Coccidioides in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in Coccidioides infection are not known.Coccidioidomycosis or Valley Fever is caused by Coccidioides posadasii infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of C. posadasii (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays.In this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of C. posadasii infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % (SEM 3.5, p < 0.001), 38 % (SEM 5.8, p < 0.001) and 4 % (SEM 1.3, p < 0.001) of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of C. posadasii protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens.We found that in lavage fluid samples of C. posadasii infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.Our results suggest that the  Coccidioides immitis or posadasii [C. posadasii or C. immitis, are the most virulent fungal pathogens enlisted in Select Agent list and pose a risk for bioterrorism [Coccidioides [Coccidioides.Coccidioidomycosis or Valley Fever is a fungal disease caused by the biphasic, highly virulent, soil-fungus osadasii . It is eosadasii . C. posaerrorism . The prierrorism -4. Previidioides ,6. HowevStreptococci [Pseudomonas aeruginosa [At alveolar level in the lung, the innate immune system is composed of many cell types and chemical mediators, including surfactant. The pulmonary surfactant is a complex mixture of lipids 88–90%) and proteins 10–12%), synthesized by type II epithelial cells and Clara cells. It lines the alveoli, and helps in maintaining normal lung function . Among f8–90% and–12%, synptococci , Pseudomruginosa . Also, iruginosa . Both SPruginosa . Recent ruginosa . HoweverC. posadasii and C. posadasii protected mice after immunization with protective formalin killed spherule (FKS) vaccine, and binding of pulmonary collectins to Coccidioidal antigens.This study focuses on analyzing the changes in amounts of the SP-A and SP-D in the bronchoalveolar lavage fluid (BALF) samples from mice infected with lethal dose of C. posadasii infection. The BALB/c mice were used to study the changes in pulmonary surfactant after intranasal challenge with C. posadasii. And, the C57BL6 mice were used to study the changes in pulmonary surfactant after vaccination with protective FKS vaccine. Mice were housed in Biosafety Level-3 animal facility at UTHSCSA and provided with food and water ad libitum. All experimental animal care and treatment protocols were reviewed and approved by Institutional Animal Care and Use Committee.BALB/c and C57BL6 mice  from Jackson Laboratory (Bar Harbour, ME) were used in this study. Both mouse strains are susceptible to C. immitis (now posadasii) Silveira strain, cultured on 1 % glucose-0.5 % yeast extract agar (GYE), was used for infecting the mice [C. posadasii were carried out in Biosafety Level-3 facility at UTHSCSA.C. posadasii arthroconidia) suspended in endotoxin-free 0.15 M NaCl  using sterile pyrogen-free microtip. Mice were held in an upright position for 1–2 min to resume normal breathing after injection. Control mice were challenged with equal volume of endotoxin-free 0.15 M NaCl.Mice were anaesthetized after intramuscular injection of ketamine- xylazine (75 μg/g body weight ketamine and 10 μg/g body weight xylazine) and were then challenged intranasally with a lethal dose of arthroconidia (n = 30, fresh harvest of C. posadasii (strain Silveira)arthroconidia were used to prepare FKS as described earlier [2 incubator. The spherules were collected from the harvested culture, washed in endotoxin-free water and killed with 1 % formalin. FKS preparation was checked for sterility and lyophilized. C57BL6 female mice (age 6 weeks old) were immunized intramuscularly twice and subcutaneously once at one week interval with FKS (0.7 mg/dose each time). The mice in FKS immunized, infected group were then challenged intranasally with 30 C. posadasii arthroconidia, 15 days after last immunization, as described above. earlier . BrieflyMice were anaesthetized as mentioned above, prior to sacrifice on day 10, post intranasal infection. This standard procedure was used for intranasal injection since it does not cause respiratory depression during anaesthesia. The lung and spleen tissues were collected in sterile 0.15 M NaCl for studying fungal load.The fungal burden was studied by plating ten fold dilutions of lung and spleen homogenates in 0.15 M saline on Mycosel agar plates (BD Biosciences, Franklin Lakes, NJ) and incubating for 72 h at 30°C. The cfu counts were recorded and normalized with organ weight.At the time of necropsy, we collected BALF by injecting 1 ml endotoxin-free 0.15 M NaCl solution  three times, via an angiocatheter (BD Biosciences, San Diego, CA) placed in the trachea. The volume of the input solution was kept constant  and approximately, 90–95 % of the solution was recovered consistently. The BALF was centrifuged at 500 rpm for 10 min at 4°C to remove cells. The cell free BALF supernatant was filtered through 0.2 μm syringe filters  and stored at -80°C for further analysis.The total protein concentration was measured in BALF specimens using micro bicinchonic acid protein assay kit (Pierce, Rockford, IL) against bovine serum albumin (BSA) standard protein. The total phospholipid content in lipid extracts of BALF specimens was determined using the method of Stewart, against Dipalmitoyl-phosphatidylcholine (DPPC, Avanti Polar Lipids, Alabaster, AL) standard solutions ,18. BrieThe BALF and lung tissue homogenate samples  were run on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) running gel and transferred on nitrocellulose membranes (Schleicher & Schuell, Keene, NH) overnight at 15 mA current. The nonspecific sites on the membrane with transferred proteins were blocked by 15% nonfat milk in Tris-buffered saline containing 0.05% tween 20 (TBST). The membrane was washed and incubated for 1 h with diluted (1:500) primary anti-human SP-A polyclonal antibody raised in rabbit (obtained from Dr. Richard J. King, UTHSCSA, San Antonio, TX) or anti-mouse SP-D antibody . After washing the membrane with TBST, the membrane was incubated for 1 h with 1:10,000 diluted alkaline phosphatase conjugated anti-rabbit IgG raised in goat . The immunoreactive bands were observed by alkaline phosphatase conjugate system (Biorad, Hercules, CA). Purified human SP-A (kindly provided by Dr. Richard J. King, UTHSCSA, San Antonio, TX) and recombinant human SP-D (kindly provided by Dr. Erika C. Crouch, Washington University in St. Louis, St. Louis, MO) were run with the samples.C. posadasii mycelia , prepared as a toluene-induced lysate of young nio, TX, ) were al3, pH 9.6. The wells were washed three times with deionized water, and nonspecific sites were blocked with a buffer containing 0.25% BSA, 0.05% tween 20, 0.17 M boric acid and 0.12 M NaCl, pH 8.5. The wells were washed and incubated for 2 h with rabbit anti-human SP-A or rabbit anti-mouse SP-D antibody. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent . The reaction was stopped by adding 50 μl of 2 N H2SO4 and read at 450 nm spectrophotometrically. The regression coefficient for a least-square linear fit to the standard curve of SP-A and SP-D was 0.99. The limits of detection for SP-A and SP-D were 2 ng/ml.The concentrations of SP-A and SP-D were measured in BALF samples as described earlier . The ant3 buffer, pH 9.6) or CDN-filtrate (10 μg/ml diluted in 0.1 M NaHCO3 buffer, pH 9.6) or BSA (10 μg/ml diluted in 0.1 M NaHCO3 buffer, pH 9.6) at room temperature. The nonspecific binding was blocked with phosphate buffered saline (pH 7.4) containing 0.1% triton-X 100 and 3% nonfat milk (buffer A). The purified human SP-A and recombinant human SP-D diluted in 20 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2 and 1 mg/ml BSA were then added to the wells and incubated for 3 h at 37°C. The wells were then washed with buffer A and incubated for 1 h at room temperature with diluted (1:1000 in buffer A) anti-SP-A and anti-SP-D antibodies. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent . The reaction was stopped by adding 2 N H2SO4 and read at 405 nm spectrophotometrically.A microtiter well based method  was usedThe coating of Coccidioidal antigens (CDN-lysate and CDN-filtrate) to the plates was confirmed using a positive control antibody that recognizes Coccidioidal antigens as described earlier . The alkStatistical analyses of the data (t-test or ANOVA) were done using Prism Software (Graphpad Software, San Diego, CA). The p value <0.05 was considered significant.C. posadasii infected mice survived till the day of sacrifice (day 10 post infection). However, the mice were lethargic and lost body weight (Table C. posadasii infected mice.All of the C. posadasii infected mice (58 μg) when compared to non-infected saline injected controls (165 μg, p < 0.05).The mean protein content of BALF samples from infected mice was 788 μg versus 326 μg protein in BALF samples from non-infected saline injected control mice, after 10 days of intranasal infection  .The anti-human-SP-A and anti-mouse SP-D antibodies recognized 34 kDa and 43 kDa monomer bands of SP-A and SP-D in BALF and lung tissue samples Fig . The upp001) Fig . No signC. posadasii were recovered from both lung and spleens of infected animals indicating the presence of active infection  C57BL6 mice when compared to FKS immunized, non-infected mice  versus FKS immunized, non-infected mice (538.4 μg).No significant changes were seen in SP-A or SP-D levels in BALF samples of protected  coated onto microtiter wells Fig . Both SPC. posadasii infected mice, but were intact in lungs of C. posadasii protected mice after immunization with protective FKS vaccine. Furthermore, our results suggest that both SP-A and SP-D bind to Coccidioidal antigens. This is the first study where the amounts of SP-A and SP-D were measured in BALF samples from mice infected with lethal dose of C. posadasii and the binding of pulmonary collectins to Coccidioidal antigens was assessed. Since lung is the first organ of the body that comes into contact with air-borne C. posadasii arthroconidia, we hypothesized that the pulmonary surfactant may play an important role in regulating the immune response against C. posadasii. Among four surfactant proteins, SP-A and SP-D, interact with most of the clinically important fungal pathogens including Pneumocystis carinii [Cryptococcus neoformans [Aspergillus fumigatus [Candida albicans [C. albicans and directly inhibit growth by aggregation of the organism without involvement of macrophage dependent phagocytosis [A. fumigatus conidia and C. neoformans by alveolar macrophages and neutrophils [A. fumigatus [C. posadasii infection versus the unaltered amounts in the lungs of protected mice and binding of collectins to Coccidioidal antigens indicate that pulmonary collectins may be involved in uptake/phagocytosis of C. posadasii by antigen presenting cells and downstream immune regulation.In the present study we found that the levels of pulmonary surfactant collectins were altered in the lungs of  carinii Cryptococoformans , Aspergiumigatus  and Candalbicans . SP-D haocytosis . On the trophils ,29. In sumigatus . The decC. posadasii infected mice  and growth factors are released during infection and inflammation. These cytokines and growth factors affect the synthesis and secretion of pulmonary surfactant by pulmonary epithelial cells [C. posadasii protected mice, but not of SP-A (Fig Besides changes in amounts of SP-A and SP-D, a decrease in the amount of BALF phospholipids was also observed in ce Table . Earlier carinii ,30. To d carinii . Likewisal cells ,33. In tSP-A Fig . We specC. posadasii infected patients with disseminated disease is not very effective [Coccidioides infection in murine model of coccidioidomycosis, but failed in humans [C. posadasii and C. immitis are highly virulent organisms, cause endemic infection, and pose a risk for bioterrorism, there is an urgent need for discovery of improved therapeutic drugs and regimens or preventive vaccines [At present, the treatment of ffective . Treatmen humans . More prvaccines ,35.in vivo administration of artificial or natural surfactant in C. posadasii infected mice may be useful in determining their therapeutic usefulness. We speculate that the findings from our present study would initiate similar studies to understand the role of pulmonary innate immune components in infectious diseases caused by C. posadasii or other virulent respiratory pathogens.In future, clearance experiments after SA designed and co-ordinated the study, performed assays and statistical analysis and drafted the manuscript. JJC assessed lung pathology. DMM prepared and immunized mice with FKS. All authors read and approved the final manuscript.


