Dynabeads have been used to remove involved proteins from chanisms ,45. Rapichanisms .A combination of mechanical cell disintegration and magnetic batch affinity adsorption was used to simplify the isolation of intracellular proteins. Magnetic glass beads were used because of their hardness and rigidity .Dictyostelium discoideum were separated after magnetic isolation of the vesicles that was accomplished by feeding the amoebae with dextran-stabilized iron oxide particles. The cells were broken, the labelled vesicles were magnetically separated and then disrupted to release proteins which were resolved by SDS-PAGE. After „in-gel“ digestion with endoproteinase Lys-C or Asp-N the generated peptides were used for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles [An example of quite different protein purification strategy can also be mentioned. Proteins associated with the endocytic vesicles of vesicles . Analogovesicles .Standard liquid column chromatography is currently the most often used technique for the isolation and purification of target proteins and peptides. Magnetic separation techniques are relatively new and still under development. Magnetic affinity particles are currently used mainly in molecular biology , cell biology and microbiology (separation of target cells) and as parts of the procedures for the determination of selected analytes using magnetic ELISA and related techniques . Up to now separations in small scale prevail and thus the full potential of these techniques has not been fully exploited.It can be expected that further development will be focused at least on two areas. The first one will be focused on the laboratory scale application of magnetic affinity separation techniques in biochemistry and related areas (rapid isolation of a variety of both low- and high-molecular weight substances of various origin directly from crude samples thus reducing the number of purification steps) and in biochemical analysis (application of immunomagnetic particles for separation of target proteins from the mixture followed by their detection using ELISA and related principles). Such a type of analysis will enable to construct portable assay systems enabling e.g. near-patient analysis of various protein disease markers. New methodologies, such as the application of chip and microfluidics technologies, may result in the development of magnetic separation processes capable of magnetic separation and detection of extremely small amount of target biologically active compounds .In the second area, larger-scale  systems are believed to be developed and used for the isolation of biologically active compounds directly from crude culture media, wastes from food industry etc., integrating three classical steps  into a single unit operation . It is nIn the near future quite new separation strategies can appear. A novel magnetic separation method, which utilizes the magneto-Archimedes levitation, has been described recently and applied to separation of biological materials. By using the feature that the stable levitation position under a magnetic field depends on the density and magnetic susceptibility of materials, it was possible to separate biological materials such as haemoglobin, fibrinogen, cholesterol, and so on. So far, the difference of magnetic properties was not utilized for the separation of biological materials. Magneto-Archimedes separation may be another way for biological materials separation .It can be expected that magnetic separations will be used regularly both in biochemical laboratories and biotechnology industry in the near future.


Studies conducted in developed countries using economic models show that individual- and household- level variables are important determinants of health insurance ownership. There is however a dearth of such studies in sub-Saharan Africa. The objective of this study was to examine the relationship between health insurance ownership and the demographic, economic and educational characteristics of South African women.The analysis was based on data from a cross-sectional national household sample derived from the South African Health Inequalities Survey (SANHIS). The study subjects consisted of 3,489 women, aged between 16 and 64 years. It was a non-interventional, qualitative response econometric study. The outcome measure was the probability of a respondent's ownership of a health insurance policy.2 test for goodness of fit indicated satisfactory prediction of the estimated logit model. The coefficients of the covariates for area of residence, income, education, environment rating, age, smoking and marital status were positive, and all statistically significant at p ≤ 0.05. Women who had standard 10 education and above (secondary), high incomes and lived in affluent provinces and permanent accommodations, had a higher likelihood of being insured.The χPoverty reduction programmes aimed at increasing women's incomes in poor provinces; improving living environment  for women in urban informal settlements; enhancing women's access to education; reducing unemployment among women; and increasing effective coverage of family planning services, will empower South African women to reach a higher standard of living and in doing so increase their economic access to health insurance policies and the associated health services. A health system in any country performs instrumental functions of stewardship (oversight), creation of resources (investment and training), delivering services (provision), and financing  . UltimatPrior to the 1994 democratic elections in South Africa, the health system was built on the apartheid ideology, which was characterized by racially segregated health services, geographical disparities, fragmentation, duplication and specialized hospital-centred services favouring the urban populations . The heaIn an attempt to overcome the above-mentioned public health challenges, the post-apartheid democratic governments have introduced several reforms in the health sector: creation of a quasi-federal structure with one national and nine provincial departments of health ; establishment of a district health system, including expansion and upgrading of the primary care infrastructure; and health care financing . The latIn spite of the abovementioned policy interventions, primary health care still remains under funded. For example, in 2003/2004 financial year, South Africa spent a total of 36.9 billion Rands (1 Rand = US$6) on health: 61.3% was spent on hospitals curative care, 16.1% on primary health care, 1.8% on HIV/AIDS treatment, 2.3% on nutrition, 4.0% on emergency services, 4.3% on administration, 2.5% on training, 1.6% on support services and 6.1% on other services .According to Doherty and McLeod  and McInIn 2001, there were 146 registered medical schemes  and eight Bargaining Council schemes (i.e. those granted exemption from certain provisions of the Act), all covering a total of 7 million people .The objective of this study was to examine the relationship between individuals' demographic, economic and educational characteristics, and their likelihood of being insured.There are two kinds of risks involved in health care: (i) the risk of becoming ill, with the accompanying loss in the quality of life, cost of medical care, loss of productive time during illness and, in more serious cases, death; and (ii) the risk of total or incomplete or delayed recovery .Welfare economics of uncertainty predicts that individuals would like to insure against both forms of risks. The theory of expected utility, on which this study is based, assumes that each individual strives to maximize the expected value of a utility function; individuals are normally risk-averse, meaning that they have a diminishing marginal utility of income; and health risks for different individuals are basically independent, so that pooling them reduces the risk to the insurer to relatively small proportions .In the South African National Health Inequalities Survey (SANHIS) , the res1) and expected utility without insurance (EU2). We need to analyse the effect of changes in the independent variables on the difference in the level of the expected utility of the two prospects, i.e. EU1 minus EU2. If the difference were equal to zero, the consumer would be expected to be indifferent between the two prospects. However, if the difference were greater than 0, then the risk-averse consumer would be expected to opt for insurance [The potential consumers of insurance are assumed to make decisions based on the magnitude of the perceived difference between the level of expected utility with insurance  for independence was undertaken to test the relationship between health insurance ownership and the individual independent variables. The null hypothesis is that the two variables are independent of one another . The alternate hypothesis is that the two variables are dependent on each other (because there is not a significant difference). Thus, for health rating, the hypotheses are as follows: (i) H0: A person's health insurance ownership status and their health rating are independent or unrelated; and (ii) HA: A person's health insurance ownership status and their health rating are related (dependent on each other).A Chi-Squared