People are unpredictable. One night you may crave Italian food, but another only Thai will do. One day you might finish a crossword puzzle in record time, and the next not a single clue prompts an answer. Such behavioral variation has been found in laboratory studies, too: a person's ability to find a faint image on a screen varies widely from one viewing to the next. Similarly, when an animal repeatedly receives the same stimulus—for example, a faint image—a neuron in a region of the animal's visual brain might be very active upon one presentation and relatively quiet the next.Across the cerebral cortex—the brain region that integrates the senses and controls voluntary movement—neurons are notorious for their unpredictable behavior. The neurons themselves don't create this noise; when directly stimulated with an electrode multiple times, neurons will give the same response every time. Most neurons, however, receive signals from a host of other neurons. These various signals combine to form a seemingly noisy electrical input, which shows up as fluctuations in the recipient neuron's membrane potential—a difference in electrical charge between the inside and outside of the cell's membrane. Neuron function is intimately tied to the membrane potential, which is usually maintained within a narrow range, called the resting potential. But incoming signals can push the resting potential higher or lower. If the membrane potential rises above a certain threshold, the neuron fires, sending an electrical signal down its length. In this way, the brain relays and processes information.PLoS Biology, Matteo Carandini addresses this longstanding mystery of neuron variability and comes up with a different answer. Carandini simultaneously measured the membrane potentials and firing patterns of individual neurons in the cat visual cortex. He found, surprisingly, that the membrane potentials varied much less than the firing patterns, ruling out noisy inputs as the cause of neurons' noisy outputs. Instead, the neurons amplified noise in the signals they received.Since the 1960s, neuroscientists trying to account for the cortex's variable responses have pointed to noisy inputs from other parts of the brain as the prime suspect. In this issue of Carandini then used a simple model of neuron behavior to explain why this would occur. He started with a tried-and-true approximation of neuron behavior, called the rectification model: a neuron doesn't fire until its membrane potential rises above a threshold, but once it crosses this threshold, its firing rate is correlated with the strength of incoming signals. Then he added the assumption that the neurons receive signals with some randomness. Given these minimal assumptions, Carandini showed that neurons fed a noisy signal will tend to amplify the noise in the signal. Importantly, his model reproduced a well-known phenomenon: as cortical neurons' average firing rate goes up, their firing rate also becomes more variable—that is, they get noisier.Carandini's model also predicted something new: as the firing rate continues to increase, the firing rate should become more consistent and less noisy—which he calls saturation of variability. Carandini's measurements in cats showed neurons actually behave this way, a key validation of his model.It's not clear whether this amplification of variability is something that helps or hampers the brain. Despite being a nuisance to neuroscientists, such fluctuations could be crucial to how the brain functions, Carandini speculates. Without some variability in their cortex, animals would act like cameras or other simple machines that respond the same way each time to a stimulus. It's advantageous for behavior, and hence brains, to be adaptable. But amplifying noise in a signal seems to run counter to relaying and processing the information in the signal. Carandini suggests that what appears as noise in the experiments are signals from other parts of the cortex—that is, noise is in the eye of the beholder. Now that the source of the variability is clear, neuroscientists can study whether it serves a function in the brain.


Lamptey and Wilson discuss the implications of a new study showing that combining treatment with prevention is the best approach to tackling the HIV pandemic. There has been a recent and dramatic rise in global funding for HIV/AIDS, from US$2.1 billion in 2001 to US$6.1 billion in 2004 [The WHO “3 by 5” Initiative: This initiative aims to place 3 million people in low- and middle-income countries on ART by the end of 2005.The US President's Emergency Plan for HIV/AIDS Relief: This initiative aims to treat 2 million HIV-infected people with ART, to prevent 7 million new infections, and to care for 10 million HIV-infected individuals and AIDS orphans in five years (2004–2009).The Global Fund to Fight AIDS, Tuberculosis, and Malaria: In the five years following its inception (2002–2007), the Global Fund aims to provide 1.6 million people with ART and 52 million people with VCT, and to support more than 1 million orphans with medical services, education, and community care.The scale of the proposed increase in the number of patients receiving ART raises numerous questions about the treatment itself. Which drugs will be used? How much will it cost? How will their quality be monitored and assured? How will they be distributed? Who will be eligible? How will the desired level of treatment be sustained? Is there adequate infrastructure and human resources to support the expanded services?The commitment of substantial funding to treatment in resource-poor countries also has implications for the prevention efforts in those same countries. In many Western countries and Brazil (the sources of the majority of the available data on the subject), the impressive drop in mortality due to HIV following increased access to ART is coupled with a disheartening rise in the number of new cases of HIV, as emphasis and funding are shifted from prevention to treatment . CountriOf course, prevention and treatment are not mutually exclusive. Successful prevention efforts mean fewer patients will need the costly drug treatment programs, helping extend the sustainability of ART. In turn, the success of ART in prolonging healthy living helps prevention efforts by reducing the stigma associated with self-education and responsible behaviors.PLoS Medicine, “Integrating HIV Prevention and Treatment: From Slogans to Impact,” Salomon and colleagues use mathematical modeling to assess the epidemiologic impact of treatment and prevention efforts, and to quantify the opportunities and potential risks of large-scale treatment roll-out. Using a variety of different scenarios, they propose methods for establishing the most effective balance between spending on prevention and spending on treatment.In their study in the January 2005 issue of Modeling is a technique used by many scientists, including epidemiologists and statisticians, to create a mathematical equation that can be used to determine which variables affect an outcome of interest, and to what extent. Once the influential variables are determined, a baseline model is established that includes those variables and reflects their relative importance to the outcome. The effect of changing the value of any of these variables, or several of them, can then be tested, and new outcomes projected. HIV modeling is inexact and requires far better data but can nevertheless provide important insights.Salomon and colleagues used mathematical modeling to assess the effect of changing aspects of the HIV/AIDS “equation” on the future course of the HIV/AIDS epidemic. First, a baseline model was created to fit expected HIV/AIDS projections for the year 2020 if there were to be no change in the current epidemiologic trends—no ART scale-up, and no changes in prevention efforts or behavior. Heterosexual contact is the predominant mode of HIV transmission across Africa, and Salomon and colleagues' study modeled the disease only within the heterosexual population. The model was also tailored to take into account epidemiologic, demographic, and sociologic patterns in the eastern, central/western and southern regions of Africa. Using the baseline models tailored to each region, the effects of prevention and treatment efforts were then measured.Two treatment-centered scenarios were tested in which the World Health Organization's “3 by 5” initiative see  was achiBaseline projections in Salomon and colleagues' study showed that without any behavioral change or ART scale-up, the HIV/AIDS prevalence rate would remain relatively stable, but the number of new infections would increase by 52.3 million by 2020. Treatment-centered scenarios reduced the total number of new infections through 2020 by a maximum of 3 million, or 6%, while indicating that the number of AIDS deaths through 2020 would decline by 13%, to 32.4 million. A prevention-centered strategy would provide greater reductions in incidence (36%) and similar mortality reductions by 2020, but more modest mortality benefits over the next five to ten years.The scenarios in which all of these statistics were most improved, however, were those that combined both prevention and treatment efforts. In the scenario in which treatment enhanced prevention, Salomon and colleagues projected 29 million averted infections (55%) and 10 million averted deaths (27%) through the year 2020. However, if a narrow focus on treatment scale-up leads to reduced effectiveness of prevention efforts, the benefits of a combined response would be considerably smaller—9 million averted infections (17%) and 6 million averted deaths (16%) .Combining treatment with effective prevention efforts could reduce the resource needs for treatment dramatically in the long term. In the various scenarios the numbers of people being treated in 2020 ranges from 9.2 million in a treatment-only scenario with mixed effects, to 4.2 million in a combined response with positive treatment–prevention synergies.The authors have demonstrated through mathematical modeling that the integration of treatment and prevention is epidemiologically sound. However, an integrated and comprehensive program  is not oEffective prevention makes treatment more affordable and sustainable. Effective prevention can lead to a substantial reduction in the number of new infections and therefore ultimately will lead to a reduction in the number of people who will need treatment. The reduction of adult HIV/AIDS prevalence in Uganda from 18.5% to 6% over the last several years has reduced the number of those eventually needing treatment by nearly 68% [arly 68% . Unless arly 68% . SalomonSuccessful treatment and care can make prevention more acceptable and effective. Widespread access to treatment could bring millions of people into health-care settings, providing new opportunities for health-care workers to deliver and reinforce HIV prevention messages and interventions [ventions . Improveventions .Prevention can make treatment more accessible. The early establishment of community-based prevention services in rural Ghana was instrumental in reducing the stigma of AIDS and improving the knowledge and attitude of the community prior to the development of ART and VCT services . This process also made it easier for community and implementing agencies to identify and refer patients needing treatment services.Expanded care and prevention activities have synergistic effects. Continued effective treatment, care, and prevention programs will reduce the number of orphans and vulnerable children, reduce mother to child transmission of the virus, and improve the lives of families and the strength of communities.Integration ensures that prevention activities are not neglected. The world has a unique opportunity, as ART services are launched and expanded, to simultaneously bolster prevention efforts [ efforts . Experie efforts . In addiIntegration can provide opportunities to address vulnerable groups more effectively. A commitment to providing large-scale treatment helps to focus attention on communities at greatest risk, particularly in lower prevalence contexts. This provides an opportunity to address the prevention and treatment needs of vulnerable groups more effectively.Treatment resources can help improve infrastructure for prevention and other health services. The training of health providers and improvements in laboratory services, pharmacy, logistics, commodity management, and health information systems can benefit both treatment and prevention services. Further, in many countries, a large number of health-care workers are themselves infected. Treatment can help to preserve the lives and productivity of these critically needed AIDS prevention and treatment workers, as well as those of other health professionals.A long-term decline in AIDS deaths may be preventing new infections. The short-term decline in AIDS deaths is driven by effective care and treatment programs, but a long-term decline may be driven by the prevention of new infections. Integrated and comprehensive strategies are more likely to lead to affordable, sustainable programs.Success requires dramatic expansion of both ART and prevention. Globally, fewer than one in five people at high risk of infection have access to proven HIV prevention interventions [ventions  and lessventions . Unless The results of Salomon and colleagues' model need to be validated. Further operational research is needed to validate the findings of this study.


Imbalance in the health workforce is a major concern in both developed and developing countries. It is a complex issue that encompasses a wide range of possible situations. This paper aims to contribute not only to a better understanding of the issues related to imbalance through a critical review of its definition and nature, but also to the development of an analytical framework. The framework emphasizes the number and types of factors affecting health workforce imbalances, and facilitates the development of policy tools and their assessment. Moreover, to facilitate comparisons between health workforce imbalances, a typology of imbalances is proposed that differentiates between profession/specialty imbalances, geographical imbalances, institutional and services imbalances and gender imbalances. Imbalance in the health workforce is a major challenge for health policy-makers, since human resources – the different kinds of clinical and non-clinical staff who make each individual and public health intervention happen – are the most important of the health system's inputs . ImbalanAlthough imbalance in the health workforce is an important issue for policy-makers, various elements contribute to obscuring policy development. First, many reports of shortages are not borne out by the evidence. Rosenfeld and Moses  show thaThe objective of this paper is to contribute to a better understanding of the issues related to imbalance through a critical review of its definition and nature and the development of an analytical framework.There are various approaches to defining imbalances . From anOne of the key questions regarding imbalances is how long these last: Is the imbalance temporary or permanent? In a competitive labour market, we should expect most imbalances to be resolved over time. Imbalances will tend to disappear faster the greater the reaction speed and also the greater the elasticity of supply (or demand) . This tyIn contrast, a static imbalance occurs because supply does not increase or decrease; market equilibrium is therefore not achieved. For instance, wage adjustments may respond slowly to shifts in demand or supply as a result of institutional and regulatory arrangements, imperfect market competition (monopoly, monopsony) and wage-control policies. Another example is physicians' education: because of the length of time required to educate physicians, changes in available supply take a long time to react significantly. Lack of information on the state of the various labour markets can also be a factor in the speed of market adjustment. To make proper labour market decisions, households and firms must be informed of the existing market conditions across markets. They must therefore know what wages are paid and the nature and location of job openings and available workers.Moreover, we should also differentiate between qualitative and quantitative imbalance. In a tight labour market, employers might not find the ideal candidate, but will still recruit someone. Under these conditions, the issue is the quality of job candidates rather than the quantity of people willing and able to do the job . From thTo better understand the role of factors affecting health workforce imbalances and to facilitate the development of policy tools, a conceptual framework is presented in this section.Factors affecting health workforce imbalances are numerous and complex, but focusing on crucial elements should permit insight into the issue of health workforce imbalances. The framework is depicted in Fig. Central to this framework are the demand for and supply of health labour. Also included in the framework is the health care system, and in particular, some of its features that are likely to have an impact on health workforce imbalances. Policies constitute another crucial element of the framework. In effect, health policies but also non-health-oriented policies can have an impact on health workforce imbalances. The framework also incorporates financial, physical and knowledge resources that contribute to model the health workforce.Finally, "global" factors such as economic, sociodemographic, political, geographical and cultural factors are included. These elements contribute directly or indirectly to shaping and transforming the entire society and hence the health workforce.The first element of the framework to be examined is the demand for health personnel. The demand for health personnel can be considered as a derived demand for health services. Accordingly, we should consider factors determining the demand for health services. Personal characteristics – such as health needs, cultural and sociodemographic characteristics – and economic factors play an important role.It has often been proposed that the planning of human resources for health be based solely on estimates of health needs in the population . HoweverSeveral studies have attempted to estimate the impact of economic factors on the demand for health care. In particular in the United States, studies have attempted to estimate price and income elasticities of demand for medical services -13. MeasAnother element influencing the demand for health care is the value of a patient's time, such as travel time and waiting time. Acton  found thOther factors affect the demand for health labour. In particular, some specific features of the health care system and its features, policies, resources and environmental factors do have an impact on the demand for health labour. Their respective role will further discussed later.After reviewing factors affecting the demand for health labour, we shall now turn to those affecting the supply of the health workforce. In particular, we shall consider the following elements: factors affecting the choice for a health professional training/education, participation in the health labour market and migration.The availability of a renewed health workforce, as well as the type of profession and specialty chosen by individuals, is a major concern for health decision-makers. These issues are of particular relevance, especially since the number of younger people, predominantly women, choosing a nursing career is declining in some countries and since in professional training/education, individuals' choices do not always match the absorptive capacity of the market.From an economic perspective, the decision to undertake professional training/education is considered an investment decision. To emphasize the essential similarities of these investments to other kinds of investments, economists refer to them as investment in human capital . Since iBased on the human capital approach, rate of return on education can be estimated. An average rate of return that is high and rising for a given profession will attract more individuals to that profession. On the other hand, a lower and decreasing average rate of return will discourage individuals from choosing that profession. Nowak and Preston , using tBesides the human capital approach, the choice of a profession can also be explained by sociopsychological factors. For instance, individuals may choose a profession because it is highly valued by the society or for family tradition. In the health sector, the satisfaction afforded by caring for people and assisting them to improve their health is an important element used by nursing schools to attract new enrollees. In the light of this approach, the decline in the number of individuals choosing nursing as a career might also be explained by the fact that this profession is now less socially valued than before ,20.The economic theory of the decision to work views the decision as a choice concerning how people spend their time. Individuals face a trade-off between labour and leisure. They decide how much of their time to spend working for pay or participating in leisure activities, the latter being activities that are not work-related.An issue that has drawn a lot of attention recently is the impact of wage increases on labour participation, in particular for nurses. In the short term, higher wages can have at least two effects on the labour supply of current qualified nurses: first, qualified nurses who are working in other occupations may return to nursing activities; second, nurses now in practice may respond by working more hours. In the long run, higher wages in nursing relative to other occupations make nursing an attractive profession and will draw more people into nurse training programmes.In their literature review of wage elasticity of nursing labour supply, Antonazzo et al.  and ChihMoreover, hospitals are also using a variety of strategies to recruit new staff. A survey of hospitals in the United States shows that richer benefits, such as health insurance and vacation time, are the most common incentives used. In addition, hospitals may offer other recruitment and retention benefits, such as tuition reimbursement, flexible hours and signing bonuses based on experience or length of commitment . Many coEconomic factors also play a role in physician's participation in the labour market, as demonstrated by the impact of cost-containment policies in Canada, where most provincial governments have implemented an assortment of controls of health care expenses. Threshold reductions were introduced, so that fees payable to individual physicians were reduced as billing exceeded an agreed threshold. As a consequence, physicians who had billed at the threshold level chose to take leaves of absence rather than receive a level of reimbursement they considered inadequate .When health personnel choose an alternative or additional occupation, this is likely to have consequences on health labour supply. In developing countries, and particularly in Africa, attempts to reform the health care sector have frequently failed to respond to the aspirations of staff concerning remuneration and working conditions. Salaries are often inadequate and may be paid late, and health workers try to solve their financial problems in a variety of ways . PrivateParker and Rickam  examinedThe relative importance of wage is also emphasized by studies investigating job satisfaction. There is support in the empirical literature for the existence of job dissatisfaction among nurses, and the link between job dissatisfaction and job exit ,31. In tMigration of health personnel can have a serious impact on the supply of human resources in health, because it may exacerbate health personnel imbalances in "sending" countries. It is suggested that migration is an "individual, spontaneous and voluntary act" that is motivated by the perceived net gain of migrating – that is, the gain will offset the tangible and intangible costs of moving . DecisioThe reality for many health workers in developing countries is to be underpaid, poorly motivated and increasingly dissatisfied and sceptical . The relAs the health workforce is part of the health care system, we shall also consider features of the health care system that are likely to have an impact on the demand and supply of health labour. In particular, we shall examine market failures, the diversity of stakeholders, the supply-demand adjustment time lag and hospitals' potential monopsony power.From an economic perspective, the health care market is characterized by market failures – that is, the assumptions for perfect competition are violated. From a societal perspective, in the presence of market failures such as externalities – imperfect knowledge, asymmetry of information and uncertainty – market mechanisms lead to a non-optimal demand and/or supply in health services. In other words, shortages and surpluses are likely to result from the health care market.Most markets are characterized by market failures, but what is unique to the health services market is the extent of these market failures . GovernmThe health care system is characterized by a wide range of institutional stakeholders involved in shaping human resources for health, all of whom may have different objectives ,42. The In the case of Mozambique, whereas the policy of employing national professionals by cooperation agencies has met with warm support from national cadres, its effect on the health sector is problematic . The proMoreover, adjustments between the demand and supply for health personnel may take a long time. In the health care field; the time lag between education and practising may be quite substantial. To obtain licensure to practise medicine requires lengthy education and training, and the long lag time between a changed student intake and a change in supply has been noted : supply monopsony. One example is the potential monopsony power of hospitals in hiring nurses or the ministry of health in hiring the health workforce. The amount of labour demanded will influence the price the monopsonist must pay for it. In contrast to the