test  of 1998.2 Rand. The sub-sample of the households without health insurance had an average income of 853 Rand (STD = 1073) compared to 3172 Rand (STD = 2623) in the group with insurance. Seventy-seven percent of the households with at least one member with health insurance had a monthly income of more than 951 South African Rand, compared to only 28% among the group without health insurance.The average and median household size in the overall sample was five members, with a standard deviation of about 3. The group with health insurance had an average household size of four people (STD = 2.1) compared to 5 people (STD = 2.7) in the group without insurance. Fifty-seven percent of the sub-sample without health insurance had a household size of more than five members compared to 38% among the group with health insurance.The group with health insurance had an average age of 40 years (STD = 12.5) compared to 41 years (STD = 14.1) in the group without insurance. The average age in the whole sample was 40.9 years (STD = 13.6) and the median was 39 years.A) that a person's health insurance ownership status and their health rating are related (dependent on each other).As alluded to in the methods section, a Chi-Squared test for independence was undertaken to test the relationship between health insurance ownership and the individual independent variables. The results are presented in the last column of Table 0: β = 0) about individual regression slope coefficients. The 't' values for individual variables are obtained by dividing their coefficients (e.g. βINCOME) by their standard errors (e.g. SEINCOME). For example, the coefficient for income is 0.0004727 and the standard error is 0.0000339, and given H0: β = 0, the relevant t-value is indeed 13.946 as specified in Table Table INCOME = (βINCOME)/(SEINCOME) = (0.0004727)/(0.0000339) = 13.946t0: β = 0) if the calculated t-value, tk, is greater than the critical t-value, tc, as long as the sign of tk is the same as the sign of the coefficient implied in the alternative hypothesis (HA: β≠0). Otherwise, accept the null hypothesis (H0) that the estimated regression coefficient in question is not significantly different from zero. In the above example, the coefficient of income is statistically significant at 95% level of confidence, based on a two-sided test, since the computed t-value (13.946) is greater than the critical t-value (1.960).The decision rule is: reject the null hypothesis (Hi) on the log of odds of a health insurance policy ownership, holding other explanatory variables constant. The coefficients for environment rating, residence, income, education, age, smoking and marital status are statistically significant at 95% level of confidence, and have positive signs. The latter result implies that an increase in any of these variables spontaneously impacts positively on the log of odds of health insurance policy ownership, holding other factors constant. Contrastingly, the coefficients for household size, alcohol, contraceptive use, age-squared, occupation, employment and health rating are statistically significant, and have got a negative effect on the log of odds of health insurance policy ownership.The coefficient (β) of the estimated binary logit model measures the impact of a one-unit change in an explanatory variable  and health-related quality of life  ,23. The An individual's stock of health determines the total amount of time he/she can spend producing commodities and money earnings . Health The adverse selection problem can be curbed in two main ways without compromising equity objectives, namely: (i) compulsory social health insurance  for a defined population through legislation – not an option for private health insurance which is voluntary by definition; and (ii) the government could step in and provide health insurance to all those at exceptionally high risk (e.g. the elderly and those with chronic diseases) and the poor who cannot afford the premiums. Commercial health insurance firms often curb adverse selection by introducing experience rating, i.e. linking insurance premium to the degree of assessed risk of falling sick (this action may have negative equity implications); and/or subjecting all those who apply for insurance cover to a thorough medical examination .There were three economic variables, namely, income (+), occupation (-) and employment (-), where (.) is the hypothesized sign of the coefficients. The coefficients of the three variables were statistically significant and had the expected signs. High incomes, white-collar occupations and being gainfully employed are significant predictors of health insurance ownership. The proportion of people with health insurance rises considerably as one moves up the household income distribution ladder, with the coverage going from 6.3% of those in the income range 1–950 Rand to over 90.7% among those earning 7600 Rand and above per month. The trend is similar to that reported by Harmon and Nolan  in IrelaThe demographic factors include: age (+), age squared (-) and household size (-), where (.) is the postulated sign of the coefficient. The coefficients for age and age squared were statistically significant at the 5% level. Economic theory predicts that as individuals advance in age, their inherited health stock depreciates at an increasing rate  and they tend to increase investments in health  in an attempt to decrease the rate of depreciation. This is consistent with Grossman's findings  that becThe coefficient for household size variable had a statistically significant negative effect on the likelihood of health insurance policy ownership. This finding is intuitively sensible since any increase in the household size, while holding the income constant, reduces the per capita income.The social factors include: education (+) and marital status (+). The coefficient for education was statistically significant, and had the expected positive sign. Respondents with at least a matriculation (secondary) level of education were two times more likely to be in possession of a health insurance policy than those with a lower level of education. This could be attributed to a positive relationship between a person's: (i) educational level and propensity to acquire skills; (ii) stock of knowledge and his/her market and non-market productivity  and earnMarital status had a statistically significant positive effect on health insurance ownership. Married persons are more likely to have insurance cover than those who are single, separated or divorced. This finding is consistent with the result obtained by Harmon and Nolan , Rhine eThe spatial and environmental factors – residence (+) and environment rating (+) – had a statistically significant effect on health insurance ownership. The respondents living in formal urban settlements or rural white-owned farms had a seven times higher odds of owning a health insurance policy than those living in informal urban settlements or former rural homelands. This could partly be a reflection of the economic well being of the former group.vis-à-vis informal settlements .The respondents who felt that the environment they lived in was good, very good or excellent were twenty-seven times more likely to have a health insurance cover than those who lived in fair or poor environments. This phenomenon may be a reflection of a better socio-economic status of those living in relatively affluent, formal (and cleaner) residential areas The behavioural factors included in the analysis were: contraceptive use (-), alcohol use (-) and smoking (+). The three had a statistically significant effect on the demand for health insurance. The coefficient for contraceptives assumed a negative sign. This implies that the use of contraceptives may not necessarily be linked with individuals' attitudes toward risk.The coefficient for alcohol use also took a negative sign, which implies that those people who drank alcohol were less likely to purchase health insurance. On the contrary, the parameter for smoking assumed a positive sign, meaning that being a cigarette smoker, the probability of a person demanding health insurance coverage increases. In the context of the health insurance market, the latter finding could be a source of concern if it were a signal for the presence of moral hazard. Moral hazard is a potential cost of insurance in which the presence of insurance increases the tendency for losses to occur through careless, irresponsible or perhaps illegal behaviour .For example, the fully insured individuals may embark on risky behaviours, such as smoking, and by so doing expose themselves to a high risk of developing various forms of cancer . Insurers attempt to control moral hazard by careful underwriting of applicants for insurance and by various policy provisions, such as deductibles  and co-insurance/co-payment  ,29.This study used consumption of contraceptives, alcoholic drinks and cigarettes as proxies for consumers' attitudes toward health risks. The three may not be ideal proxies for risk attitudes; however, there were no better alternatives in the data set. If this were a study primarily designed to analyse the demand for health insurance, it would have been preferable to either proxy risk attitudes using a qualitative scale variable ranging from 1 (extremely risk-averse) to 10 (risk-lover) , directlAppendix), leading to specification bias or omitted variable bias [The main weakness of the current study is that since the data set upon which the analysis was based was gathered for a different purpose , it did not contain insurance-specific attributes, e.g. premiums, co-payments, deductibles, benefits covered and the quality of care in the health facilities where the insured sought care. Thus, we had a situation where important explanatory variables were left out of the estimated regression equation 2  and household socio-economic characteristics (including attitudes toward risk).• The willingness and ability to pay for various forms of health insurance, including voluntary and non-voluntary insurance schemes .• The economic viability of various forms of health insurance, e.g. social health insurance and community-based prepaid schemes.• Design of innovative health insurance schemes; for example, within farmers' cooperative societies , savings• Whether the expansion of private health insurance under the current health care delivery system would yield significant public sector cost savings, and improved targeting of subsidies for the poor and preventive services .• Optimal ways of curbing health insurance problems of moral hazard and adverse selection.• Benefit-incidence analysis of alternative health insurance arrangements.The environment rating, residence, income, education, age, smoking and marital status variables were all found to have a statistically significant (at 95% confidence level) positive relationship with ownership of health insurance schemes. Contrastingly, the other covariates, namely: health rating, age squared, household size, occupation, employment, alcohol use and contraceptive use had a significantly negative relationship with health insurance ownership.There are a number of policy implications of this study:• High incomes, white-collar occupations and being gainfully employed are significant predictors of health insurance ownership. Thus, economic development (or poverty reduction) programmes geared at: (i) improving incomes of the vulnerable segments of the South African population; (ii) reducing involuntary unemployment; and (iii) creating white-collar job opportunities will empower South African women to reach a higher standard of living and in doing so boost their economic access to health insurance policies and the relevant health services.• Policies aimed at ensuring that the majority of South Africans attain a matriculation (i.e. secondary) education level will increase by almost two-fold, the probability of acquiring health insurance.• The self-assessed health status was found to have a statistically negative effect on the demand for health insurance. This implies existence of adverse selection. However, this problem can be reduced through compulsory social health insurance ; or throThe author(s) declare that they have no competing interests.JMK recoded the raw data and participated in the development of the conceptual framework, analysis and drafting of sections of the document. LGS, BN, GMM, RC and TM participated in the development of the conceptual framework and drafting of sections of the document. All authors read and approved the final manuscript.i) and a vector of a household's socioeconomic characteristics (Ri), plus a stochastic error term (ε). The latter component captures errors in model specification (e.g. omission of relevant variables) and errors in data measurement.A binary logit model was used in the analysis of individual household's choice between having no health insurance and having health insurance. We assumed that the expected utility associated with each health insurance option is a function of a vector of its attributes  + ε .................................... (1);ij is the utility that ith household expects to derive from choosing jth health insurance option; j = 1 if a household has health insurance; j = 2 if a household has no health insurance; and X, R and ∈ are as defined above.where: EUth household opts for 'health insurance' if EUi1 > EUi2, prefers 'no health insurance' if EUi1 < EUi2, and is indifferent between the two options if EUi1 = EUi2. Thus, the probability that ith household prefers to have health insurance is: Pi1 = P(EUi1 > EUi2). And, conversely, the probability that ith household prefers not to have health insurance is: Pi2 = P(EUi1 < EUi2).The basic assumption is that the iTo determine the probability of health insurance ownership, the following model was estimated:Pij = (α + β1 HEALTH_RATING + β2 ENVIRONMENT_RATING + β3 RESIDENCE + β4 INCOME + β5EDUCATION + β6 AGE + β7 AGE_SQUARED + β8 RACE + β9 HOUSEHOLD_SIZE + β10 OCCUPATION + β11 EMPLOYMENT + β12 SMOKING + β13 ALCOHOL_USE + β14 CONTRACEPTIVES_USE + β15 MARITAL_STATUS + εi........................ (2)ij = 1 if individual 'i' owns insurance (j = 1) and equals zero otherwise (j = 0); (α) is the intercept term; (β's) are the estimated coefficients; and εi is the stochastic error term. The explanatory variables included in the model are defined in Table where: PThe pre-publication history for this paper can be accessed here:


Published February 15, 2005PLoS Biology, volume 2, issue 12.In 10.1371/journal.pbio.0020449The art credits were missing from the image accompanying this synopsis. The image caption should read as follows:


Results of previous autopsy studies indicate that increased hepatic iron stores or hepatic iron overload is common in African Americans dying in hospitals, but there are no reports of hepatic iron content in other cohorts of African Americans.We investigated the prevalence of heavy liver iron deposition in African American adults. Using established histochemical criteria, we graded Perls' acid ferrocyanide-reactive iron in the hepatocytes and Kupffer cells of 341 consecutive African American adults who were autopsied in the coroner/medical examiner office. Heavy staining was defined as grade 3 or 4 hepatocyte iron or grade 3 Kupffer cell iron.There were 254 men and 85 women ; gender was unstated or unknown in two subjects. Approximately one-third of subjects died of natural causes. Heavy staining was observed in 10.2% of men and 4.7% of women. 23 subjects had heavy hepatocyte staining only, six had heavy Kupffer cell staining only, and one had a mixed pattern of heavy staining. 15 subjects had histories of chronic alcoholism; three had heavy staining confined to hepatocytes. We analyzed the relationships of three continuous variables  and two categorical variables ) in all 341 subjects using a correlation matrix with Bonferroni correction. This revealed two positive correlations: hepatocyte with Kupffer cell iron grades (p < 0.01), and male sex with hepatocyte iron grade (p < 0.05). We also analyzed the relationship of steatosis, inflammation, and fibrosis/cirrhosis in 30 subjects with heavy iron staining using a correlation matrix with Bonferroni correction. There were significant positive correlations of steatosis with inflammation , and of inflammation with fibrosis/cirrhosis .The present results confirm and extend previous observations that heavy liver iron staining is relatively common in African Americans. The pertinence of these observations to genetic and acquired causes of iron overload in African Americans is discussed. Hepatic iron overload was detected by Perls' acid ferrocyanide staining and atomic absorption spectrometry at autopsy in more than one percent of African American adults who died in hospitals ,2. In onHFE-associated hemochromatosis [HFE-associated hemochromatosis.In contrast, screening programs that included cohorts of African Americans presumably representative of the general African American population identified a much lower proportion of subjects with possible iron overload -5 than iomatosis -5. In thomatosis -5. TakenThus, we graded Perls' acid ferrocyanide-reactive iron in the livers of 341 consecutive African American adults who underwent autopsy in the coroner/medical examiner office. We selected this population for study because such subjects are more representative of the general African American population than persons who died in hospital. We then compared these observations with autopsy results reported previously in African Americans who died in hospitals . The perThe performance of this study was approved by the Institutional Review Board of the University of Alabama at Birmingham and by the Coroner/Medical Examiner's Office of Jefferson County, Alabama. We evaluated formalin-fixed tissue obtained in 361 consecutive, unselected coroner/medical examiner autopsy cases of African-American adults (age >18 years) in Jefferson County, Alabama; deaths in all of the present cases occurred during the interval 1998 – 2002. Each subject was identified as African American by his/her previous medical histories or legal records, or by the coroner/medical examiner staff. Most autopsies were performed on the same day that the respective bodies were available for evaluation at the Coroner/Medical Examiner's Office; other autopsies were completed within ~ 18 hours. All tissues were placed directly in fixative at the time of collection at autopsy. Liver was not available or was not