situation in a competitive market, the monopsony is a price maker, not a price taker. Monopsony results in lower wages and lower employment of nurses compared to a competitive market.A single entity that is the sole purchaser of labour is a A number of studies have tested whether or not hospitals possess monopsony power with respect to nurses, and the results are contradictory. Sullivan  and StaiIn contrast, providers' power may enable the latter to restrict the supply of human resources for health. Seldon, Jung and Cavazos  suggest The type off regulation associated with a profession plays an important role regarding the supply of members of a profession. Regulation has, by tradition, been achieved through a combination of direct government regulation and, to a large extent, through rules adopted by professional associations, whose self-regulatory powers enable them to establish both entry requirements and rules regarding professional conduct .Such barriers to entry exist in particular for doctors, but also in other health professions, such as dentistry. Some argue that these barriers constitute a means to limit entry into the profession, and hence maintain high incomes. Muzondo and Pazderka  establisHealth and non-health policies contribute to shaping the health care system and have an influence on the demand and supply of health labour.Health policy can be defined as a formal statement or procedure within institutions (notably government) that defines priorities and the parameters for action in response to health needs, available resources and other political pressures. Health policy is often enacted through legislation or other forms of rule-making that create regulations and incentives for providing health services and programmes and access to them. For instance, the decision to introduce or expand health insurance coverage is likely to have an impact on the demand for health services. This is illustrated by the RAND Health Insurance Experiment, a controlled experiment that increased knowledge about the effect of different insurance copayments on use of medical services. Insurance copayments ranged from zero to 95%. The RAND study concluded that as the co-insurance rose, overall use and expenditure fell for adults and children combined .Non-health policies reflect state interventions in areas such as employment, education and regional development that contribute to shaping the health workforce. These policies do not directly address health issues, but have an indirect impact on such issues. In France, a controversial new regulation was introduced that reduced the workweek to a maximum of 35 hours in an attempt both to create hundreds of thousands of new jobs and to achieve greater flexibility in the labour force. Unions responded by demanding the creation of more posts in public hospitals.Financial, physical and knowledge resources are crucial to any type of health care workforce. The level of resources attributed to the health care system, and how these resources are used, will have a significant impact on health workforce issues.In terms of financial resources, human resources account for a high proportion of national budgets assigned to the health sector . Health Physical resources include human resources within the health sector and other sectors; buildings and engineering services such as sanitation, water and heating systems for community use and for the use of medical care institutions; and equipment and supplies. Finally, the health workforce is also constrained by its human capital. This human capital can be associated with the qualification and education of the health workforce. Education of the health workforce is the systematic instruction, schooling or training given in preparation for work.Economic, sociodemographic, cultural, and geographical factors contribute to shaping and transforming society and hence have a direct or indirect impact on health workforce issues.From an economic perspective, for instance, there is evidence of a correlation between the level of economic development of a country and its level of human resources for health. Countries with higher GDP per capita are said to spend more on health care than countries with lower income, as demonstrated by cross-sectional studies,  and hencMoreover, both the demand and supply are likely to be affected by sociodemographic elements such as the age distribution of the population. On the demand side, the ageing of the population is giving rise to an increase in the demand for health services and health personnel, especially nurses for home care.On the supply side, the ageing of the health workforce, and in particular of nurses, has serious implications for the future of the nursing labour market. For example, the Institute of Medicine noted that older registered nurses have a reduced capacity to perform certain tasks . It was Geographical and cultural factors also play a role in determining the demand and supply of human resources. Geographical characteristics affect the organization of health services delivery. For instance, a country with many islands or with isolated population groups will face particular challenges in terms of health workforce issues. Similarly, significant climatic changes are likely to give rise to changes in health needs, which in turn will call for changes in health services and in the health workforce. Finally, both cultural and political values also affect the demand for and supply of human resources for health.This section considers a typology of imbalances, and differentiates between the following:• Profession/specialty imbalances: Under this category, we consider imbalance in the various health professions, such as doctors or nurses, as well as shortages within a profession, e.g. shortage of one type of specialists.• Geographical imbalances: These are disparities between urban and rural regions and poor and rich regions.• Institutional and services imbalances: These are differences in health workforce supply between health care facilities, as well as between services.• Gender imbalances: These are disparities in female/male representation in the health workforce.Imbalances have been reported for almost all health professions, and in particular for nurses. The United States General Accounting Office  reports Major variations occur in the number of health care workers per capita population and in the skill mix employed across countries, as depicted in Fig. Virtually all countries suffer from a geographical maldistribution of human resources for health, and the primary area of concern is usually the physician workforce . In bothUnderstandably, most health care professionals prefer to settle in urban areas, which offer opportunities for professional development as well as education and other amenities for themselves and their families. But it is in the rural and remote areas, especially in the developing countries, that most severe public health problems are found.The geographical maldistribution of doctors has been the object of particular attention. In general there is a higher concentration of general practitioners in the inner suburbs of the metropolitan areas. According to the Australian Medical Workforce Advisory Committee , the rea• historical• lifestyle-related: access to amenities• spouse/husband-related: greater employment opportunities• child-related: better access to secondary and tertiary education services• professional, family and social ties and professional ambitions.The geographical distribution of health care personnel is an important issue in many countries. Managua, the capital of Nicaragua, contains one-fifth of the country's population but around half of the available health personnel . In BangIn Indonesia the geographical distribution of physicians is a particular concern, since Indonesia's vast size and difficult geography present a tremendous challenge to health service delivery . It is dTo improve the geographical distribution of physicians, governments often have used combinations of compulsory service and incentives. So far, there is virtually no country in the world that has solved the problem of a rural/urban imbalance of the physician workforce . This doInstitutional imbalances occur when some health care facilities have too many staff because of prestige, working conditions, ability to generate additional income, or other situation-specific factors, while others are understaffed . InstituImbalance between the types of health services provided may also arise. In particular, we can consider the issue of curative versus preventive care. In effect, it has been estimated that most diseases (80%) and accidents are preventable through known methodologies, yet at present there is an imbalance in the funding of medical research, with only 1%-2% going to prevention and 98%-99% spent on curative approaches . This imIn many countries, women still tend to concentrate in the lower-status health occupations and to be a minority among more highly trained professionals and managers. In Bangladesh, the distribution by gender of the health workforce shows that the total proportion of women accounts for little more than one-fifth in health services . The disFemale general practitioners have been shown to practise differently from males, managing different types of medical conditions, with some differences due to patient mix and patient selectivity, and others inherent in the sex of physician. In some more traditional areas, some women will not seek care for themselves or even for their children because they do not have access to a female provider .This framework can be used to assess policy reforms and their impact on health workforce imbalances; it also provides a common framework for cross-country comparisons. This framework emphasizes the number and type of factors affecting health workforce imbalances, illustrating the complexity of this issue. From a policy perspective, it is particularly interesting to identify factors that policy-makers can influence in order to remedy imbalance problems.Various monetary and non-monetary incentives are used to influence the supply and/or demand for the health workforce. For example, subsidies, grants and scholarships are examples of incentives that can be used to attract more nursing students, whereas wage increases, additional benefits and working hours flexibility are examples of commonly used incentives to attract or retain the health workforce.The numerous factors and actors involved in the health workforce imbalance issue call for a coherent health workforce vision and policy. In that context, health planning plays an important role since it contributes to shaping the health care system. Moreover, since from a societal perspective market mechanisms alone do not allow an adequate demand/supply of health personnel to be reached, public interventions such as human resources planning are a means to correct for market failures. Health planning involves a time horizon. Forecasting the future number of health personnel needed and developing policies to meet such figures are common to any health care system. Physicians represent the profession for which more planning effort has been expended to achieve a workforce of appropriate size than for any other health profession. Countries' desire to meet population health needs and to avoid social welfare losses resulting from a shortage or an oversupply are factors explaining, to a large extent, the importance attributed to planning in the context of public health policies.The policy implications of forecasting either a shortage or a surplus of health care personnel are different, and hence attempts at projections must be rigorous. For instance, referring to previous studies predicting significant surpluses, Cooper  notes thAs a result of forecasted physician surpluses, various policy recommendations have been formulated. The United States Institute of Medicine  publisheThis framework also apprehends the different types of imbalances. This is important since the choice of a policy will also depend on the type of imbalance. Significant disparities in human resources for health between health occupation, regions, gender or health services are recognized as classic problems of imbalance. However, the question of a public/private imbalance is more debatable. One the one hand, we can argue that for equity and access, a health care system should have a strong public component. On the other hand, we can imagine a private-sector oriented health care system with mechanisms to ensure access to the poor.In an attempt to contribute to a better understanding of imbalances in the health workforce, this paper has discussed a framework for human resources for health and proposed a typology of imbalances. Although the term "imbalance" is commonly used with respect to the health workforce, it is clear that imbalance in the health workforce encompasses a wide range of possible situations and is a complex issue. The use of a framework should facilitate the development of policy tools and their assessment.None declared.All authors participated in writing the original text and read and approved the final manuscript.