interpretable due to autolysis in 20 cases. Available records in each case were reviewed; age at death, sex, summary of known illnesses, and cause of death were tabulated for all cases for which liver tissue was available.Tissues obtained at autopsy were routinely fixed in 10% neutral buffered formalin. Triplicate sections of paraffin-embedded liver were prepared. One section was stained with hematoxylin and eosin, another with Perls' acid ferrocyanide technique to demonstrate non-heme ferric iron , and a tAll slides were simultaneously reviewed by three investigators, and the iron grades assigned in each case represent their consensus opinions. Hepatocellular iron was graded according to these criteria: grade 0 – no visible iron; grade 1 – iron visible in very few hepatocytes; grade 2 – iron visible in 5 – 10% of hepatocytes; grade 3 – iron visible in ≥ 40% of hepatocytes; and grade 4 – abundant iron visible in most hepatocytes . KupfferWe designated a gradient of stainable iron in hepatocytes from the periportal area decreasing towards the hepatic venule as present or absent; visualization of a gradient required hepatocyte iron staining of grade ≥ 2 ,10. The ®, Microsoft Corp., Redmond, WA), and a statistical program . Descriptive data are displayed as enumerations, percentages, mean ± 1 S.D., medians, and ranges. Frequency values were compared using chi-square analysis or Fisher exact test, as appropriate. Mean values were compared using student t-test. Some data were analyzed using a correlation matrix with Bonferroni correction. Values of p < 0.05 were defined as significant.The present data set consisted of observations in 341 subjects and their respective livers. Analyses were performed with a computer spreadsheet , and 85 women . The mean age of men was lower than that of women (p = 0.0255). There were two subjects for whom gender was unstated or unknown. Approximately one-third of subjects died of natural causes . However, the mean hepatocyte iron grade was 0.83 ± 0.96; the mean Kupffer cell iron grade was 0.32 ± 0.32.Increased hepatocyte iron grades were observed in 64 men 25.2%) and 8 women (9.4%) (p = 0.0021). Increased Kupffer cell iron grades were observed in 20 men (7.9%) and one woman (1.2%) (p = 0.0266) (Tables 5.2% and Heavy iron staining was observed in 30 subjects (8.8%), including 10.2% of men and 4.7% of women (p = 0.1202). The mean age of men and women with heavy iron staining was similar: 45 ± 15 y and 47 ± 9 y (p = 0.7389). 24 subjects (7.1%) had grade 3 or 4 hepatocyte iron; of these, one also had grade 3 Kupffer cell iron. Seven subjects (2.1%) had grade 3 Kupffer cell iron; of these, one also had grade 3 hepatocyte iron. Altogether, 23 subjects had heavy iron staining in hepatocytes only  and two categorical variables ) using a correlation matrix with Bonferroni correction. This revealed two significant positive correlations: hepatocyte iron grade with Kupffer cell iron grade (p < 0.01), and male sex with hepatocyte iron grade (p < 0.05).These subjects were comprised of 26 men and 4 women Table . HepaticWe analyzed the relationship of hepatocyte and Kupffer cell iron grades, steatosis, inflammation, and fibrosis/cirrhosis in these 30 subjects .Three persons who had histories of chronic alcoholism also had hepatic cirrhosis. A 36 year-old man had grade 0 hepatocyte and grade 0 Kupffer cell iron. A 46 year-old man had grade 2 hepatocyte iron and grade 0 Kupffer cell iron. A 50 year-old woman had grade 4 hepatocyte iron and grade 0 Kupffer cell iron . Three had histories of chronic alcoholism (described above). One of these three subjects, a 50 year-old woman, had grade 4 hepatocyte iron and grade 0 Kupffer cell iron  [HJV on Ch1q [ALAS2 on ChX [Primary iron overload in African Americans is often associated with preferential deposition of iron in macrophages in multiple organs ,7,21. InFPN1 ,21,22. T2Y/H63D) ,21, or w on Ch1q  or the e2 on ChX . Other p2 on ChX . HoweverAcquired disorders account for increased hepatic iron deposition in some African Americans. In the present study, we did not observe any subject who had heavy liver iron staining and fibrosis or cirrhosis who did not also have hepatic steatosis or inflammation. Chronic viral hepatitis C occurs in approximately 1.8% of the overall U.S. population , and theIron overload sometimes develops spontaneously or after repeated erythrocyte transfusion in African Americans with heritable or acquired anemia, or with myelodysplasia or acute leukemia ,15,33-41Three of the five present subjects with micronodular cirrhosis had a history of chronic alcoholism. African Americans are at greater risk than whites for developing several alcohol-related conditions, including hepatic cirrhosis ,43. In tWe conclude that heavy liver iron staining is common in African American adults who were autopsied in the coroner/medical examiner office. The different histologic patterns of heavy liver iron staining we observed in the present subjects are consistent with the phenotypic and genotypic heterogeneity of primary iron overload in African Americans  and withThe author(s) declare that they have no competing interests.JCB conceived and designed the study, reviewed and graded the liver specimens, contributed to the statistical analyses of data, and contributed to writing the manuscript. RTA reviewed and graded the liver specimens, contributed to the statistical analyses of data, and contributed to writing the manuscript. AKR reviewed and graded the liver specimens and contributed to writing the manuscript. RMB provided information on the autopsy cases, provided the liver specimens, reviewed the histology of selected liver specimens, and contributed to writing the manuscript. All authors approved of the manuscript in its final form.The pre-publication history for this paper can be accessed here:


Timely and accurate information about the onset of malaria epidemics is essential for effective control activities in epidemic-prone regions. Early warning methods that provide earlier alerts  may permit control measures to interrupt transmission earlier in the epidemic, perhaps at the expense of some level of accuracy.th-degree polynomial distributed lag model. For each week, the numbers of malaria cases were predicted using coefficients obtained using all years except that for which the prediction was being made. The effectiveness of alerts generated by the prediction system was compared against that of alerts based on observed cases. The usefulness of the prediction system was evaluated in cold and hot districts.Expected case numbers were modeled using a Poisson regression with lagged weather factors in a 4The system predicts the overall pattern of cases well, yet underestimates the height of the largest peaks. Relative to alerts triggered by observed cases, the alerts triggered by the predicted number of cases performed slightly worse, within 5% of the detection system. The prediction-based alerts were able to prevent 10–25% more cases at a given sensitivity in cold districts than in hot ones.The prediction of malaria cases using lagged weather performed well in identifying periods of increased malaria cases. Weather-derived predictions identified epidemics with reasonable accuracy and better timeliness than early detection systems; therefore, the prediction of malarial epidemics using weather is a plausible alternative to early detection systems. Malaria epidemics are reported frequently and have caused high morbidity and mortality among all age groups in the African highlands -4. Earlyth percentile of cases from the same week in previous years [Early detection systems, which are used to detect epidemics once they have begun, can correctly identify periods that are defined by expert observers as "epidemic," albeit with varying specificity. A number of such systems have been proposed or implemented. For example, WHO has advocated the use of alerts when weekly cases exceed the 75us years  and otheus years -12. Howeus years  and if tus years .While early detection systems appear to provide timely information about the onset of severe epidemics, they intrinsically trigger alerts only when unusual transmission is already underway. Another approach, known as "early warning," attempts to predict epidemics before unusual transmission activity begins, usually by using weather variables that predict vector abundance and efficiency, and therefore, transmission potential ,15-18. THere, an attempt to combine these avenues of previous work is described, using modified versions of previously described models based on weather factors to provide predictions of Plasmodium falciparum cases in these 10 districts of Ethiopia, and evaluating thresholds that trigger warnings. The hypothesis tested here is that the use of predicted cases  would reduce the precision of the alert thresholds , as the price of obtaining the alerts with greater advance notice. In fact, the early warning system based on predicted cases performed slightly worse in most cases than the early detection system, but the performance was rarely much worse and occasionally slightly better. These comparisons are described and their implications for the choice of malaria prediction/detection systems in epidemic-prone areas of Africa are discussed.P. falciparum. The original data collected on the basis of Ethiopian weeks (where the number of days in each week varies between 5 and 9) were normalized to obtain mean daily cases for each Ethiopian week [Microscopically confirmed malaria cases were collected from a health facility in each of ten districts of Ethiopia over an average of 10 years; this data set has been previously described . Each ofian week .Daily meteorological data  recorded at the local weather stations nearest to the health facility were obtained from the National Meteorological Services Agency (NMSA) for the same period. These daily data were collapsed into weekly data to correspond with the weekly malaria cases. The weekly mean for minimum and maximum temperatures and the total weekly rainfall were calculated from the daily records.th-degree polynomial distributed lag (PDL) model [The expected case numbers for a given week were modeled using a Poisson regression with lagged weather factors, an autoregressive term, a time trend and indicator variables for week of the year. Biological considerations about the interrelationship between weather, mosquito and malaria parasite suggest that malaria cases should follow periods of increased temperature and increased rainfall, at defined intervals -28. ThusL) model  was fittE(stY) denotes expected value for the daily average number of malaria cases at site s on week t; , , t-iR, and st-i Yare the weekly minimum and maximum temperatures, rainfall and autoregressive term i weeks previously; s tand s Wdesignate time trend and week in a year at site s; s αrepresent the intercept, at site s.where For each week at each location in the data set, the number of cases was predicted using equation (1) and data available four weeks prior to the week for which the prediction is made. Coefficients of equation (1) were obtained using all years except that for which the prediction was being made, to avoid circularity. The prediction for week t was then made using this all-but-current-year model with weather and case data for the weeks up to week t-4. The predicted number of cases is thus estimated using the following model: represent the predicted cases for year j; , , , , ,  and  are parameter estimates  from all years except j; , , , jt,  and  are minimum and maximum temperatures, rainfall, time, week and autoregressive term respectively from year j.where In early detection algorithms, actual cases in a given time period are typically compared against some threshold level of cases to determine whether excess cases have been observed. Often, the threshold level represents an upper bound on "normal" case numbers from previous years. If this threshold level is crossed , an alert is generated . Such syIn this study of the usefulness of prediction systems for generating alerts, historically based thresholds were similarly used – weekly percentile (defined as a given percentile of the case numbers obtained in the same week) or weekly mean with standard deviation (defined as the weekly mean plus a defined number of standard deviations) algorithms as threshold levels  – but ge1. Weekly percentile, where sij T= psijQ, where psij Qrepresents the pth  percentile of observations from week i at facility s in years other than j.Threshold is exceeded when 2. Weekly mean with standard deviation., where sij T= sij μ+ Ysijβσ, where β = 0.5, 1.0, 1.5, 2.0, 2.5 or 3.Threshold is exceeded when The effectiveness of alerts generated by our four-week-ahead prediction system was compared against that of alerts based on a detection system using actual cases. Since the prediction system generates predicted numbers of cases four weeks ahead of time, this permits implementation of control measures four weeks earlier than under a detection system. On the other hand, one would expect that the accuracy of prediction might be less than that of detection. The comparisons were designed to assess this trade-off between the ability to act earlier in possible epidemics and the possible loss of accuracy.A method previously described  was usedFor each value of each type of threshold (applied to either the predicted and observed number of cases) at each health facility, the number of PPC was transformed into a proportion (percentage), by adding the number of PPC for the alerts obtained and dividing this sum by the sum, over all weeks in the data set, of the number of potentially prevented cases. Proportion rather than actual cases were used because the numbers of malaria cases vary from district to district. To compare the performance of the predicted and observed cases on a single scale, a curve was plotted for each algorithm showing the mean percent of PPC (%PPC) over all districts versus the average number of alerts triggered per year, with each point representing a particular threshold value. Better methods of generating a warning were those that potentially prevent higher numbers of malaria cases using smaller numbers of alerts.The performance of the alerts provided by both the predicted and observed cases was compared with random and optimally timed alerts. PPC was estimated for alerts chosen on random weeks during the sampling period. To estimate the performance of optimally-timed alerts , the optimal timing of alerts were identified by retrospectively going through data if one had perfect predictive ability; the optimal week for one alert was chosen; then by going through the remaining weeks, the optimal week for a second alert was chosen, and so on. The optimal alert would serve as an upper bound curve for the best choice of alert times, given a defined alert frequency .The relative importance of weather factors in determining malaria transmission significantly depends on the climate of the area. It has recently been shown that although rainfall was significantly associated in cold and hot districts, minimum temperature contributed only in the cold districts of Ethiopia . FurtherThe prediction algorithm indicates the overall pattern of cases well, yet underestimates the height of the largest peaks. Comparisons of the predicted and observed malaria cases, for each week in six of the ten districts, are shown in Figure th percentile or mean + 1.50 standard deviations). The horizontal axis gives the number of alerts per year triggered by the particular threshold value, while the vertical axis shows the %PPC associated with that threshold value. Each point represents the mean across all 10 districts. Two different choices of the function for determining PPC  and the choice of window of effectiveness  were considered. The performance of the predicted number of malaria cases using the mean plus  standard deviation algorithm (for an eight-week window of low-effectiveness) reveals that it prevented 29%/0.9 alerts, 27.3%/0.6 alerts and 24.2%/0.43 alerts per year, which compares with 31.4%/0.85 alerts, 29.8%/0.65 alerts and 27.5%/0.52 alerts per year respectively when the observed cases are used to trigger alerts  indicate that for most  districts, the model performed well qualitatively, in the sense of predicting more cases than expected from the weekly mean when such excess cases occurred, and predicting fewer when in fact fewer cases than the weekly mean occurred. This finding focuses attention on the fact that a system can give timely and accurate alerts for epidemic control, even if it is unable to provide accurate predictions of case numbers Figure . The iniStudies have shown that temperature affects transmission in cold environments more than it does in hot environments ,32. ThusNon-climatic factors such as population immunity, migration and drug resistance are believed to influence malaria transmission and have been cited as causes of malaria epidemics -36. The The model chosen for the prediction of malaria cases was based loosely on a model previously evaluated for its ability to explain seasonal variation in malaria incidence in the same data set . The forP. falciparum cases using lagged weather and case incidence data performed well in identifying periods of increased malaria cases. Furthermore, the prediction system allowed recognition of epidemic periods at an early stage, thereby facilitating interventions making epidemics preventable with adequate lead time. However, this study indicated that early warning system using weather and other predictor variables are more reliable in relatively cold than hot districts. In conclusion, it has been demonstrated that weather derived predictions identified epidemics with reasonable accuracy and better timeliness compared to early detection systems. Therefore, warning systems based on predictions derived from lagged weather variables may be a useful alternative to early detection systems for targeting resources against incipient falciparum malaria epidemics.This study showed that short-term (four-week-ahead) predictions of HDT, ML and JS conceived the study. HT and ML undertook statistical analysis. HT drafted the manuscript, which was revised by ML. JS participated in designing of the study and statistical analysis. AT initiated the study and made data available in collaboration with WHO and Ministry of Health of Ethiopia. All authors contributed to the writing of the manuscript and approved the submitted version of the manuscript.