Scoparia dulcis on the occurrence of oxidative stress in the brain of rats during diabetes by measuring the extent of oxidative damage as well as the status of the antioxidant defense system.The aim of the study was to investigate the effect of aqueous extract of Scoparia dulcis plant was administered orally (200 mg/kg body weight) and the effect of extract on blood glucose, plasma insulin and the levels of thiobarbituric acid reactive substances (TBARS), hydroperoxides, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and reduced glutathione (GSH) were estimated in streptozotocin (STZ) induced diabetic rats. Glibenclamide was used as standard reference drug.Aqueous extract of Scoparia dulcis plant extract (SPEt) and glibenclamide for 6 weeks. Both the treated groups showed significant decrease in TBARS and hydroperoxides formation in brain, suggesting its role in protection against lipidperoxidation induced membrane damage.A significant increase in the activities of plasma insulin, superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and reduced glutathione was observed in brain on treatment with 200 mg/kg body weight of Scoparia dulcis plant extract. Hence, in addition to antidiabetic effect, Scoparia dulcis possess antioxidant potential that may be used for therapeutic purposes.Since the study of induction of the antioxidant enzymes is considered to be a reliable marker for evaluating the antiperoxidative efficacy of the medicinal plant, these findings suggest a possible antiperoxidative role for  The neurological consequences of diabetes mellitus in the Central Nervous System (CNS) are now receiving greater attention. Cognitive deficits, along with morphological and neurochemical alterations illustrate that the neurological complications of diabetes are not limited to peripheral neuropathies . The cenOxidative stress, leading to an increased production of reactive oxygen species (ROS), as well as lipidperoxidation, is increased in diabetes  and alsoIncreases in superoxide production are observed in the serum of Type 1 diabetic patients and was reduced with improved glycemic control . LipidpeScoparia dulcis L (Scrophulariacae), a folk-medicinal plant known as sweet broomweed, has been used as a remedy for diabetes mellitus in India [Scoparia dulcis include scoparic acid A, scoparic acid B and scoparic acid D [Scoparia dulcis and obtained a glycoside, amellin from fresh plant and reported that it brought relief in other complications accompanied with diabetes (ie., pyorrhoea, retinopathy, joint pain, susceptibility to cold etc.) within a very short period [Nowadays, the use of complementary/alternative medicine and especially the consumption of botanicals have been increasing rapidly worldwide, mostly because of the supposedly less frequent side effects when compared to modern western medicine . Scopariin India  and for in India ,15. A nuc acid D , scopaduc acid D  and Scopc acid D  that havc acid D , gastricc acid D . In addic acid D . Becauset period .Scoparia dulcis to STZ diabetic rats led to reduction in blood glucose [Scoparia dulcis in STZ diabetic rats after 6 weeks treatment and the effect produced by Scoparia dulcis was compared with Glibenclamide.Administration of  glucose . In Rece glucose  for 3 wead libitum with normal laboratory pellet diet (Hindustan Lever Ltd., India) and water. Animals were maintained under a constant 12 h light and dark cycle and at an environmental temperature of 21–23°C.Adult male albino Wistar rats (8 weeks), weighing 180–200 g bred in the Central Animal House, Rajah Muthiah Medical College, Annamalai University, were used. All animal experiments were approved by the ethical committee (Vide. No: 73, 2002), Annamalai University and were in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India. The animals were fed All the drugs and biochemicals used in this experiment were purchased from Sigma Chemical Company Inc., St Louis, Mo, USA. The chemicals were of analytical grade.Scoparia dulcis L. (40 – 60 cm in height) were collected from Neyveli, Cuddalore District, Tamil Nadu, India in September 2001. The plant was identified and authenticated at the Herbarium of Botany Directorate in Annamalai University. A voucher specimen (No.3412) was deposited in the Botany Department of Annamalai University.Whole plants of Scoparia dulcis plants were extracted with 1.5 l of water by the method of continuous hot extraction at 60°C for 6 h according to Jain (1968) and the filtrate was concentrated at 40°C to constant weight in a rotavapor apparatus (Buchi Labortechnik AG, Switzerland). The residue collected (yield 31 g) were thick paste, green in color and gumaceaous in nature and stored at -20°C, when needed the extract was dissolved in sterile water and used in the investigation [Five-hundred grams of fresh whole tigation .STZ, freshly prepared in 10 mmol/l citrate buffer, pH 4.5, was injected to experimental animals (30 rats) intraperitoneally at a dose of 45 mg/kg body weight . 48 h afScoparia dulcis plant extract (SPEt) (200 mg/kg body weight) in aqueous solution daily using an intragastric tube for 6 weeks. Group 3: Diabetic control rats. Group 4: Diabetic rats given SPEt (200 mg/kg body weight) [In the experiment, a total of 30 rats  were used. The rats were divided into 5 groups of 6 rats each. Group 1: Normal rats. Group 2: Normal rats given  weight)  in aqueo weight) .th week, all the rats were killed by decapitation (Pentobarbitone sodium) anaesthesia (60 mg/kg). Blood was collected in two different tubes (i.e.,) one with anticoagulant – potassium oxalate and sodium fluoride for plasma and another without anticoagulant for serum separation. Plasma and serum were separated by centrifugation. Whole Brain was immediately dissected out, washed in ice cold saline to remove the blood. The brains were weighed and 10% tissue homogenate was prepared with 0.025 M Tris – HCl buffer, pH 7.5. After centrifugation at 200 rpm for 10 min, the clear supernatant was used to measure thiobarbituric acid reactive substances (TBARS), hydroperoxides and GPx activity. For the assay of SOD, CAT, GST and GSH, the brains were weighed and 10% homogenate was prepared with 0.2 M, phosphate buffer pH 8.0. After centrifugation, the clear supernatant was used for the assay of enzyme activities.All doses were started after 48 h STZ injection. No detectable irritation or restlessness was observed after each drug or vehicle administration. No noticeable adverse effect  was observed in any animals after the drug administration. Blood samples were drawn at weekly intervals till the end of study (ie. 6 weeks). At the end of 6Blood glucose was determined by the O-toluidine method . Plasma Lipid peroxidation in brain was estimated colorimetrically by thiobarbituric acid reactive substances TBARS and hydroperoxides by the method of Niehius and Samuelsson  and Jian0.1 ml of tissue homogenate was treated with 0.9 ml of Fox reagent (88 mg butylated hydroxytoluene (BHT), 7.6 mg xylenol orange and 9.8 mg ammonium ion sulphate were added to 90 ml of methanol and 10 ml 250 mM sulphuric acid) and incubated at 37°C for 30 min. The color developed was read at 560 nm colorimetrically. Hydroperoxides was expressed as mM/100g tissue.2O2 consumed/min/mg protein as described by Sinha [2O2. The reaction was stopped by the addition of 2.0 ml of dichromate-acetic acid reagent .CAT was assayed colorimetrically at 620 nm and expressed as μmoles of Hby Sinha . The reaSOD was assayed utilizing the technique of Kakkar et al.  based onGPx activity was measured by the method described by Rotruck et al. . BrieflyGSH was determined by the method of Ellman . 1.0 ml -1 cm-1.The GST activity was determined spectrophotometrically by the method of Habig et al. . The reaProtein was determined by the method of Lowry et al.  using BoAll data were expressed as mean ± SD of number of experiments (n = 6). The statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS version 7.5 (SPSS, Cary, NC, USA) and the individual comparison were obtained by Duncan's Multiple Range Test (DMRT). A value of p < 0.05 was considered to indicate a significant difference between groups .Table Table For studying the effect of SPEt on antioxidant status, the activities of enzymic antioxidants SOD, CAT, GPx, GST and non-enzymic antioxidant GSH were measured Table  and 4. TThis work is one of a series of studies showing that chronic hyperglycemia causes an imbalance in the oxidative status of the nervous tissue and that the resulting free radicals damage the brain through a peroxidative mechanism. The STZ diabetic rat serves as an excellent model to study the molecular, cellular and morphological changes in brain induced by stress during diabetes . Under nScoparia dulcis showed significant antihyperglycemic activity. The antihyperglycemic activity of this plant may be, at least in part, through release of insulin from the pancreas in view of the measured increase in the plasma insulin concentrations.In the present study, treatment with aqueous extract of 2O2 forming hydroxyl radical:Earlier studies in this lab have demonstrated a defective metabolism of lipid peroxides in other tissues of diabetic animal ,36. TBAR3+ + O2•- → Fe2+ + O2Fe2+ + H2O2 → Fe3+ + OH* + OH-Fe2O2 by SOD or CAT would ameliorate STZ toxicity, as would substances able to scavenge the hydroxyl radical [The destruction of superoxide radical or H radical ,40. Vuln radical .2O2 and O2 by the autoxidation of glucose and non-enzymatic glycation [Scoparia dulcis has been reported to be rich in alkaloids and terpenoids [2•- anions as these anions have been shown to inactivate CAT and GPx [The altered balance of the antioxidant enzymes caused by decrease in CAT, SOD, GPx, GST and GSH activities may be responsible for the inadequacy of the antioxidant defenses in combating ROS mediated damage. The decreased activities of CAT and SOD may be a response to increased production of Hlycation . These elycation . Treatmerpenoids -18,43,44 and GPx .Under in vivo conditions, GSH acts as an antioxidant and its decrease was reported in diabetes mellitus . We haveScoparia dulcis may be anticipated to have biological significance in eliminating reactive free radicals that may otherwise affect the normal cell functioning. The disfunctioning of these antioxidant enzymes has been implicated in several disorders including rheumatoid arthritis, reperfusion injury, cardiovascular diseases, immune injury as well as diabetes mellitus [Significantly lower levels of lipid peroxides in brain of SPEt treated diabetic rats and increased activities of enzymic and non-enzymic antioxidants in brain suggest that the extract reduce oxidative stress by quenching free radicals. Terpenoids and alkaloids were reported to have free radical scavenging activity and antioxidant capacity in diabetes ,50. SPEtmellitus .Scoparia dulcis plant possesses antioxidant activity, which could exert a beneficial action against pathological alterations caused by the presence of free radicals in STZ diabetes.It may be concluded that in diabetes, brain tissue was more vulnerable to oxidative stress and showed increased lipidperoxidation. The above observation shows that the aqueous extract of Scoparia dulcis plant extract significantly decreased the lipidperoxidation and significantly increased the antioxidant status. Since the study of induction of the antioxidant enzymes is considered to be a reliable marker for evaluating the antioxidant efficacy of the medicinal plant, these findings are suggestions of possible antioxidant role played by Scoparia dulcis plant extract in addition to its antidiabetic effect.The brain exhibits numerous morphological and functional alterations during diabetes. Oxidative stress, a factor implicated in the pathogenesis of diabetic complications may contribute towards some of these alterations. Treatment of diabetic rats with The author(s) declare that they have no competing interestsLP – supervised the design and co-ordination of the studyML – Practically conducted the design of the study and drafted the manuscript.The pre-publication history for this paper can be accessed here:


In the spirit of good health, cells are constantly subjecting their protein contents to immunological surveillance by cytotoxic (killer) T cells. Tens of thousands of major histocompatibility complex (MHC) class I molecules cradle peptides (bits of proteins) on cell surfaces, and T cells detect any suspicious peptides with extreme sensitivity. If a cell is infected with a virus, peptides created from viral DNA will end up on the cell's surface as antigens, triggering immunological red flags.Most—but not all—of the peptides presented by MHC class I molecules are created by conventional cellular mechanisms: with the help of a ribosome, three mRNA nucleotides (a codon) are decoded into a corresponding amino acid, which is strung as the next link on an elongating peptide. Most peptides begin with the amino acid methionine, coded by the mRNA nucleotide triplet A-U-G (AUG). But some peptides are “cryptic,” arising from normally untranslated regions of mRNA or initiated with codons other than AUG.not begin with methionine.Previous studies suggested that an unconventional translation mechanism creates some cryptic peptides. But how? And why? Only one type of translation initiator, transfer RNA (tRNA), specific for AUG and loaded with a methionine molecule, is known. Protein synthesis beginning at alternate codons has been attributed to imprecise pairing between the methionine translation initiator and mRNA. This, however, does not explain proteins that do Only two mechanisms for building non-methionine-initiated peptides have been discovered. In a new study, Susan R. Schwab et al. characterize one of them, the CUG-initiated translation of a peptide starting with leucine instead of methionine.The authors explored cellular translation by engineering cells to create peptides of interest and present them through matching MHC molecules on the cells' surfaces. Then, by harnessing the exquisite sensitivity of T cells to probe for antigens on MHC molecules, they could identify which peptides were created under different experimental conditions.Their findings point to a unique translation mechanism. In the other known example of a non-methionine-initiated peptide, translation beginning at GCU or CAA is guided by a specific folded structure of mRNA nucleotides called the internal ribosome entry site. Schwab et al. have found that no similar structure is necessary for CUG-initiated translation. However, similar to the standard mechanism of AUG initiation, they found that ribosomes do scan for CUG. Additionally, the presence of a specific ribosome-binding sequence in mRNA (the “Kozak context”) near a CUG site can enhance the efficiency of initiation there.Schwab et al. have also suggested a possible purpose for this translation mechanism. Under stress, cells can down-regulate conventional translation, which curbs the production of viral proteins in the event of an infection but also suppresses the creation of antigens needed to flag down T cells for an immune response. Here, Schwab et al. report that peptides starting with leucine were produced in the absence of the protein eIF2, which normally aids in AUG-initiated peptide synthesis. Cells under stress slow conventional translation by restraining the function of eIF2. Therefore, CUG-initiated translation, which works without eIF2, might provide an out for stressed cells needing to create peptides. This alternative could be a great way to avoid pumping out viral proteins and still create antigens for T cell surveillance—unless, of course, viruses take advantage of the loophole for their own peptide production.


Over 4 million single nucleotide polymorphisms (SNPs) are currently reported to exist within the human genome. Only a small fraction of these SNPs alter gene function or expression, and therefore might be associated with a cell phenotype. These functional SNPs are consequently important in understanding human health. Information related to functional SNPs in candidate disease genes is critical for cost effective genetic association studies, which attempt to understand the genetics of complex diseases like diabetes, Alzheimer's, etc. Robust methods for the identification of functional SNPs are therefore crucial. We report one such experimental approach.Luciferase based plasmid reporter system. Two out of the 4 SNPs exhibited differential promoter activity in several human cell lines.Sequence conserved between mouse and human genomes, within 5 kilobases of the 5-prime end of 176 GPCR genes, were screened for SNPs. Sequences flanking these SNPs were scored for transcription factor binding sites. Allelic pairs resulting in a significant score difference were predicted to influence the binding of transcription factors (TFs). Ten such SNPs were selected for mobility shift assays (EMSA), resulting in 7 of them exhibiting a reproducible shift. The full-length promoter regions with 4 of the 7 SNPs were cloned in a We propose a method for effective selection of functional, regulatory SNPs that are located in evolutionary conserved 5-prime flanking regions (5'-FR) regions of human genes and influence the activity of the transcriptional regulatory region. Some SNPs behave differently in different cell types. The SNP databases in public domain, like NCBI/dbSNP and HGVbase, have facilitated this by highlighting all nsSNPs and also further classifying the location of the amino acid within the encoded proteins [Single nucleotide polymorphisms (SNPs) are the most common form of genomic variations occurring on average every 1000 nucleotides. The vast majority of SNPs are neutral allelic variants, however the few that do influence a phenotype in a measurable way, are important for understanding the underlying genetics of human health. SNPs are the focus of a large number of human genetics studies attempting to understand their impact on complex diseases like Alzheimers, Parkinsons, diabetes, etc. Most SNPs, by the virtue of their location within genes (introns, 3'-UTRs, etc) or between genes, are considered most likely to be benign and not to contribute to a phenotype, whether it may be the manifestation of a disease or quicker metabolism of a drug. Among the group of SNPs located within coding regions of genes and causing a change in the peptide sequence (non-synonymous SNPs or 'nsSNPs') or among SNPs located within promoters (regulatory SNPs or rSNPs), a majority may not influence the overall activity of the protein or the gene expression. With the per-SNP validation and genotyping cost relatively high, it is increasingly important to develop strategies to predict functionally relevant SNPs proteins -6. Of thproteins , an estiproteins .in vivo function [Transcriptional regulatory regions in the 5'-FR of human genes encode short (often < 25 bp) ,10 sequefunction , mainly function -19.The 7TM (7 trans-membrane domain proteins), also known as the hetero-trimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) are members of a large family with an estimated 700 genes in the human genome . By someThe lists of the full names and Ensembl ENSG numbers  of the 1For EMSA-positive markers with no validation information at dbSNP, HGVbase or Celera Discovery Systems™ we did a validation analysis using RFLP (restriction fragment length polymorphism) on DNA samples from 25 healthy Nordic individuals. The three SNPs which failed to show positive gel-shift results  were not investigated any further. For dopamine receptor D1 (DRD1) polymorphism (rs267412) the genotype distribution was found to be TT = 30%, AA = 20% and AT = 50%, and for calcitonin receptor promoter (CT-R) polymorphism rs2528521, it was GG = 40%, AA = 30% and GA = 30%. While rs267412 was found to be in Hardy Weinberg Equilibrium (HWE), rs2528521 was not. A larger population sample should be genotyped to accurately measure HWE for both these loci. Bradykinin B2 (BK-2) promoter polymorphism rs1799722 is noted to be polymorphic  at NCBI/dbSNP. The polymorphic nature of this locus (rs1799722) was also confirmed by crosschecking at Celera Discovery Systems™. Also for the second BK-2 SNP rs945032, the allele frequency information was found at dbSNP . NCBI's dbSNP provided no allele frequency information about rs1538251 adenosine-A3 receptor (ADORA3), and on sequencing 20 DNA samples this marker proved