Ambystoma mexicanum. Analysis of this data has uncovered an unusual phylogenetic distribution of the cyclin dependent kinase inhibitor 1 gene family in amphibians.An EST database has been generated for the axolotl  Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum.The ambystomatid salamander, Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians.Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. Xenopus laevis and Silurana tropicalis.The Caudata  are a major focus of work in vertebrate evolution and speciation ,2. The sDesmognathus monticola  to nearly 70 billion base pairs for Plethodon vandykei  [Ambystoma mexicanum, a species important for studies in evolution, regeneration and development, has an estimated genome size between 21.9 billion and 48 billion base pairs [A salamander EST project is particularly important as these organisms have extremely large genomes, making a genome project unwieldy and unlikely without specialized approaches such as methylation filtration . Genome amander) . The ambse pairs ,6 and mec [A. mexicanum. We chose this species because of its role in evolutionary, developmental and regeneration studies. A. mexicanum is easily bred in the laboratory, and animals can be obtained from a large, NSF-funded colony [A. mexicanum EST information. This database is also being implemented for EST sequences from a full-length X. laevis cDNA library, and for sequences from a Canis familiaris EST project.Molecular evolution studies of salamanders have relied primarily on mitochondrial genes such as those for ribosomal RNAs and cytochrome c . The lacd colony . We haveTo generate a diverse set of sequences involved in organizing and regenerating the primary body axis, two independent cDNA libraries were used for sequencing. One was derived from dorsal regions of stage 18-22 embryos containing neural tube, somite and notochord - called the 'neural tube' library - the other from 6-day post-amputation regenerating tail blastema. From 18,432 sequencing attempts 17,522 high-quality sequences were obtained after Phred analysis . All seqThe blastema and neural tube libraries were unnormalized and unamplified. We assessed library quality and diversity on the basis of the number of redundant clones in the library. Redundancy was estimated by performing BLASTN searches  against c oxidase subunit I (469 ESTs), 12S rRNA (445 ESTs), nuclear factor 7 Zn-binding protein A33 (332 ESTs), type II keratin (274 ESTs), keratin (211 ESTs) and cytoplasmic beta-actin (206 ESTs) (Table To identify ESTs belonging to the same open reading frames (ORFs), sequences were assembled into contigs using TIGR-Assembler version 2 . The 17,A. mexicanum in the National Center for Biological Information (NCBI) EST databases prior to this work, most of which originate from a sequencing effort of the Voss laboratory , as the top hit with an E-value of 4e-07. This assignment was based on the carboxy-terminal 120 amino acids of the protein, which represents the less conserved region. When we isolated a full-length clone for Am_4671 from our library, we could confirm that it is indeed the axolotl ortholog of cyclin-dependent kinase inhibitor 1B (p27Kip1), as discussed later.To identify putative homologies to known proteins, we subjected the contigs to BLASTX searches against the non-redundant protein database  where a cutoff E-value of 1e-05 was used for parsing output files. In our annotation, we used an E-value of 1e-20 as an upper limit to assign significant homology. We note that this does not imply that such sequences are true orthologs. In addition, in cases where no significant homology was found, we used an E-value limit of 1e-05 to designate weak homology. We find this additional category of 'weak homology' useful for data mining. As most contigs do not represent full-length sequences, it is possible that only a highly divergent region of a gene sequence is available in our collection. The category of weak homology allows us to find potential homologs in such situations. For example, the BLAST search for contig Am_4671 yielded the GenBank entry NP_004055, cyclin-dependent kinase inhibitor 1B (Mus musculus), rat (Rattus norvegicus), frog (X. laevis), zebrafish (Danio rerio), fugu (Takifugu rubripes), fruitfly (Drosophila melanogaster), mosquito (Anopheles gambiae), worm (Caenorhabditis elegans), newts and the yeast species Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans were collected and the closest homolog from the above species was used to assign a putative identity.Taken together, a total of 3,718 (58%) sequences shared homology with a protein from selected model organisms in the non-redundant database and could be assigned a putative identity. The E-value distribution of the top hits in the non-redundant database is shown in Figure To estimate how many of the clones are full length we examined the BLAST alignments for the position of the alignment in respect to the database sequence. Of the 3,718 sequences with homologs, 1,107 (29.8%) could be aligned in the amino terminus . As the library was poly(dT) primed, many of these clones are likely to represent full-length inserts. Of these 199 (5.4%) could be aligned from the amino terminus to the carboxy terminus and are potential full-length sequences.A. mexicanum contigs. With the remaining set of contigs, we first performed BLASTN searches against the nucleotide non-redundant (NT) database and BLASTX searches against the EST database. Finally, we performed BLASTX searches against the fugu and human proteomes. In all cases, an E-value of 1e-05 was used to assign potentially homologous sequences. Sequences in the NT database identified an additional 134 contigs and a further 220 contigs found a hit in the EST databases. A homolog was found for 3,340 (52%) contigs in the fugu proteome and 3,698 (58%) contigs shared homology with a protein from the human proteome. In total, an additional 468 contigs identified a homolog in the selected databases beyond the original assignment from the non-redundant protein database  contigs in any of the databases searched. Because the library was poly(dT) primed, many of these sequences could represent 3' untranslated regions (3' UTRs). We determined that 953 sequences (43% of non-homologous contigs) contained no ORF and were therefore potential untranslated regions. Thirty of the sequences shared homology to an existing 4% contigFrom the homologous proteins found, contigs were assigned a biological process, molecular function and cellular component from the Gene Ontology (GO) database . The cloThe largest fraction of the contigs was assigned a cellular process in the GO category biological process (87% of annotated contigs) Figure . We spliA. mexicanum. We also compared the occurrence of those domains in other vertebrate species. For most of the common protein domains, only a fraction were found in our dataset; many of these are quite abundant compared to X. laevis or Gallus gallus. The RNA-binding domains KH and RRM especially showed high abundance in our contigs. A complete list of domains is downloadable from [To identify potential domains in the axolotl contigs, we performed RPS-BLAST searches against the conserved domain database  ,18 usingble from .We assigned cellular functions to the identified domains and analyzed the output according to the functional distribution of contigs Figure . The mosin silico differential display has been performed for ESTs in the NCBI collection, and, as with the NCBI differential display data, we have assessed the statistical significance of the differences using Fisher's exact test. A total of 104 contigs met the cutoff value of 0.005 in Fisher's exact test and can therefore be considered differentially expressed.We were interested to see if there were strong differences in the sequence representation of the libraries that reflect the different biological processes taking place in each tissue. To this end, we compared the representation of ESTs in the two libraries. This type of Table A total of 168 contigs annotated under RNA metabolism  were more frequently sequenced or uniquely sequenced in blastema . This group included RNA metabolism, RNA processing, splicing, editing, nuclear export, binding, catabolism, cleavage, capping, rRNA modification, rRNA transcription and tRNA aminoacetylation. Forty-five contigs assigned a process in RNA metabolism were upregulated or unique in neural tube . After Fisher's exact test analysis, 24 of the clones were considered differentially regulated in the two libraries; 22 out of the 24 contigs were enriched or unique in blastema  were assigned as cell-cycle genes  and were more frequently sequenced or uniquely sequenced in the blastema library, compared with 52 in the neural tube library . This category included regulation of mitosis, mitosis, cell-cycle regulation, regulation of cyclin-dependent kinase (CDK) activity, cell proliferation, DNA replication, M phase, mitotic spindle checkpoint, mitotic spindle assembly, chromosome segregation and cytokinesis. As an example, 10 different types of cyclins were found, from various stages of the cell cycle. Seven of the contigs found in cell-cycle regulation met the cutoff criteria of statistical significance in Fisher's exact test. Five out of the seven contigs were more highly represented or unique in blastema Table .Whereas proliferation-associated genes were found with a higher sequence representation in the blastema library, genes that had been electronically annotated as involved in 'cell differentiation' had a higher representation in the neural tube library. A total of 28 contigs were electronically assigned the biological process 'differentiation'. After Fisher's exact test, five contigs showed differential regulation in this group. Three out of the five contigs were found in neural tube Table . Taken tX. laevis [As mentioned earlier, the Mexican axolotl is an important model organism for a number of reasons. First, it is the premier vertebrate model for studying regeneration. Second some aspects of caudate development, for instance mesoderm involution and notochord formation, more closely resemble those found in higher vertebrates than do those in other amphibian embryological models such as . laevis . FinallyKip1, the protein phosphatase Cdc25A, as well as the anaphase-promoting complex (APC) activator proteins Cdc20 and Cdh1. Representing genes involved in developmental processes, we found transcription factors such as HoxA2, B12, C4 and C8, Pax6, as well as Cdx1 and Cdx2. Furthermore we found several genes for proteins that are part of the transforming growth factor-beta (TGF-β) signaling pathway, such as TGF-β, bone morphogenetic protein 1 (BMP-1), BMP and activin membrane-bound inhibitor, activin receptor type II, as well as the transcription factors Smad5 and Smad8. Genes for proteins such as Smad8 and BMPs might be of especial interest to the research field of embryonic development, as they have been associated with mesoderm involution [A. mexicanum [We examined our dataset for genes that are potentially useful for studying regeneration features or developmental processes. To this end, we analyzed our data for genes that are either involved in regulating the cell cycle - as would be expected for the highly proliferative tissue of a regenerating body structure - or could play an essential role during development and metamorphosis from the larval to the adult stage. A list of genes that could be assigned to either cell-cycle regulation or development is shown in Table exicanum -23. We iX. laevis and the caudate A. mexicanum grouping together compared to other vertebrates such as fish, birds and mammals  and p57 (CDKN1C). In X. laevis, however, only a single family member called p28Kix1 , which shows unusual sequence features compared to the p27 sequences from any other vertebrate species, had been described in the literature [A. mexicanum harbored the 'canonical' p27Kip1 or a p28Kix1 similar to that of Xenopus. We initially searched our A. mexicanum data for CDKN1 orthologs and, in contrast to Xenopus, we found a bona fide p27Kip1 sequence that clusters closer to vertebrate p27Kip1 sequences compared to the Xenopus p28Kix1 , p27terature ,25. We w1 Figure . Considea et al. .Kix1 orthologs were only found in amphibians . We were not able to identify a p28Kix1-like gene in any other database. These p28 orthologs group as a distinct branch in an unrooted phylogenetic tree  and CDKN1C (p57) were present in the A. mexicanum databases but were not found in either X. laevis or X. tropicalis, which have far more EST and genome sequence information  and the highly derived p28Kix1 are the only CDKN1 family members found in both X. laevis and X. tropicalis. In contrast, the ambystomatids appear to have all the members of the CDKN1-family - including p28Kix1 - assuming that the p21 gene is missing purely as a result of lack of sequence information. In addition, our data suggest that p28 is an amphibian-specific variant of the CDKN1 family. Two major questions arise from these data: first, does the amphibian-specific p28 fulfill a cellular function that is unique to this phylogenetic lineage; and second, does the genotypic difference in the gene set of the CDKN1 family in the two amphibian species account for the macroscopic differences observed in developmental mechanisms. The fact that the CDKN1 family is an essential regulator of the cell cycle opens new possibilities for experimental research along these lines.The salamander, and in particular the species tebrates . This anA. mexicanum genes are homologous to genes present in other vertebrates. Sixty-six percent of contigs gave a significant match in either the non-redundant protein or nucleotide databases, the EST databases or the human and fugu protein databases. Thirty-four percent of contigs could not be assigned a homolog in any of the searched databases, and 44% of those could not be assigned a coding sequence and are therefore considered to be part of the UTR. Nineteen percent of the contigs seem to represent novel genes that have not been found in any other organism so far.Given the estimates in the number of genes present in the human genome  , we estiThe expressed sequence tags generated in this study also provide a large source of sequence information for developmental and regeneration studies. For example, an examination of the database yielded 194 genes involved in cell proliferation, including pivotal cell-cycle genes such as those for Cdc2, 10 different cyclin family members, Cdk4 and p27. A search for developmental molecules involved in intercellular communication yielded Wnt8, Wnt5B, FGF receptor 4a (FGFR4a), Sonic hedgehog, BMP receptor (BMPR) and BMP-1, while a search for homeodomain-containing proteins yielded 11 members, including Cdx1, Cdx2, HoxA2, HoxC8 and HoxB13.The ESTs were derived from two cDNA libraries, stage 18-22 embryonic neural tube/notochord/somite tissue, and day-6 regenerating tail tissue. The embryonic library represents a developmental stage where tissue specification is occurring, whereas the blastema library represents a tissue that is undergoing dedifferentiation, rapid proliferation and cell respecification. Accordingly, we find differences in transcript representation in the two libraries. The blastema library is particularly enriched in cell-cycle genes and RNA metabolism genes, presumably reflecting the high proliferative index of the early regenerating blastema.A. mexicanum was generated to provide a comprehensive sequence dataset for the community of biologists. Forty percent of genes could still be found in singlets, which reflects a high diversity of sequences in our cDNA set. Annotation of the assembled contigs revealed a substantial difference in gene representation in the two sequence libraries, reflecting their biological source - regenerating blastema being in a highly proliferative state and embryonic neural tube being a tissue undergoing differentiation. Sequence analysis of assembled contigs revealed that 64% of genes had a putative homolog in other species; 19.4% of the contigs contained a putative coding sequence and can be considered novel genes. From this, we conclude that A. mexicanum does not contain an unusually high number of organism-specific genes. The CDK inhibitor family CDKN1 was selected for comparative phylogenetic analysis. Unlike the frogs X. laevis and X. tropicalis, ambystomatids most probably contain all members of the CDKN1 family, including the amphibian-specific protein p28Kix1/p27Xic1, which shows unusual sequence divergence compared to CDKN1 members in other vertebrate species. Such data would support the contention that A. mexicanum is closer to a basal tetrapod compared to X. laevis. The EST sequences and annotated contigs presented in this paper will be a publicly available and useful resource for research in various fields.This set of 17,352 ESTs from A. mexicanum embryos. Total RNA quality was assessed by determining the relative brightness of the 28S:18S rRNA bands (2:1). For library construction mRNA was purified and size fractionated, then poly(dT)-primed cDNA was synthesized and directionally cloned into the NotI-SalI sites of the pCMVSport6 vector. DNA was transformed by electroporation into EMDH10B-TONA bacteria (library construction performed by Invitrogen). Two separate, unnormalized libraries were produced. The blastema library contained an average insert size of 1.67 kb and 2.67 × 107 independent transformants and the neural tube library had an average insert size of 1.5 kb and 1.9 × 107 transformants. From each library 100,000 clones were arrayed into 384-well plates .Total RNA was purified using Trizol (Invitrogen) from 6-day regenerating tail blastemas and from neural tube-somite-notochord-containing tissue dissected from stage 18-22 For sequencing, single-pass reads from the 5' end of the library inserts were performed using a custom-designed SP6 primer: GCACATTAGGCCTATTTAGGTGACA. DNA from bacterial library clones was amplified using the Templiphi reaction, based on φ29 rolling-circle replication of DNA (AP Biotech). Briefly, approximately 0.5 μl of bacterial glycerol stocks were picked up using 96-pin plastic replicators (Genetix) and centrifuged into 96-well PCR plates. Five microliters of denaturing buffer was added, and samples heated to 95°C for 3 min. After cooling, 5 μl Templiphi enzyme was added and samples incubated overnight in a 30°C incubator. The Templiphi reaction provides two advantages for large-scale sequencing projects on capillary sequencers. First, the reaction proceeds to an endpoint where all nucleotide is incorporated, yielding uniform quantities of DNA from varying amounts of starting bacteria (or DNA). Second, the rolling-circle reaction results in large pieces of DNA that, in contrast to plasmid DNA, do not enter the capillary and interfere with the sequencing run.For sequencing reactions, the DNA preparation was diluted fivefold with distilled water. Sequencing reactions were performed using the DYEnamic ET Dye terminator kit diluted twofold with DYEnamic ET dilution buffer (AP Biotech). Five microliters of DNA was added to 5 μl of sequencing reaction mix with primer and cycled 30 times under the following conditions: 95°C 20 sec, 60°C 1 min. Sequencing was performed on a MegaBACE 1000 (AP Biotech). Runs were either performed at injection: 3 kV 60 sec, run: 8 kV 120 min, or injection: 3 kV 60 sec injection, run: 3 kV 360 min.The redundancy of the arrayed libraries was tested by performing BLASTN searches  against The sequences were submitted to GenBank. After quality control, individual ESTs were used to search the non-redundant protein database (release of July 2004) using the program BLASTX from the standalone NCBI-BLAST package . For ann2), the base would be false, 30 would reflect a wrongly sequenced base in 1 of 1,000 trials (103), and so forth.) Sequence and contig files can be downloaded at [Quality control of sequenced ESTs was performed using the program Phred  using a oaded at . The resoaded at . Alignmeoaded at  or Cap3 oaded at ) againstoaded at ) from thoaded at .A. mexicanum contig was an E-value of 1e-20.Based on the GO annotation of the closest annotated homolog, contigs were assigned a molecular function, biological process and cellular component from the GO database . To thisKip1 gene were sequenced in the EST collection but neither were full-length sequences. To isolate the full-length sequence, 200,000 clones of our arrayed blastema and neural tube libraries were screened by PCR. Briefly, the bacterial library clones in each 384-well plate were pooled, mini-prepped and arrayed into 96-well plates , resulting in 576 DNA pools. These DNA pools were screened by PCR using the custom SP6 primer (GCACATTAGGCCTATTTAGGTGACA) as a forward primer and a gene-specific p27 reverse primer (TGATTTCCAATGGCTGGTTT). Fifty nanograms of DNA from each pool was used for PCR reactions and PCR cycling was performed at the following conditions: 94°C 2 min, 30 cycles of 94°C 15 sec, 65.5°C 30 sec, 72°C 90 sec, followed by 72°C 7 min). The largest positive band (1.1 kb) was gel purified and sequenced on an ABI377 machine using the SP6 primer.Two EST sequences of the p27A. mexicanum contigs was used for tree construction. Trees were displayed using the program nj-plot [Multiple sequence alignments were done with the program ClustalX  using st nj-plot  for the  nj-plot  for the A relational database was created using the open source software MySQL as the database server to store and navigate through resulting sequence contigs and annotations. Scripts connecting the web-based front end to the database were written in the programming language Python.