to not be polymorphic in Nordic sample population, and was eliminated from further analysis (data not shown). The genotype frequency of muscarinic acetylcholine receptor M1 (CHRM1) SNP rs509813 was documented at Celera Discovery Systems™. The contig-position of rs2882225  was not in agreement between the three major public genome databases, i.e. NCBI, Ensembl and Santa Cruz Genome Assembly (UCSC). It was mapped within the transcript for FSHR by NCBI/dbSNP, and completely absent from Ensembl and UCSC [For expression analysis in living cells, the published promoter regions, or putative regulatory 5'FR of up to 2 Kb, were cloned and basal levels of luciferase were monitored. Repeated attempts to clone the promoter region of DRD1 failed (data not shown). Furthermore the position of rs267413 is mapped at -4446 nucleotides with respect to the transcription start site, whereas the minimum length of the genomic fragment known to drive DRD1 expression is limited to 2571 nucleotides. Considering the distal position of the marker, we decided not to examine rs267413 further in this study. The promoter regions of BK-2, CHRM1 and CT-R were cloned successfully. A total of four dissimilar human cell lines (HeLa, Hep2G and SK-N-MC, HEK293) were used to monitor the influence of the four SNPs (rs945032, rs1799722, rs2528521 and rs509813) to investigate differences in expression that are possibly due to differences in TF expression in different cell types. BK-2 promoter SNPs rs945032 (genotype = GG) and rs1799722 (genotype = AA) showed approximately 40%-60% higher activity in HeLa cells as compared to their other homozygote alleles AA and GG, respectively  from known binding sites. PWMs are probabilistic models that capture the nucleotide preference at each position of a TFBS as well as the differential contribution of positions to the overall binding energy. When a putative binding site sequence is assessed using a PWM, a score is obtained that theoretically should be proportional to the binding energy between the TF and that sequence . It has in silico prediction methods based on other in vitro or in vivo selection technologies, like 'systematic evolution of ligands by exponential enrichment' (SELEX), may not agree with the experimental outcome of EMSA procedures. Since the construction of PWMs is often concluded from published records based on SELEX enrichment approaches, it is informative to experimentally validate the predicted binding site using methods like EMSA. Therefore, we validated a subset of our predictions with in vitro electro-mobility shift. We used a stringent selection criterion, that is, qualified only alleles demonstrating an absolute binding score difference of 2.0 or more. A stringent selection criterion would no doubt decrease excessive hits and false positives at the expense of certain loss of true positives. Our results showed that approximately 60%-70% (i.e. 7 out of the selected 10 SNPs) of predicted sites (Table Electromobility Shift Assays (EMSA) produce DNA-protein binding interactions in artificial conditions. Therefore We finally attempted to correlate the EMSA findings from the 10 SNPs with influences on actual promoter activity within living cells. Due to mapping discrepancies of one SNP and failure to clone one promoter, we tested only four out of the seven EMSA-positive SNPs identified thus far. Of the four SNPs tested in a promoter-less expression vector, two (rs945032 and rs1799722) indicated significant influence on promoter activity, while two showed convincing and reproducible, yet comparatively limited influence (rs2528521 and rs509813) on promoter activity. The influence of these polymorphisms (rs945032 and rs1799722) indicate that any given functional variation within a regulatory region might exert a measurable influence within the context of a cell type determined by the TF expression profile of the cells and perhaps competitive binding of the TF to overlapping multiple binding sites.There are several factors in the current approach which indicate that there are far more rSNPs than currently detected using available technologies. The EMSA assays employ HeLa nuclear extract, thereby limiting our findings to the TF expression repertoire of only HeLa nuclei. The TFBS package used a limited collection of high quality PWMs, which unfortunately represent only a small part of the approximately 2,000 known human and mouse TFs. The theoretical thresholds set for selections of alleles which are predicted to differentially bind TF require further rigorous testing to ensure that the present selection is optimal.in situ reporter system. Finally, two of the four showed significant and reproducible influences which were dependent on the cell environment. Thus starting from a large pool of potential regulatory SNPs, we successfully identified a small fraction that actually influenced promoter activity. We therefore propose a method for effective selection of functional, regulatory SNPs, in evolutionary conserved 5'-FR regions of human genes, as a means for identification of candidate SNPs for genetic association analysis studies.From a total of approximately 200 SNPs in evolutionally conserved 5'-FR of 176 human GPCR genes, our prediction algorithm selected 36 SNPs with possible influence on TFBS. When ten of these 36 SNPs were tested for mobility shifts, seven exhibited a positive result, and four of these were further tested for influence on promoter activity using an The GPCR genes were selected from Ensembl . Human aTable 32P] dCTP with fill-in reaction using Klenow flagment. The labeled oligonucleotide were passed through ProbeQuant G-50 Micro Columns (Pfizer-Pharmacia Inc) and the concentration was adjusted to 0.035 nM. A 0.8 μl volume portion was mixed with 1.6 μl HeLa Nuclear Extract (Promega™), 1.6 μl 5x Gel Shift Binding 5x Buffer (Promega™), and 3.2 μl water. After 10 min incubation at room temperature, 0.8 μl of non-labeled competitor DNA, either one allele or the other allele, was added in varying concentrations (5-, 10-, 15-, 20- and 25-folds greater than the radio-labeled oligonucleotide). After 20 min room temperature incubation, the entire 8 μl reaction was loaded on polyaclylamide gel (5% 22.5 mM Tris/22.5 mM boric acid/0.5 mM EDTA buffer in, BIO RAD™). Thereafter, electrophoresis was performed in TBE for 20 min at 200 V. Gels were placed on Whatmann 3 MM™ filters and to facilitate drying a BIO RAD™ gel-dryer was used for 30 minutes. The dried gel were exposed to intensifying screen and analyzed by Typhoon Image Analyzer 9400 (Pfizer-Pharmacia Inc™).Double stranded oligonucleotides were synthesized with 5'-GG dinucleotide overhangs. The 3'-end of the complementary strand was labeled with [αKpnI restriction site (GGTACC) and GG-dinucleotide was added to the 5' end. The sequence of the reverse primer was 5'-TCTTTTAAGCTCTACTGTGGGTGA-3'. Calcitonin receptor promoter polymorphism rs2528521 was analyzed by RFLP (fragment length was 334 bp, restriction enzyme Tsp45I). Forward primer sequence used for PCR was 5'-ACCCCCAAGGTGTCTCTTCT-3' and reverse primer: 5'- GAGGGACCCGAGTTAGACCT-3'. The primer sequences for Bradykinin promoter SNP rs1799722 were as follows: Forward primer 5'-CCAGGAGGCTGATGACGTCA-3'. The fourth base from 3'-end was changed from A to G from original genomic sequence to create a Tsp45I restriction site for RFLP analysis.The frequency of Dopamine receptor D1 polymorphism rs267413 was determined by sequencing a 174 base-pair fragment of the promoter in 10 DNA samples from healthy Swedish individuals. The sequence of the forward primer used for PCR was 5'-GGGGTACCACTTGACCGTTCTGTTGCTTT-3' where a Reverse primer: 5'-TCAGTCGCTCCCTGGTACTG-3'. Fragment length amplified was 150 bp. PCR conditions for all RFLP and sequencing reactions were as follows: 94 C (4 min), followed by 42 cycles of 94 C (1 min), 61 C (30 sec) and 72 C (30 sec).KpnI and BglII of the plasmid vector. Primers for CHRM1: Forward: 5'-GGGGTACCGCAGGACCCACATCTCTAGG-3' Reverse = 5'-GAAGATCTTCACCAGGGCACCCAAT-3'. Primers for BK-2: Forward = 5'-GGGGTACCATCTGAGACTCTGTTTCCC-3' reverse = 5'-GAAGATCTTTCAGTCGCTCCCTGGTACT-3'. Primers for CT-R: Forward = 5'-GGGGTACCCCTTGGAATCAACTTGCCT-3' reverse = 5'-TTCTCGAGCGTCCTTGGAATCAACTTGC-3'. Genomic DNA of 27 individuals of Nordic origin were amplified and sequenced to identify the genotype of sample DNA. Cloned DNA were sequenced by using primer set GLprimer2 : 5'-CTAGCAAATAGGCTGTCCC-3' and 5'-CTTTATGTTTTTGGCGTCTTCC-3'. HeLa cells were plated in 24 well plates one day before transfection in appropriate medium with serum without antibiotics. Basic PGL3 Plasmids containing the cloned promoter region (180 ng) were co-transfected with 20 ng of pRL-TK plasmid, using Lipofectamine 2000 (Invitrogen™). Luciferase activities were determined using a dual Luciferase Reporter Assay system (Promega™) according to the manufacturer's instructions.A promoter-less luciferase vector (Basic PGL3, Promega™) was used for cloning known promoter regions between restriction sites TF: Transcription Factor; TFBS: transcription factor binding site; SNP: Single Nucleotide Polymorphism; GPCR: G-protein coupled receptors; 5'-FR: 5' flanking regulatory region.Original Concepts and supervision: CW, WWW, BL and SM-T; Bioinformatics: PGE, BL and SMT; Running costs: SMT and CW; Manus preparation: SMT, PGE, CW; Electromobility Shift Assays and RFLP assays: YM; Luciferase expression: MAF and YM.ENSG ids A list of 176 initial GPCRs considered for this study, along with the Ensembl ENSG Ids.Click here for fileAlignments Alignment information for sequence flanking rs945032 and rs1799722 in human and mouse.Click here for file


In the context of the significant risk of graft failure after allo-HSCT from alternative donors and the risk of relapse in recipients transplanted for malignancy, the precise monitoring of posttransplant hematopoietic chimerism is of utmost interest. Useful molecular methods for chimerism quantification after allogeneic transplantation, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers. Such analyses can be performed regardless of donor's and recipient's sex. Additionally, in patients after sex-mismatched allo-HSCT, fluorescent We compared different techniques for analysis of posttransplant chimerism, namely FISH and PCR-based molecular methods with automated detection of fluorescent products in an ALFExpress DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE). We used Spearman correlation test.We have found high correlation between results obtained from the PCR/ALF Express and PCR/ABI 310 Genetic Analyzer. Lower, but still positive correlations were found between results of FISH technique and results obtained using automated DNA sizing technology.All the methods applied enable a rapid and accurate detection of post-HSCT chimerism. Allogeneic hematopoietic stem cell transplantation  is performed mainly in patients with high-risk or advanced hematologic malignancies and aplastic anemias, and for some of them it is the only curative treatment. After allo-HSCT, incomplete engraftment and appearance of recipient's hematopoietic cells can lead to a coexistence of donor and host hematopoiesis – a situation known as mixed chimerism. Complete recovery of hematopoiesis of the donor origin is referred to as complete chimerism.in situ hybridization (FISH) can be applied. This technique is based on identification of Y-chromosome-specific sequences in the posttransplant sample examined. Variants of these techniques can be used for precise, quantitative assessment of the amount of donor's cells in recipient's peripheral blood and/or bone marrow after transplantation, in the long run giving a picture of the dynamics of changes in chimeric status within a hematopoietic compartment. Chimerism also reflects response to treatment [Widely accepted molecular methods for analysis of chimerism after allo-HSCT, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers, such as variable number of tandem repeats (VNTR) or short tandem repeats (STR) . Such anreatment , since ireatment .in situ hybridization (FISH) and PCR-based molecular methods with fluorescent products detected in an ALF Express DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE).Here we report results of a comparison of different techniques for analysis of posttransplant chimerism: fluorescent Investigation of hematopoietic chimerism was performed in ten children (eight girls, two boys) aged 6–16 years. They were diagnosed with acute myelogenous leukemia (n = 4), acute lymphoblastic leukemia (n = 2), chronic myelogenous leukemia (n = 1), myelodysplastic syndrome (n = 1), or Fanconi Anemia (n = 2). All recipients received hematopoietic stem cells from HLA-matched sibling donors, and sex-mismatch between donor and recipient was present in all cases. The material  for hematopoietic chimerism quantification was collected in different periods after transplantation. In all 10 children reconstitution of hematopoiesis was observed. Out of 8 children transplanted for hematologic malignancies, 4 are well and alive, in complete continuous remission (CCR), while in the other 4, leukemia relapse occurred 4–23 months after transplantation.High-molecular-weight DNA was extracted from frozen whole blood (approximately 5 ml) or bone marrow (approximately 3–5 ml) by the standard treatment with sodium dodecyl sulfate (SDS) and proteinase K, and the salting-out method. DNA was isolated from the donors' and patients' blood samples collected before and after transplantation at various intervals in order to determine the chimeric status.The PCR protocol optimized for the Qiagen polymerase and the PE 9700 thermocycler was performed as described previously . For fraThe PCR protocol optimized for the Thermal Controller MJ Research (Watertown, MA) model PTC-100™ was applied. PCR was performed in a volume of 10 μl; the PCR reaction mixture contained: 2.5 pM of each forward and reverse primer, 200 μl of each dNTP, 0.4 U Taq polymerase (Qiagen, Chatsworth, CA), 1x PCR buffer (Qiagen, Chatsworth, CA), and 40 ng of genomic DNA. Conditions for PCR were as follows: 5 min at 94°C for the first denaturation; 26 cycles of amplification with a temperature profile of 45 sec at 94°C, 1 min at 55°C, 1 min at 72°C; with additional 5 min at 72°C in the last cycle. STR loci were amplified with fluorescent PCR primers described previously [DXZ1) and Y (locus DYZ1). The FISH procedure according to Cytocell Ltd. was used [The experiments were performed on interphase nuclei obtained by standard short culturing of fresh whole blood samples, with probes specific for chromosomes X , all obtained data were analyzed by GeneScan 3.1 software and then transferred to Genotyper 2.5 software [% of recipient's DNA = (R1 + R2)/(D1+ D2 + R1 + R2) × 100,where: R1, R2 = peak areas of recipient's alleles; and D1, D2 = peak areas of donor's alleles.Only informative markers were used for the analysis. If donor and recipient were heterozygous but shared one allele, only the area of the non-shared alleles was considered for the analysis .To make sure that quantification is accurate, we performed serial dilution experiments, where standardized mixed chimeric samples were created by mixing donor's and pretransplant recipient's DNA in a range between 0 and 100 percent. The sensitivity strongly depends on the size of alleles, the detection level was around 3–5% of patient cells.The results of chimerism detection by different methods were compared by the Spearman correlation test.The results of chimerism quantification with the use of an ALF Express DNA Sequencer, ABI 310 Genetic Analyzer, and FISH are compared in Table Coefficients of Spearman rank correlation between results of chimerism quantification by the three different methods are shown in Table One of the advantages of application of automated DNA sizing techniques for detection of posttransplant chimerism is that the use of radioactivity is not necessary. It is arelatively simple and rapid method, consisting of two steps: polymerase chain reaction with fluorescent primers and automated detection of fluorescently labeled PCR products, separated by electrophoresis. The analysis of fluorescently labeled PCR products provides better accuracy and precision of measurement than traditional electrophoretic methods. The most time-consuming step, which might prolong the examination process, is the search for informative markers.Analysis of the chimerism status by amplification of STR loci can be performed regardless of donor's and recipient's sex. The most advantageous is the high sensitivity of the detection system used, so that only small amounts of DNA are needed.Fragment analysis in the ABI 310 Genetic Analyzer (PE) is performed after capillary electrophoresis. The examination of one sample requires about 6 hrs; 144 samples can be analyzed per day if three colors are simultaneously analyzed . The advIn the ALFExpress DNA Analyzer, electrophoresis is carried out in an off-vertical gel cassette specially designed for easy and safe gel casting. The number of samples to be loaded on a gel is limited to 40 per run. The ALFwin Fragment Analyzer is used afterwards for fragment analysis. It is provided with versatile application software for the control of DNA fragment separation runs and subsequent analysis of the data. Collected data are used to accurately size PCR product peaks on the basis of external and internal standards.One analysis of 40 samples takes 7 hrs, including PCR, electrophoresis in the gel cassette, and paperwork. The appropriate assembling and cleaning of the gel cassette is critical and time consuming.in situ hybridization for one patient sample lasts at least 5 hrs, including preparation of interphase nuclei, hybridization with specific probes , and analysis. The high cost of the procedure is definitely a disadvantage.It is well known that FISH is a good quantitative method of fluorescent signal detection, but requires lots of technical experience and expertise. Fluorescent PCR-based techniques allow the relative proportions of recipient's and donor's cells in the post-HSCT period to be identified and quantified and is not only limited to sex-mismatched transplants. Although when using chimerism analysis one cannot assess whether or not the population of recipient's nucleated cells contains leukemic cells, samples taken at various intervals can show if the expansion rate of the particular population is consistent with hematologic and clinical symptoms of the disease. When it is not possible to find an informative marker for PCR amplification, only FISH analysis enables assessing the chimerism status. However, cytogenetic Y chromosome probing by FISH is limited exclusively to sex-mismatched transplantations. Results obtained with the use of ALFExpress DNA Sequencer and ABI 310 Genetic Analyzer are identical or very similar. We showed that appropriate quantitative assessment of chimerism after HSCT by using microsatellite genotyping and automated DNA sizing does not depend on the sequencer model used. The high correlation between results from the PCR/ALFExpress and PCR/ABI 310 Genetic Analyzer indicate that these two methods can be used interchangeably. The superiority of the ABI 310 Genetic Analyzer is limited to the possibility of analysis of three samples at the same time in one reaction tube and the technical ease of capillary electrophoresis with no need for the time-consuming and cumbersome use of glass plates. Lower, but still positive correlations were found between results of FISH analysis and these two methodological variants of PCR. However, in some samples analyzed with PCR, no recipient's signals were found, attesting to full donor chimerism, while at the same time residual host cells turned out to be detectable by FISH. We suggest that these results are within the range of error of the method applied.Finally, we conclude that all the methods applied enable a rapid and accurate detection of post-HSCT chimerism and with due caution can be used interchangeably.The author(s) declare that they have no competing interests.J.J. carried out the molecular chimerism studies and drafted the manuscript.T.S. performed the statistical analysis.A.P and D.B. supplied clinical data.P.B. initiated quantitative analysis.J.W. supervised clinical part and final writingM.W. supervised laboratory part and final writingThe pre-publication history for this paper can be accessed here: