However, the regulatory mechanism(s) governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset.Large-conductance, calcium-activated potassium (Maxi-K) channels are implicated in the modulation of human uterine contractions and myometrial CaMyometrial biopsies were obtained at hysterectomy (non-pregnant, NP), and at Caesarean section, at elective (pregnant not-in-labour, PNL) and intrapartum (pregnant in-labour, PL) procedures. RNA was extracted from all biopsies and quantitative real-time RT-PCR was used to investigate for possible differential expression of the Maxi-K alpha subunit, and that of its splice variants, between these functionally-distinct myometrial tissue sets.RT-PCR analysis identified the presence of a 132 bp and an 87 bp spliced exon of the Maxi-K alpha subunit in all three myometrial tissue sets. Quantitative real-time PCR indicated a decrease in the expression of the Maxi-K alpha subunit with labour onset. While there was no change in the proportion of Maxi-K alpha subunits expressing the 87 bp spliced exon, the proportion of alpha subunits expressing the 132 bp spliced exon was significantly increased with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. An increased proportion of 132 bp exon-containing alpha subunit variants with labour onset is of interest, as channels expressing this spliced exon have decreased calcium and voltage sensitivities.Our findings suggest that decreased Maxi-K alpha subunit mRNA expression in human myometrium at labour onset, coupled to an increased proportion of Maxi-K channels expressing the 132 bp spliced exon, may be linked to decreased Maxi-K channel calcium and voltage sensitivity, thereby promoting enhanced uterine activity at the time of labour. The regulatory mechanisms for uterine smooth muscle contractility during human pregnancy and labour are poorly understood. Such information is essential to understanding the clinical problems associated with human parturition and particularly preterm or premature labour. It is clear however that the myometrium is transformed from a state of relative quiescence during pregnancy, to one of maximal contractile activity at the time of labour. It is also established that the state of contractility of uterine smooth muscle is intrinsically linked to cell membrane ion channel activity ,2.+) channels are functionally important in the regulation of smooth muscle tone [+ channels, large-conductance, calcium-activated K+  channels are the predominant K+ channels in myometrium, and thus have been implicated in the control of cellular excitability [slo gene family, and a regulatory β subunit [Potassium  channe subunit . What in subunit ,21.It appears that expression of different pore-forming α subunit isoforms, with associated regulatory β subunits, occurs in a tissue-specific manner, thereby providing functional specificity . Maxi-K Although multiple alternatively spliced exons of the Maxi-K α subunit have been identified ,19,21,28Patient recruitment took place in the Department of Obstetrics and Gynaecology, University College Hospital Galway (UCHG), Ireland, between October 2001 and August 2002. The study was approved by the Research Ethics Committee, UCHG, and recruitment was carried out by provision of information sheets and obtaining written informed consent.Biopsies of myometrium were excised from the midline of the upper lip of the uterine incision made at caesarean section, at elective  and intrapartum  procedures. The mean age of the women was 33.3 years (range 26–42) of whom four were primagravida and eleven were multigravida. All women were delivered between 37 and 41 weeks gestation. There was no significant difference between those undergoing elective or emergency caesarean section in terms of age, parity or gestation. Women who had received prostaglandins or oxytocin were excluded from the study. Reasons for emergency section included breech presentation, previous caesarean section and abnormal foetal position. The criteria for inclusion in the intrapartum group were regular spontaneous uterine contractions, effacement of the cervix, and cervical dilatation >3 cm prior to caesarean section.Samples of non-pregnant myometrium  were excised from the body of the uterus of hysterectomy specimens from pre-menopausal women. The mean age of women undergoing hysterectomy was 42.5 years (range 34–48). Women with malignant conditions, and those receiving exogenous hormone therapy (e.g. progestagens), were excluded from the study. Immediately upon removal, tissue samples were rinsed in sterile saline, snap frozen in liquid nitrogen and stored at -80°C until RNA extraction.® Reagent (Life Technologies, Paisley, UK) [260. To eliminate any residual contaminating genomic DNA, all RNA samples were DNase-treated with the DNA-free™ DNA removal kit (Ambion, Huntingdon, Cambridgeshire, UK), as previously described [260, after removal of DNA, and adjusted to a final concentration of 500 ng/μL.RNA was isolated from frozen tissue by homogenisation in TRIzolley, UK) . RNA conescribed . RNA con15 primer and 200 IU M-MLV reverse transcriptase (Promega, Madison, WI, USA), as previously described [Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was performed to check for mRNA expression of all potential spliced exons of the Maxi-K α subunit in non-pregnant and pregnant myometrium, prior to and after labour onset. Purified RNA samples were reverse transcribed using oligo (dT)escribed . PCR ampescribed . Primer Splice variant-specific cDNAs were prepared for each RNA sample using reverse primers as shown in Table 9 to 1 × 104 cDNA copies, in 10-fold increments) were created for each spliced exon and conserved region amplicons, and for β-actin, to enable accurate quantitation of product-specific cDNA copy numbers. Amplicon-specific PCR products were generated by RT-PCR (as described above) using primers shown in Table 260, and ligated into TA cloning vector pCR® 2.1 (TA cloning® kit, Invitrogen Ltd, Paisley, UK), according to manufacturers' instructions. Vector-ligated PCR products were transformed into One Shot® TOP10 cells, which were plated on Luria-Bertani (LB) agar plates containing 50 μg/mL kanamycin antibiotic, and incubated overnight at 37°C. Plasmid templates containing inserts in the desired orientation to transcribe sense RNA, as determined by colony PCR, were linearized by HindIII digestion. 2 μL of digestion products were electrophoresed on 1% agarose gels and visualised to ensure complete plasmid linearisation.Standards (1 × 10in vitro transcription using the MAXIscript™ In vitro Transcription Kit (Ambion, Huntingdon, Cambridgeshire, UK). 1 μg of linearized plasmid DNA was in vitro transcribed in a final volume of 20 μL containing 0.5 mmol/L each of ATP, CTP, GTP, and UTP, 2 μL 10X Transcription buffer, and 2 μL T7 Enzyme mix for 1 hr at 37°C. After transcription, samples were treated with DNA-free™ DNA removal kit (Ambion, Huntingdon, Cambridgeshire, UK) to remove plasmid DNA. The supernatant, containing purified cRNA, was pipetted onto pre-hydrated NucAway™ spin columns (Ambion, Huntingdon, Cambridgeshire, UK) to remove free nucleotides from the transcription reaction and further purify the cRNA samples. These columns were centrifuged at 1200 g for 2 min. Eluted cRNA concentration was determined by absorbance at A260. Copy number/μL of cRNA was calculated according to the following formula, available from the Roche Lightcycler™ website:Sense cRNA transcripts were generated by 9 cRNA copies/μL to 1 × 104 cRNA copies/μL, in 10-fold increments) for each product-specific cRNA molecule generated. Serially-diluted cRNA standards were reverse transcribed in a 20 μL final volume as described above, using transcript-specific reverse primers , 1.0 μL forward primer (0.5 μmol/L), 1.0 μL reverse primer (0.5 μmol/L), 1.0 μL specific probe (200 nmol/L) . Hydrolysis TaqMan probes were synthesized for each amplicon to be quantified (TIB MolBiol Syntheselabour, Berlin, Germany), and are shown in Table The experimental protocol used for TaqMan probe quantitative analysis consisted of two stages: initial denaturation (95°C for 10 mins), followed by 45 cycles of denaturation (95°C for 0 s) and annealing/extension (59–61°C for 50 s). The annealing temperature used in each experiment was dependent on the melting temperature of both the primers and probe involved. Fluorescence data were acquired at the end of each annealing/extension cycle.T) values for each individual sample using a software algorithm, which allows initial mRNA concentration in each sample to be accurately quantified based on the standards used. Using this method removes user influence, as well as any influence of background fluorescence on the data. The fluorescence display mode used was F1/F2, which is the optimal setting for use with hydrolysis TaqMan probes. PCR products were isolated from capillaries after each program had finished and were visualised by electrophoresis on 1.5% agarose gels.Data analysis was performed using Lightcycler™ Second Derivatives Method software. This method automatically determines the threshold cycle . Multiple group comparisons were made using analysis of variance (ANOVA), which were followed by individual group comparisons using the Tukey HSD test, where appropriate.RT-PCR analysis of the Maxi-K α subunit gene, using primers designed to flank predicted splice sites, produced a variety of bands in samples of non-pregnant (NP), pregnant not-in-labour (PNL) and pregnant in-labour (PL) myometrium (data not shown). From this analysis, only two potential alternatively spliced exon-containing PCR products were identified in the three tissue sets assayed Figure . SequencQuantitative analysis of alternatively spliced exon expression was performed using sequence-specific hydrolysis TaqMan probes to analyse mRNA expression of these exons in non-pregnant myometrium and pregnant myometrium, prior to and after labour onset, as outlined. In order to correct for random errors from sources such as pipetting inaccuracies, separate real-time PCR reactions were performed in triplicate for each amplicon involved. Agarose gel electrophoresis, as well as sequencing analysis, confirmed the specificity of PCR products formed, yielding single product bands of the expected size (data not shown).T) values for each sample, as determined mathematically by the "Second Derivatives" method. Mean absolute cDNA copy number values for each probed amplicon involved, in each myometrial sample, were calculated and grouped per tissue set (i.e. NP, PNL, PL), as shown in Figure Quantitative results for each amplicon were obtained by determination of the threshold cycle  are demonstrated in Figure In this study RT-PCR analyses were performed to identify mRNA expression of the Maxi-K α subunit, and alternatively spliced exons of this subunit, in human myometrium in its non-pregnant state, and at term pregnancy, prior to and after labour onset. This was followed by quantitative real-time PCR, which was performed to determine the overall pattern of expression of the Maxi-K α subunit as well as expression of alternatively spliced exons of this subunit identified in these tissue sets.+ channels provide a potent repolarizing current through the efflux of K+ ions, thereby dampening cell excitability and promoting cell relaxation [2+ and voltage sensitivity is at least partly due to decreased levels of the Maxi-K channel [2+ levels without the activation of an opposing K+ conductance [Our findings indicate a trend towards a decrease in α subunit mRNA levels with human labour onset. In order to maintain the uterus in a quiescent state during pregnancy, Klaxation . Previoulaxation  identifilaxation  indicate channel . Our finductance .Maxi-K channels derive their molecular diversity by alternative splicing of their α subunit transcript at several key sites, which generate channel variants with distinct phenotypes ,15,16. P2+ sensitivity of the Maxi-K channel [Quantitative real-time PCR was used to determine whether identified spliced exons displayed altered expression in pregnancy and/or with labour onset. The use of hydrolysis TaqMan probes provided high reaction specificity and sensitivity, and allowed for highly accurate quantification of target sequences. Our results indicate that there was no significant change in expression of the 87 bp spliced exon, in absolute terms, in non-pregnant (NP), pregnant not-in-labour (PNL) and pregnant in-labour (PL) myometrium. Furthermore, analysis of the expression of this spliced exon as a proportion of the total α subunit expressed (i.e. the 87 bp exon-containing α subunit splice variant) also showed no differences between the three tissue sets assayed. The proportion of Maxi-K α subunits expressing this variant was very low, accounting for only ~1% of Maxi-K channels expressed in the three tissues sets. Little is currently known about the physiological effects of expression of this variant of the Maxi-K channel. However, as described above, it contains a consensus sequence for protein kinase phosphorylation; therefore, it may have important consequences for post-translational modification of channel function. Also, the region into which this exon is inserted, between hydrophobic regions S8 and S9 of the α subunit protein, is thought to be involved in determining the Ca channel .2+ and voltage sensitivities of the Maxi-K channel [2+ and voltage sensitivities of Maxi-K channels after labour onset [In contrast, while there was no significant change in the mRNA levels of the 132 bp spliced exon in absolute terms between the three tissue sets, there was a significant increase (~1.7 fold) in the proportion of Maxi-K α subunits expressing this exon with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. Messenger RNA for this 132 bp variant was expressed at much higher levels in comparison to the 87 bp variant, accounting for 9% of total Maxi-K channels in NP and PNL tissues, increasing to 15% at labour onset. An increased proportion of myometrial Maxi-K α subunits expressing the 132 bp spliced exon with labour onset in the human is of interest, as the presence of this exon has been shown to decrease both the Ca channel . Thus, our onset  to reducur onset . The exa2+ and Maxi-K channel activation would permit Ca2+ levels in the cell to rise without the activation of an opposing K+ conductance, hence increasing the availability of Ca2+ for myometrial contraction [2+ and voltage levels, is responsible for enhanced uterine activity at the time of labour. Whether these variants are assembled as homo- or hetero-tetramers at the plasma membrane remains to be determined. Further investigations are required to assess factors such as the role of β-subunit attachment and modulation of the calcium bowl, and their input to regulation of Maxi-K channels during human pregnancy and labour. This study provides the first quantitative analysis of Maxi-K α subunit mRNA expression in human myometrium, and also highlights alternative exon splicing as a potentially important control mechanism by which myometrial Maxi-K channels may be modulated to suit their functional requirements during these physiological processes.Following the onset of labour, the putative disabling of the link between Catraction . Our finMC designed the quantitative RT-PCR techniques and carried out all experimental work. JJM recruited patients, organised the collection of tissues, and conceived of the study. TJS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


This article considers some of the effects of health sector reform on human resources for health (HRH) in developing countries and countries in transition by examining the effect of fiscal reform and the introduction of decentralisation and market mechanisms to the health sector.Fiscal reform results in pressure to measure the staff outputs of the health sector. Financial decentralisation often leads to hospitals becoming "corporatised" institutions, operating with business principles but remaining in the public sector. The introduction of market mechanisms often involves the formation of an internal market within the health sector and market testing of different functions with the private sector. This has immediate implications for the employment of health workers in the public sector, because the public sector may reduce its workforce if services are purchased from other sectors or may introduce more short-term and temporary employment contracts.Decentralisation of budgets and administrative functions can affect the health sector, often in negative ways, by reducing resources available and confusing lines of accountability for health workers. Governance and regulation of health care, when delivered by both public and private providers, require new systems of regulation.The increase in private sector provision has led health workers to move to the private sector. For those remaining in the public sector, there are often worsening working conditions, a lack of employment security and dismantling of collective bargaining agreements.Human resource development is gradually being recognised as crucial to future reforms and the formulation of health policy. New information systems at local and regional level will be needed to collect data on human resources. New employment arrangements, strengthening organisational culture, training and continuing education will also be needed. This paper considers health sector reform and its impact on human resources for health (HRH) in developing countries and countries in transition. Health sector reform has been defined as the "sustained purposeful change to improve the efficiency, equity and effectiveness of the health sector" . Health The introduction of new budget management systems, designed to maintain financial control throughout government, is one of the most important elements of fiscal reform. These incorporate new financial planning and control systems that emphasise what outputs a department or agency will be expected to deliver. Overall, there is a focus on the performance of public services . MechaniFiscal reform introduces new ways of allocating resources in line with government objectives . There mHealth sector employees are often part of the civil service, and so civil service reform has an impact on the employment and deployment of health workers. Civil service downsizing results from policies to cut the costs of the public sector and transfer the delivery of services to the private or non-profit sectors. These changes lead to a reduction in the size of the public health care workforce. Compensation schemes may include retraining and lump-sum severances to ease the transition of workers into the private sector. This may be accompanied by wage policy reform to limit and contain wage expenditures, again with the potential to affect health workers .Trying to improve the performance of the health sector, one of the objectives of health sector reform, has been a slow process because the savings from reducing the size of the workforce are often not enough to raise salaries for the remaining staff. Several countries, including Zambia, have set up a separate health service agency, operating as a semi-autonomous government agency, that employs staff directly. Some writers argue that agencies need to be well-managed on a limited budget rather than be seen as an escape from civil service restrictions . In addiThe decentralisation of budget management is another element of fiscal reform. In the health sector, this has been reflected in the decentralisation of service provision to semi-autonomous hospitals, because hospitals often consume the largest part of the health sector budget . Set up The motivations for introducing corporatisation may also vary from wanting to increase efficiency and achieve cost saving and quality improvements to just wanting to "free up a public function from constraints of ... red tape" . CasselsIn Mexico, new systems of financial management affected public health institutions by restricting the maintenance and upgrading of equipment and imposing cuts in the wages of health workers. This has led to the deterioration of working conditions and the quality of care provided by the public health sector .Part of a programme of fiscal reform involves the development of new systems and structures of financial management, which have organisational implications . The rolIn health systems, new forms of financing for the health sector may involve moving from a tax-based system to an insurance system. This, in turn, introduces new forms of budgetary management and control between insurance funds and health service providers as well as new systems of payment collection. Financial management is often accompanied by new information technology systems, which have the potential to change the ways in which public services are monitored .The introduction of market mechanisms is often driven by the goal of fiscal stability. Stronger systems of budget and management control are introduced that focus on results. They affect sectoral priorities and available human resources. Market mechanisms may be introduced by making health care institutions operate within an internal health care market and subjecting some health services to wider market testing.As part of developing a managed market in the public sector, the health sector is often reorganised into two separate purchasing and provider functions. The purchasing entity, typically a national or regional health authority, buys services from provider units within the government sector and is also encouraged to buy services from a range of providers in the private and NGO sectors . This haUser charges have been introduced as a way of generating income for the health sector. User fees in many countries have affected access to services and equity ,13. In NA recent World Bank report (2002) presents informal payments as a hindrance to health sector reform. Payments made to health workers are considered to draw resources away from the health care system because they are given to individuals rather than institutions. Such payments operate as a private, unregulated system and the practice is often illegal. Poor people often avoid using health care facilities because of the need to make informal payments . StrongePublic sector and health sector reform often introduce new approaches to managing staff. Perhaps the most important innovation is "thinking differently about staff", which effectively underpins other changes. The three most innovative dimensions are "flexible staffing and recruitment practices, recognising achievement and developing performance contracts" .The element of fiscal reform that emphasises outputs and outcomes of government services informs the development of performance management. It aims to address management problems relating to poor employee performance management, wage and non-wage incentives, job classification systems and ineffective payroll and personnel systems. Performance management may also be introduced as a way of improving standards within public services and making services more responsive to citizens. Wider programmes of training and capacity building for staff can accompany this. Some developing countries have experimented with performance management systems, with limited success . Often tThe delegation and decentralisation of administrative and management processes often accompany budgetary reforms. In Nicaragua, decentralisation was used to introduce market reforms. Budget cuts, loss of resources from primary health care, user fees and privatisation were introduced at the same time . In 1991In Uganda at the time of decentralisation, salaries for staff on the payroll were a central responsibility, although this has now been decentralised through a special conditional grant. In the past, professional staff were put on the national payroll and nursing aides were hired locally for work in rural health centres and health posts and paid for by the Ministry of Local Government. After the decentralisation reforms, nursing aides were supposed to be paid by local committees, but in practice this often did not happen and they were not paid for long periods .Botswana and Tanzania have had long experience of decentralisation. As a result of health sector reform, health staff were transferred to local government contracts although senior staff remained employed by the Ministry of Health. This has led to confused loyalties and management responsibilities. In some districts the "personality factor" has meant that individuals working together have managed to overcome some of these problems, in spite of the systems introduced. Senior staff who have subsequently been transferred to local government complain that that there is "little relationship between promotions/disciplinary actions and performance". In both countries there is some scope for local decision-making in relation to personnel management, but there is still resistance to distributing staff according to local needs. More incentives and other measures are considered necessary if regional imbalances of staff are to be addressed .Decentralisation may lead to a loss of resources for the health sector. In Uganda, after decentralisation, once central government stopped a block grant, primary heath care was not given the allocation at local level that had been expected by the Ministry of Finance. There were also considerable district variations in the allocation of health resources. Although some districts did increase their health allocation, in many cases decentralisation led to fewer resources for health. One of the reasons cited for the decline in allocation of resources to the health sector was that a large part of the health budget goes on salaries and wages, which do not show any dramatic change in the sector. Decentralisation in this context led to problems of financial management and corruption at local level, new problems of governance with a lack of accountability and concerns over quality of services .Some changes have run contrary to the main aims of reform, such as increased centralisation of controls over pay. Much health sector reform was to strengthen and rationalise budgeting, financial control and staff classification, but in some cases control over health sector staffing has remained at national level . Even whThe use of the private sector as a health service provider has had implications for the recruitment and retention of staff in the public sector. Some services have been privatised and are now run by local, national or international private companies. Other services have been contracted out to both private and non-profit service providers. This has resulted in movement of health workers from public to private or non-profit sectors .In the public institutions that remain, market conditions have been introduced and services are contracted out, which has resulted in a widespread decrease in job security in many countries. Health workers have moved from collective-bargaining arrangements to individual contracts. Decentralisation and privatisation have contributed to the breakdown of national collective bargaining. In Eastern and Central Europe, new organisations and professional associations and reorganised trade unions have led to a breakdown in labour relations expertise .Changes of responsibility for managing health services, from national to local level and from public to private sectors, have led to some confused accountabilities for health workers . Health The process of health sector reform has had an impact on human resources for health through new systems of financial and performance management, decentralisation and the introduction of market mechanisms. This has led to changes in the demand for health workers and in some cases the types of skills and expertise required from health workers. At the same time, the capacity of the new management systems is unable to create conditions in which a new health workforce can be developed. This can be seen particularly clearly in the process of budget decentralisation, which leads to a focus on local decision-making but where the capacity of local institutions to recruit, train and manage local health service workers is limited. This has an influence on the quality of health care delivered.Demand for human resources in health systems that have experienced health sector reform must be considered in terms of the numbers of health workers and the skills and expertise needed currently and what will be anticipated in the future. There is a growing awareness that human resource issues need to be prioritised more effectively within reforms in order to secure an adequate health care workforce to deliver services now and in the future.Although health sector reform has included elements of human resources strategies such as improved education and training, restructured salary scales and a closer link between performance and reward, it has also had a fundamental impact on organisational culture and public sector ethos, which in turn influence demand for human resources.A study of four countries in Eastern and Southern Africa concluded that "human resource development, personnel management and staff motivation are critical issues" . TanzaniBurkina Faso introduced health sector reforms in 1991 but they have not been fully implemented. It has recently introduced civil service reform, which "aims at a more flexible management and better performances of personnel". In a country where services are centralised, with an imbalance in personnel and low staff motivation and poor standards of care, there is resistance to the new reform. There has been a decline in standards of service between 1986 and 1997 . Poor fiMatheson (2002) points out that "the least systemically orientated area of recent public management reforms has been human resource management.... There is a danger that the constitutional, legal, cultural and leadership factors, which together create what is important and distinctive about public services, are not reflected on, or are dismissed as the bureaucratic problem which must be 'reformed' " .Some national health sector reforms reduced the numbers employed in the health sector, as in Chile and Latvia. Others, as in Mexico and Zambia, led to a rise in employment . In almoHealth services have usually been seen as "essential services" and so health workers had the legal status of public servants. They had to account to both employers and professional bodies subject to strict regulation and registration rules. The effect of privatisation has been to change the pay and terms of employment and the legal status of health workers . The pubThe introduction of flexible contracts and fall in full-time permanent contracts has been a characteristic of most reforms, leading to a reduction in long-term employment security. Some of these changes in terms and conditions have led to health workers' taking on second jobs. This may be caused by the increase in part-time employment and low and erratically paid wages . In EastLow pay levels have led to staff leaving the public sector and moving to the private sector, NGOs and aid agencies . Low payThese changes in organisational culture have often had a negative impact on workers' motivation. Important informal factors – for example, staff commitment – have "become the prime means of direction, motivation, coordination and control" . When stThe aim of introducing market mechanisms to the public sector has been to improve economic efficiency. New skills are needed to implement commissioning and contracting of services. For example, contracts can be a powerful form of regulation if drawn up and monitored effectively, but increased expertise is required to establish this form of regulation.Understanding the process of reform is important for understanding how changes have taken place but also what the critical factors are for successful policy implementation in future. "The process of reform offers numerous opportunities to alter the political equations that impede change" . This isIn Latin America, health sector reform has been characterised by various forms of privatisation, competition among providers, new insurance systems, management autonomy for hospitals and increased community participation. The goals were efficiency, accountability and improved quality of services. A recent study looked at how groups, such as unions, play different roles in relation to reforms with some opposing and others supporting reform. Some public sector health workers have played an important role in supporting change . The impCommunities also influence health worker motivation through their expectations of services. As health sector reform also aims to empower service users, this focus will have a significant impact on the individual health worker in future . The eviHowever, health workers do play an important role in the implementation of health sector reform policies. The lack of consideration of the value of human resources in health sector reform programmes has meant that this has often been ignored. Gilson et al. (2003) recommended that: "Technical analysts might consider working with middle managers and health workers to ensure adequate consideration is given to implementation realities in proposal development" .The process of health sector reform is not complete. More research is needed to monitor changes still taking place as well as the outcomes of the reforms. One of the major changes is the role that health workers play in both the public and private sectors. How health workers perceive their roles in the different sectors and what the implications are for motivation, particularly in the public sector, will need further exploration.The role of the public sector is changing and this is reflected in public sector institutions and the public sector ethos. How this changed public sector can demonstrate a commitment to health workers as well as harnessing their own commitment, still needs to be explored.A further area of research needs to improve the understanding of human resources in health sector appraisal studies "by incorporating functional, institutional and policy dimensions. Only then will human resources become in practice the most valuable resource within any national system" .There is a growing awareness that human resources for health (HRH) must be addressed more effectively within public sector reform. Stein thinks that HRH strategies need to be a "primary objective for public organisations" .Public sector reforms have sometimes been characterised as containing the paradox of aiming to reward performance and empower staff whilst at the same time implementing downsizing and redundancy – the "human costs of reform" . Issues To address some of these issues, action at strategic, regional and local levels will be needed to strengthen skills, expertise and analysis of HRH and to strengthen the integration of HRH issues into health policy making and with relevant agencies . New humAdditional data on existing employment, retention and deployment issues must be made available to decision makers so as to relate them to health equity issues . An incrThe working conditions of health workers need to be improved. This might be achieved through developing more flexible employment arrangements that are employee-focused. The public sector needs to be encouraged to establish a "living wage" and other forms of worker security so that terms and conditions of public sector workers are better than those of private sector workers . Health The role of central government in setting standards for professional practice and legal requirements for registration needs to be strengthened so that human resources policies, registration and regulation are mutually supportive. Registration requirements that include experience in rural or remote areas would help to address uneven distribution of health workers.The author(s) declare that they have no competing interests.


Saccharomyces cerevisiae can respond in a complex way to the presence of different energy sources. While yeast can metabolize many different sugars, glucose provides the highest energy yield. To achieve this energy efficiency, yeast cells rely on specialized enzymes and metabolic states that mediate glucose metabolism by sensing and responding to this sugar. The presence of glucose triggers a rapid and dramatic change in the expression of about a quarter of S. cerevisiae's 5,500 genes.An organism's survival depends on developing effective strategies for identifying and adapting to available sources of food. Even organisms as small as the budding yeast Many of the cellular participants in the glucose “sense-and-response” pathway have been identified, but their exact relationships remain unknown. The gene expression response to glucose has been previously characterized through the use of a cDNA microarray, which allows simultaneous assessment of the transcriptional state of every gene. In research reported in this issue, James Broach and his colleagues at Princeton University have attempted to connect the individual components in this complex pathway by performing microarray analysis on a series of mutants (yeast strains with defects in specific proteins). They link certain portions of the response to known proteins and begin to understand how the pieces of this pathway fit together.The researchers initially focused on two proteins, called Ras2 and Gpa2. These proteins have been previously implicated in the transcriptional response to glucose and are members of a well-established family of signaling proteins. To investigate the roles of these proteins in the response, the researchers used mutants of Ras2 and Gpa2 that could be activated on demand and then performed microarray analysis to see which genes responded to activation of these proteins. They found that even in the absence of glucose, activation of either protein induced a transcriptional profile almost identical to the profile generated in yeast exposed to glucose. This shows that the complex and dramatic transcriptional response to glucose can be recapitulated by the activation of a single protein.The Ras2/Gpa2 pathway, however, is not the whole story. The group then went on to show that not all glucose-responsive genes are regulated in the same manner. They found that another pathway, independent of Ras2 and Gpa2, is able to elicit a portion of the transcriptional response to glucose. The partial redundancy of these pathways is a curious phenomenon and bears further investigation.The authors have achieved an initial step in mapping the topology of the intricate signaling pathway (or pathways) involved in the response to glucose. Furthermore, the approach—that of using microarray analysis of mutants in a pathway to deduce the mechanisms of regulation—will be useful in efforts to map other complex responses in yeast as well as in higher organisms.


Dr. Gerberding outlines critical steps for arresting the HIV/AIDS epidemic


Enterococcus faecalis and Enterococcus faecium and is associated with a high mortality rate. Enterococcus casseliflavus has been implicated in a wide variety of infections in humans, but never in meningitis.Enterococcal meningitis is an uncommon disease usually caused by E. casseliflavus. The patient was successfully treated with meropenem and ampicillin-sulbactam.A 77-year-old Italian female presented for evaluation of fever, stupor, diarrhea and vomiting of 3 days duration. There was no history of head injury nor of previous surgical procedures. She had been suffering from rheumatoid arthritis for 30 years, for which she was being treated with steroids and methotrexate. On admission, she was febrile, alert but not oriented to time and place. Her neck was stiff, and she had a positive Kernig's sign. The patient's cerebrospinal fluid was opalescent with a glucose concentration of 14 mg/dl, a protein level of 472 mg/dl, and a white cell count of 200/μL with 95% polymorphonuclear leukocytes and 5% lymphocytes. Gram staining of CSF revealed no organisms, culture yielded E. casseliflavus can be inserted among the etiologic agents of meningitis. Awareness of infection of central nervous system with Enterococcus species that possess an intrinsic vancomycin resistance should be increased. Enterococcal meningitis is an uncommon disease accounting for only 0.3% to 4% of cases of bacterial meningitis which is nevertheless associated with a high mortality rate. It has been described most frequently in patients with neurosurgical conditions , although it can also occur as a "spontaneous" infection complicating remote enterococcal infections such as endocarditis or pyelonephritis .Enterococcus faecalis and Enterococcus faecium are the two species most frequently isolated during the course of meningitis (76%–90% and 9–22% respectively). Enterococcus casseliflavus, first considered as a subspecies of E. faecium, is a motile enterococcus that produces a yellow pigment in agar and often has a VanC phenotype determining an intrinsic low level resistance to vancomycin. It has been implicated in a wide variety of infections in humans, especially immunocompromised hosts, but to the best of our knowledge it has never been associated to meningitis [ningitis .E. casseliflavus that was believed to originate from the gut in an old patient with bowel erosions.We describe here a case of enterococcal meningitis caused by A 77-year-old Italian female presented for evaluation of fever, stupor, diarrhea and vomiting of 3 days duration. She had no urinary symptoms. There was no history of head injury nor of previous surgical procedures. She had been suffering from rheumatoid arthritis for 30 years, for which she was being treated with steroids and methotrexate; other medical problems were insulin-dependent diabetes and moderate renal failure.On admission, she was febrile (temperature, 38.0°C), alert but not oriented to time and place. Her neck was stiff, and she had a positive Kernig's sign. A CT brain scan showed an increase in subarachnoid space and in the volume of the ventricular system.Laboratory examinations revealed a white blood cell count of 15,100/μL with 70% neutrophils and 23% lymphocytes. Results of urinalysis were unremarkable. Cultures of blood and urine were drawn and subsequently resulted negative.The patient's cerebrospinal fluid (CSF) was opalescent with a glucose concentration of 14 mg/dl, a protein level of 472 mg/dl, and a white cell count of 200/μL with 95% polymorphonuclear leukocytes and 5% lymphocytes. Gram staining of CSF revealed no organisms. Pending the culture results the patient was empirically treated with intravenous meropenem, cotrimoxazole, acyclovir and dexamethasone.E. casseliflavus that was identified using the Vitek-2 system (bioMérieux-Vitek) on the basis of 6.5% NaCl tolerance, bile-esculin hydrolysis, and growth rate at 45°C, arginine hydrolysis, methyl-a-D-glucopyranoside testing, and acid production from ribose, motility testing, and yellow pigmentation testing.Culture of CSF yielded The isolate was sensitive to penicillin, ampicillin, ampicillin-sulbactam, imipenem, teicoplanin, tetracyclines and linezolid; it exhibited intermediate sensitivity to vancomycin (MIC, ≥8 μg/mL), trimethoprim-sulfamethoxazole (MIC, ≥10 μg/mL), levofloxacin (MIC, 4 μg/mL), norfloxacin (MIC, 8 μg/mL), ciprofloxacin (MIC, 2 μg/mL) and quinupristin-dalfopristin (MIC, 2 μg/mL); it was resistant to clindamycin (MIC, 4 μg/mL) and showed high resistance to gentamicin, streptomycin and kanamycin (MIC, ≥2000 μg/mL).The patient became afebrile 48 hours after the beginning of antibiotic therapy with rapid improvement of her mental status and disappearance of meningeal signs (within 36 hours).Once the organism was identified (4 days later), trimethoprim-sulfamethoxazole and acyclovir were discontinued and ampicillin-sulbactam (3 g every 6 hours) was added. After 2 weeks of antibiotic therapy the patient was discharged in good health with sterilization of the CSF culture. An echocardiogram revealed no vegetations whereas a colonoscopy examination showed two ulcerative lesions associated with two polyps, oedema and multiple punctuate erosions.Enterococcus identified as E. gallinarum have been previously documented; they occurred in patients with ventriculoperitoneal shunts for hydrocephalus [E. casseliflavus and E. gallinarum are responsible for 1–2% of all enterococcal infections and are characterized by the fact that they possess intrinsic low-level vancomycin resistance [E. gallinarum, and the VanC-2/3 ligase is specific for E. casseliflavus [Enterococcal infections of the central nervous system are quite rare and according to a MEDLINE search of the English literature only three cases of CNS infection by a motile cephalus ,7. E. casistance . The Vanliflavus . Organisliflavus ,9,10.E. casseliflavus it is important to remember that most strains are susceptible to penicillin and ampicillin. Combination therapy of ampicillin with an aminoglycoside such as gentamicin or streptomycin is considered the standard therapy of enterococcal meningitis due to ampicillin-susceptible strains [E. casseliflavus are beta-lactamase negative. There was not therefore a clear indication for the use of combination therapy with meropenem and a beta-lactamase inhibitor in the case reported.Despite the intrinsic low-level vancomycin resistance exhibited by  strains . MeropenE. casseliflavus.Our patient had gastrointestinal signs and symptoms, and colonoscopy revealed multiple erosions, which probably were the portal of entry of In fact, enterococcal meningitis may appear as a complication of diverse gastrointestinal diseases such as enterocolitis, peritonitis, abdominal surgery, or bowel carcinoma. A case of bowel erosions as the portal of entry of enterococcal meningitis has been previously reported .E. gallinarum and E. casseliflavus colonize the gastrointestinal tracts of both hospitalized individuals and nonhospitalized healthy ones [E. gallinarum and E. casseliflavus in healthy subjects who were administered oral vancomycin [E. gallinarum and E. casseliflavus/flavescens are part of the normal stool flora of the general population; this has perhaps impacted the ability of researchers to detect specific risk factors [Several studies have demonstrated that thy ones ,12,13. Tncomycin . Our pat factors .Although our patient had significant underlying conditions this case of meningitis was mild and had most of the typical features of "spontaneous" enterococcal meningitis (community-acquired infection, severe underlying diseases, and immunodepression).E. gallinarum and E. casseliflavus has been associated with high mortality, but it is difficult to attribute their mortality directly to infection or to the underlying conditions of the patient.The clinical significance of the enterococci that are intrinsically resistant to vancomycin has not been fully established yet. Infection with E. casseliflavus meningitis and it is the fourth documented so far with a motile Enterococcus species. Awareness of infection of central nervous system with Enterococcus species that possess an intrinsic vancomycin resistance should be increased.This case is the first report of The author(s) declare that they have no competing interests.IC and RDL carried out the clinical study of the patient and conceived of the study. GS and GBC carried out the microbiologic studies. AT and AC carried out the clinical study of the patient, conceived of the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The evidence base of clinical interventions in paediatric hospitals of developing countries has not been formally assessed. We performed this study to determine the proportion of evidence-based therapeutic interventions in a paediatric referral hospital of a developing countryThe medical records of 167 patients admitted in one-month period were revised. Primary diagnosis and primary therapeutic interventions were determined for each patient. A systematic search was performed to assess the level of evidence for each intervention. Therapeutic interventions were classified using the Ellis score and the Oxford Centre for Evidence Based Medicine Levels of EvidenceAny dehydration due to diarrhoea (59 cases) and pneumonia (42 cases) were the most frequent diagnoses. Based on Ellis score, level I evidence supported the primary therapeutic intervention in 21%, level II in 73% and level III in 6% cases. Using the Oxford classification 16%, 8%, 1% and 75% therapeutic interventions corresponded to grades A, B, C, and D recommendations, respectively. Overall, according to Ellis score, 94% interventions were evidence based. However, out of the total, 75% interventions were based on expert opinion or basic sciences. Most children with mild to moderate dehydration (52 cases) were inappropriately treated with slow intravenous fluids, and most children with non-complicated community acquired pneumonia (42 cases) received intravenous antibioticsMost interventions were inappropriate, despite the availability of effective therapy for several of them. Diarrhoeal dehydration and community acquired pneumonia were the most common diagnoses and were inappropriately managed. Existing effective interventions for dehydration and pneumonia need to be put into practice at referral hospitals of developing countries. For the remaining problems, there is the need to conduct appropriate clinical studies. Caution must be taken when assigning the level of evidence supporting therapeutic interventions, as commonly used classifications may be misleading Previous studies have shown that medical interventions based on scientific evidence range from 10 to 80% . These sIn paediatric practice, the proportion of evidence based interventions reported ranges from 75 to 91% . These sThe assessment of quality of care at referral level for severely ill children is an important component of the efforts for reducing the child mortality rate in poor countries, for reducing the burden on health systems and for investing money in favour of high priority health interventions ,10. ThusReferral care provided to children hospitalized in the paediatric department of the Instituto Especializado de Salud del Niño (IESN) was assessed for evidence base. IESN is a national paediatric hospital with more than 500 inpatient beds, serving mostly patients from deprived socioeconomic areas of Lima and inner cities of the country.The clinical records of 195 children aged 1 month through 16 years old and hospitalized during January 2003 were initially revised.One of the investigators (NYC) assessed the clinical records of children and assigned to each one a primary diagnosis and one or more primary therapeutic interventions, on the basis of the main clinical features and/or definitive diagnostic laboratory aids. Patients in whom a primary diagnosis was not possible to determine or those without a clear diagnosis were excluded.The primary intervention was defined as the treatment or other manoeuvre that represented the most important attempt to cure, alleviate, or care for the patient in respect of his or her primary diagnosis .To determine the level of evidence for each primary intervention, Cochrane reviews were searched. If there was not such a systematic review, a search through PubMed (MEDLINE) was performed by one of the investigators (NYC). All Cochrane reviews were searched through their own search tools. For PubMed, the period of search was 1966 through 2002. The key words used included those related to the primary diagnosis (e.g., pneumonia). Limits: "Title", "All child: 0–18 years". Publication type was sequentially searched for "Practice Guideline", "Meta-Analysis", "Randomized Controlled Trial" and "Review". Articles in English or Spanish were included. The National Guideline Clearinghouse was additionally visited for additional references. Published recommendations for judging the quality of guidelines were used for deciding the selection of the guidelines . In addiThe level of evidence assigned to interventions was based on Ellis score (levels I, II and III) and the Oxford Centre for Evidence Based Medicine Levels of Evidence (grades A, B, C and D) ,12. ElliAs a next step to our study, we planned the dissemination of the results among the hospital policy makers and the suggestion of corrective courses of action for those interventions needing improvement.Results of the search strategies for PubMed are included as an appendix See . A guideOverall, one hundred and ninety five clinical records were revised. Twenty eight clinical records were excluded because of undefined primary diagnosis Table . One hunOut of 167 primary interventions, 21% were supported by level I evidence, 73% were level II, and 6% were ranked as level III, according to Ellis classification .Most assessed interventions were considered as level II Table . DiarrhoOvertly unsubstantiated therapy according to Ellis classification is shown in Table Comparison of grade of recommendation of the prescribed intervention with the appropriate one is shown in Table In this study 94% of therapeutic interventions were evidence-based by Ellis score. It may seem encouraging that more than 90% of therapeutic decisions in a referral paediatric hospital of a developing country are evidence based. However, the level II of evidence from Ellis includes interventions based in cohort studies, case-control studies, case series, expert's opinion, and even those based in basic sciences. We attempted therefore to classify the primary interventions according to more specific criteria. Using the Oxford classification, 75% of therapeutic interventions were based in expert opinions or in basic sciences (Grade of Recommendation D).Some limitations of our study include the possible author' bias when assigning the primary diagnosis and primary intervention. The assignment of diagnosis by the clinician may have been influenced by both the choice of treatment and the available evidence. Only one of us classified the primary intervention. In addition, we evaluated only a primary intervention for a single primary diagnosis. Actually, many patients had more than one diagnosis and obviously more than one therapeutic intervention.We used as evidence-base for rating the interventions assessed in our study, guidelines and evidence-based resources published in the developed world. This raises the issue of whether they are fully applicable to our setting. The most prevalent conditions found were diarrhoeal dehydration and community acquired pneumonia. For both of them we used British produced guidelines ,21 becauRegarding community acquired pneumonia, the British guidelines recommend antibiotic treatment for all children with pneumonia, due to the difficulties in identifying the aetiology, and they also specify criteria for hospitalization . These rWe acknowledge that the evidence derived from studies performed in developed countries should be translated with caution to developing settings. However, when the native research is scarce or of low quality, we think that the transfer of knowledge from the developed countries is an acceptable approach, as far as the particular characteristics of patients in developing countries are considered on an individual basis.Dehydration due to diarrhoea and pneumonia were the most frequent diagnoses. Oral rehydration for diarrhoeal dehydration and antibiotics for pneumonia are considered as interventions with sufficient evidence for implementing them widely ,10. In oIn addition to their enormous potential for saving lives, outpatient antibiotic therapy for pneumonia and outpatient oral rehydration can drastically reduce the rate of hospitalizations, the hospital stay, the hospital mortality rate, and the costs incurred. At our hospital, the mean stay time for hospitalized children is 4.7 days, and the mean crude mortality rate is 3.6%. We estimated the cost of managing hospitalized children with pneumonia and diarrhoeal dehydration as US$ 10.6/day and US$ 8.6/day, respectively. These costs are referred only to hospital bed and laboratory tests. A substantial amount of money could be saved treating these conditions on an outpatient basis.We planned the dissemination of our results among the hospital policy makers. The tools that will be suggested for improving the standards include the development and systematic application of locally produced guidelines and/or the adaptation of published guidelines. A useful alternative that has been experienced for several years at our inpatient ward unit is to make available personal computers connected to Internet for attending physicians, residents and interns, and to encourage the use of online evidence-based resources. This last alternative may work better, particularly where there are motivated physicians who are able to lead the efforts for improving the health care standards. However, the ultimate decision to systematically introduce and monitor the suggested changes will rest on hospital managers. Such changes should also depend on taking into account the role of several other determinants of the clinical decision making by individual practitioners, such as continuous training, motivation, time, availability of drugs, equipments and supplies, supervision, and long-term health system strengthening strategies.Caution must be taken when assigning the level of evidence that supports therapeutic interventions, as commonly used classifications may be misleading. Existing effective interventions for dehydration and pneumonia are not being implemented in developing countries, even at referral level, and thus there is the need to change the current medical interventional behaviours. For the remaining diagnoses, the majority of assessed interventions were based on weak or non-existent evidence, highlighting the need to conduct appropriate clinical studies.The author(s) declare that they have no competing interests.NYC and LH conceived and designed the study. All authors analyzed and interpreted the data and contributed to earlier drafts of the manuscript.The pre-publication history for this paper can be accessed here:"Appendix: Literature search results" details of the Medline (PubMed) search strategy and of the results are provided in this additional file. The selected articles are in black, bold characters. In addition, the search results from the National Guideline Clearinghouse website are provided for painful crisis in sickle cell anaemia.Click here for file


The amino acid composition of a low molecular weight chromium binding peptide (LMWCr), isolated from bovine liver, is reportedly E:G:C:D::4:2:2:2, though its sequence has not been discovered. There is some controversy surrounding the exact biochemical forms and the action of Cr(III) in biological systems; the topic has been the subject of many experimental reports and continues to be investigated. Clarification of Cr-protein interactions will further understanding Cr(III) biochemistry and provide a basis for novel therapies based on metallocomplexes or small molecules.Oryza sativa). A broader search for pentameric sequences (and assuming a disulfide dimer) corresponding to the stoichiometric ratio E:D:G:C::2:1:1:1, within the set of human proteins and the set of proteins in, or related to, the insulin signaling pathway, yields a match at an acidic region in the α-subunit of the insulin receptor (-EECGD-, residues 175–184). A synthetic peptide derived from this sequence binds chromium(III) and forms a metal-peptide complex that has properties matching those reported for isolated LMWCr and Cr(III)-containing peptide fractions.A genomic search of the non-redundant database for all possible decapeptides of the reported composition yields three exact matches, EDGEECDCGE, DGEECDCGEE and CEGGCEEDDE. The first two sequences are found in ADAM 19  proteins in man and mouse; the last is found in a protein kinase in rice (The search for an acidic decameric sequence indicates that LMWCr may not be a contiguous sequence. The identification of a distinct pentameric sequence in a significant insulin-signaling pathway protein suggests a possible identity for the LMWCr peptide. This identification clarifies directions for further investigation of LMWCr peptide fractions, chromium bio-coordination chemistry and a possible role in the insulin signaling pathway. Implications for models of chromium action in the insulin-signaling pathway are discussed. EECGD-, residues 175–184) and conforms to the predicted stoichiometry. Further, visible absorption data and electron paramagnetic measurements of the synthesized disulfide linked dimeric Cr-peptide complex are identical to that of the biologically isolated fractions.Type II diabetes continues to grow as a worldwide epidemic; it is expected that this disease will surpass 300 million cases by 2025 -5. Undernr) protein database for their occurrence. Based on a simple model where all amino acids are equally possible and about 1.5 million sequences of average length 200 present in the nr database, the E-value for an independent exact match is about 0.55 [(0.05)10 * 200 * 1.5 * 106 * 18,900].Recent reports ,21,24, unr database corresponding to 3 unique permutations: EDGEECDCGE, DGEECDCGEE and CEGGCEEDDE. One match, CEGGCEEDDE, was found only in one sequence annotated as a putative protein kinase in rice (Oryza sativa). The peptide sequences, EDGEECDCGE and DGEECDCGEE, both matched the same region of the disintegrin domain of the human and mouse versions of ADAM 19  [bos taurus. This acid-rich sequence region (Table We found perfect matches in the D: 8728) . Since tnr database is much higher, ~104. Restricting this search to the human proteome gave rise to 439 (expected: ~102) matches, with no reason to prefer one over the other.Noting that the reported stoichiometry consists of even numbers of amino acids, we further considered the possibility that LMWCr might be a disulfide-linked dimer, with each monomeric unit having the composition, E:D:G:C::2:1:1:1. Such a hypothesis is consistent with the limited resolution of the experimental data used to derive the stoichiometry  and yielConsidering that LMWCr might be a subsequence of a protein related to "insulin" or known to be involved in the insulin signalling pathway, we compiled a set of 96 such sequences. This set comprised two components: 1) proteins playing a role in the insulin signaling pathway (23 protein sequences were selected) derived from pathway charts, and 2) the set of all protein sequences derived from SwissProt for the search, "insulin + human" (78 entries). The two components were compared for redundancy and the duplicates removed (5 instances).-1). Comparison of the pentapeptide set to a more detailed insulin signaling pathway construct [A cross comparison of the resulting 439 entries using the BLAST query method versus the set of 96 pathway and insulin-related proteins results in a unique match for one of the 60 pentapeptides, EECGD, within the insulin receptor (INSR), residues 180–184  complexes, i.e. proteolysis may lead to a group of similar peptides that differ by one or more amino acid residues on either side of the Cr binding site. This interpretation differs significantly from reports on the isolation of LMWCr, which attempt to avoid a proteolytic product.This sub-sequence lies in the extracellular α-subunit of INSR in an acid-rich region (-NKDDNEECGD-) towards the end of the L1 domain, and at the start of the "cysteine-rich region". A BLAST query  for all KECGDLCPGTL-. The cysteine in -KECGD- forms a disulfide bond with another cysteine residue 28 positions away in the crystal structure. However, we do not know if this is due to the crystallization conditions or representative of the natural form. Further, the insulin receptor possesses 3 additional cysteine residues compared to the insulin-like growth factor receptor, thus the pattern of pairing of cysteine residues to form disulfide bonds may be different in the two receptor types. The observed difference may be great enough to preclude Cr(III) acting on the insulin-like growth factor receptor.A crystal or solution structure of this region of the insulin receptor has not been determined. However, the crystal structure of a homologous molecule, the insulin-like growth factor receptor (IGR1, GeneID: 124240) has been published ,31. Sequ2) and generated the disulfide-linked dimer via air oxidation. This peptide was subjected to conditions for the reconstitution of apo-LMWCr to generate a holo-peptide complex. Incubation of the peptide with fresh solutions of CrCl3 results in a clear gray-green solutions of a Cr-peptide complex with visible electronic spectra 2 and (AcNEECGD-CONH2)2 indicate that Cr exists in trinuclear arrays in these complexes. A preliminary spectrum is shown in Figure The identified sequence in hand, we synthesized the pentapeptide (AcEECGD-CONHBarring the discovery of a novel, unsequenced or unidentified protein or peptide, these data point to the possible sequence of LMWCr fractions and may point to new strategies in therapeutic design. In addition, the question of sequence specificity in Cr(III)-peptide complexes must be fully addressed, along with thermodynamic and kinetic aspects of Cr(III) binding and transfer.in vitro studies suggest that Cr(III) replacement in the physiological iron transport and storage apparatus may lead to some small beneficial outcome for certain diseases [in vitro experiments (including our own) may be finally ascertained only after the fact.Models of non-toxicological action of Cr(III) in biological systems may broadly fall into three categories: structural, redox, and iron homeostasis. The earliest models , and thodiseases . The bioextracellular model for Cr(III) biochemistry with respect to insulin signaling may be plausible (see Supporting Information). Such a structural model would include the known aspects of INSR cycling and insulin degradation [intracellular mechanisms for LMWCr action [Although unexpected, the results in this report and a critical review of other literature ,11-18, sradation , and incradation  of a terr action ,18, redor action , and ther action . In addir action  may satiThis model is a parsimonious alternative to current proposals of Cr action in the insulin signaling pathway. However, this model points directly back to significant kinetic and a thermodynamic questions about Cr(III) in biological systems. For instance, what is the physical form of Cr in the bloodstream? How is Cr(III) transported and exchanged between ligands in the serum? Is transport specific? What structure/activity relationship exists in Cr(III) complexes to allow their transport across biological membranes? Thermodynamically, a hydrolyzed, multinuclear Cr cluster should predominate at neutral pH, but transport by transferrin would presumably be in the mononuclear Fe binding sites. Alternatively, Cr(III) clusters may be transported non-specifically in serum by proteins, possibly including transferrin and serum albumin. At this point, there exist significant gaps in understanding the possible biochemistry of Cr(III) and what molecular processes it may affect.The proposed extracellular model of Cr(III) action in this report is upstream of IRS1, a therapeutic target of White and others ,8, and mA bioinformatic search for an acidic decameric sequence matching reported stoichiometries of LMWCr amino acid composition indicates that the peptide may not be a contiguous sequence. An expanded search localized a pentameric sequence in the insulin receptor and suggests a possible identity for the Cr(III)-containing peptide fractions derived from liver. Disulfide linked penta- and hexameric peptides based on the identified sequence bind Cr(III) in a similar fashion to LMWCr fractions reported in the literature.nr database was downloaded and Perl scripts used to search for exact matches corresponding to all unique permutations of EEEEGGCCDD. In a separate search, a set of 78 unique protein sequences from Swiss-Prot were obtained as a result of using the query "insulin human." This set was searched for exact matches corresponding to all unique permutations of EEGCD. In addition, the human proteome was also downloaded and searched for matches to the same set of pentameric peptides.The 2, was synthesized by continuous-flow automated solid-phase synthesis on a Perseptive Biosystems Pioneer Peptide Synthesis System. Peptide synthesis was performed by using standard Fmoc-protection strategies with TBTU/DIEA activation strategy. Typically, a 0.5 mmol scale, using Rink amide resin (loading ~0.65 mmol/g) and four times excess of the other reagents (TBTU, protected amino acid) were used. After the solid-phase synthesis was complete, acetylation at the amino terminus was carried out for 2 hours (50:50:1:: acetic anhydride:dimethylformamide:pyridine), and the resulting peptide cleaved from the resin. The peptide was cleaved from the solid phase resin by adding a slurry of 94% trifluoroacetic acid, 2.5% of water, 2.5% of ethanedithiol, and 1% of triisopropylsilane to the reaction vessel and shaking it for 5 h. The trifluoroacetic acid, containing the peptide, was filtered off by vacuum, and the resin was washed 2–3 times with trifluoroacetic acid. The filtrate was evaporated under nitrogen gas until the volume was reduced to 15 mL. 30 mL of ice-cold diethyl ether was added to the filtrate, causing the peptide to precipitate, and the mixture centrifuged to form a pellet of the peptide. The diethyl ether was decanted and the peptide pellet was washed with diethyl ether three times. Finally, the peptide was dissolved in 20 mL water containing 0.1% trifluoroacetic acid and extracted with diethyl ether three times. The aqueous portion was collected and freeze-dried to give the synthetic peptide. AcEECGD-CONH2, has a retention time of 5.3 minutes on a 250 mm × 4.5 mm C-18 reversed phase HPLC column using a gradient of 5 to 20% acetonitrile in 0.1% trifluoroacetic acid/water mobile phase running at 1 mL per minute and detected at a wavelength of 220 nm.The peptide, AcEECGD-CONH2, was dissolved to 10 mg/mL in a 0.1 M solution of ammonium carbonate at pH 7 and allowed to oxidize under ambient air for 48 hours. The product was isolated by lyophilization and analyzed by HPLC. The disulfide peptide dimer, (AcEECGD-CONH2)2, has a retention time of 6.0 minutes using the above conditions. The product, (AcEECGD-CONH2)2, has a calculated mass of 1183.35141 and exhibits a mass of 1183.3477 Daltons when analyzed by electrospray mass spectrometry.The synthetic peptide, AcEECGD-CONH2)2 was weighed and dissolved in 30 mL of water. A portion of this solution (5 mL) was taken up in a 25 mL tube, and 4 equivalents of chromium(III) chloride were added as a solid or in an aqueous solution. The reaction of the two components took several minutes and was monitored by ultraviolet-visible spectrophotometry. The chromium peptide complex, Cr3O(AcEECGD-CONH2)2, exhibits characteristic spectral absorbance features at 432 nm and 615 nm. The features are consistent with chromium bound to oxygen atom donors and similar to the reported spectrum of LMWCr [Fifteen milligrams of  complexes and drafted the manuscript, tables and graphics. All authors read and approved the final manuscript.2 and proposed Cr(III) model at INSR. Supporting tables (2) include FASTA sequences for insulin signaling map (20 proteins) and pentameric peptides found in genomic search (439 entries)Supporting figures (3) include HPLC and MS data for AcEECGD-CONHClick here for file


DJ-1 were recently described in two families with autosomal recessive Parkinsonism, one of which is a large deletion that is likely to lead to loss of function. Here we show that embryonic stem cells deficient in DJ-1 display increased sensitivity to oxidative stress and proteasomal inhibition. The accumulation of reactive oxygen species in toxin-treated DJ-1-deficient cells initially appears normal, but these cells are unable to cope with the consequent damage that ultimately leads to apoptotic death. Furthermore, we find that dopamine neurons derived from in vitro–differentiated DJ-1-deficient embryonic stem cells display decreased survival and increased sensitivity to oxidative stress. These data are consistent with a protective role for DJ-1, and demonstrate the utility of genetically modified embryonic stem cell–derived neurons as cellular models of neuronal disorders.The hallmark of Parkinson's disease (PD) is the selective loss of dopamine neurons in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in rare cases, indicating a genetic factor. Furthermore, pathological analyses of PD substantia nigra have correlated cellular oxidative stress and altered proteasomal function with PD. Homozygous mutations in  Dopaminergic neurons, derived from DJ-1-deficient embryonic stem cells, display decreased survival and increased sensitivity to oxidative stress Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by rigidity, slowed movement, gait difficulty, and tremor at rest . The patα-synuclein lead to a rare familial form of PD  . Of note, a mutation that encodes a similarly truncated protein (at the human DJ-1 exon 7 splice acceptor) has been described in a patient with early-onset PD  cell clone, F063A04, that harbors a retroviral integration at the .gsf.de) . This inonset PD .DJ-1 allele, clone F063A04 was exposed to a high dose of the antibiotic G418, which selects cells that are homozygous for the neomycin resistance gene insertion (DJ-1 locus) were identified by Southern blotting -2,5-diphenyltetrazolium bromide [MTT] by metabolic enzymes) in triplicate polymerase-1 (PARP) in a pattern indicative of an apoptotic death program  and FACS analysis. Initial ROS accumulation (at 15 min after stimulation) appeared unaltered in the DJ-1-deficient cells in comparison to control heterozygous cells , a DN-specific toxin that enters DNs through the dopamine transporter and leads to oxidative stress and apoptotic death . DJ-1-deDJ-1. DJ1-shRNA virus-infected cells displayed efficient silencing of DJ-1 gene expression to 10%–20% of control vector-infected cultures .To confirm the role of DJ-1 in primary midbrain DNs, DJ-1 expression was inhibited by RNA interference (RNAi) in embryonic day 13.5 (E13.5) murine primary midbrain cultures by lentiviral transduction of short hairpin RNAs (shRNAs)  Rubinso. E13.5 mblotting Q). TransDJ-1 shRNA virus and with control vector displayed similar numbers of TH-positive neurons in the absence of exposure to H2O2   . Total esurvival T. These 2O2, but they are unable to cope with the consequent toxicity, culminating in apoptosis. Additionally, we find that DJ-1 deficiency sensitizes cells to the proteasomal inhibitor lactacystin but not other toxic stimuli such as tunicamycin. Proteasomal inhibition induces the accumulation of short-lived and misfolded cytoplasmic proteins, leading to oxidative stress and apoptosis  for 18–36 h according to the manufacturer's instructions . The antiserum was affinity-purified on a peptide-coupled Sulfolink column (Pierce Biotechnology, Rockford, Illinois, United States) according to the manufacturer's instructions. Antibody was used at a dilution of 1:200 for immunohistochemistry and Western blotting as described . Immunoh4 cells/well) were treated for 15 h with H2O2 in ES cell medium deficient in β-mercaptoethanol . Dopamine uptake assays were performed as described (ES cells plated in 96-well format (2.3 × 10oethanol . Cell vioethanol . AV/PI (oethanol , cells wescribed . ReporteFor 6- hydroxydopamine (6-OHDA, Sigma) treatment, the drug was diluted in the differentiation medium  and mediDJ-1 shRNA vector was generated by insertion of annealed oligonucleotides 5′-TGTCACTGTTGCAGGCTTGGTTCAAGAGACCAAGCCTGCAACAGTGACTTTTTTC-3′ and 5′-ACAGTGACAACGTCCGAACCAAGTTCTCTGGTTCGGACGTTGTCACTGAAAAAAGAGCT-3′ into the LentiLox vector . For expression of DJ-1 in ES cell rescue experiments, DJ-1 was cloned into the expression vector pcDNA3.1  containing a Flag peptide sequence at the N-terminus using standard cloning techniques. Flag-L166P DJ-1 (pcDNA3) was generated by PCR-mediated mutagenesis.The cDNA for human 5 cells per well), grown for 24 h, and then treated with 10 μM H2O2 as indicated. Cells were lysed in 200 μl lysis buffer and cleared lysate was prepared as described above. An aliquot of 40 μl from each time point was added to 2 M HCl (120 μl) with or without 10 μM 2,4-dinitrophenyl-hydrazine and incubated for 1 h at 24 °C with shaking. Proteins were then TCA-precipitated and resuspended in 200 μl of 6 M guanidinium chloride. Absorbance was measured at 360 nm, and DNP-conjugated samples were normalized for protein concentration with the underivitized control samples.For protein carbonyl quantitation , cells wFigure S1DJ-1 cDNA in WT (+/+), heterozygous (+/–), and knockout (–/–) cultures. Each expression value was normalized to that of β-actin and expressed relative to the respective value of the WT (+/+) control. These gene expression patterns were replicated in at least three independent PCR experiments. Total RNA from ES cells differentiated with the SDIA method for 18 days was isolated using the Absolutely RNA Miniprep kit . Synthesis of cDNA was performed using the SuperScript first strand synthesis system for RT-PCR (Invitrogen). Real-time PCR reactions were optimized to determine the linear amplification range. Quantitative real-time RT-PCRs were performed (Stratagene MX3000P) using the QuantiTect SYBR Green PCR Master Mix  according to the manufacturer's instructions. DJ-1 primer sequences were 5′-CGAAGAAATTCGATGGCTTCCAAAAGAGCTCTGGT-3′ and 5′-CAGACTCGAGCTGCTTCACATACTACTGCTGAGGT-3′; primers used for β-actin were 5′-TTTTGGATGCAAGGTCACAA-3′ and 5′-CTCCACAATGGCTAGTGCAA-3′. For quantitative analyses, PCR product levels were measured in real time during the annealing step, and values were normalized to those of β-actin.(A) Real-time PCR analyses of DJ-1 (625 bp) and β-actin (350 bp).(B) Ethidium bromide staining of the PCR products obtained after 29 cycles for (704 KB EPS).Click here for additional data file.Figure S2DJ-1 heterozygous cells (solid bar) and DJ-1-deficient knockout clone 32 (open bar) after exposure to CuCl2 or staurosporine at the doses indicated.(A and B) Cell viability of (C) MTT values of untreated DJ-1-deficient ES cell clones and the control heterozygous cells. Assays were performed exactly as in (D) MTT values of untreated DJ-1-deficient ES cells transfected with vector alone or various DJ-1-encoding plasmids. Transfection and expression of WT DJ-1 or mutant forms of DJ-1 does not alter the basal metabolic activity or viability of the cells.DJ-1 or the L166P mutant.(E) Western blotting of extracts from ES cells transfected with vectors harboring WT human (545 KB EPS).Click here for additional data file.Figure S3Both cell cultures were differentiated by SDIA for 18 DIV. Cells were fixed with 4% paraformaldehyde and stained with mouse monoclonal antibodies against HB9 (gift from T. Jessell, dilution 1:50) and rabbit polyclonal antibodies against GABA (Sigma, dilution 1:1000) as in (5.5 MB TIF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession numbers of the genes discussed in this paper are α-synuclein (NM_000345), Parkin (AB009973), and DJ-1 (AB073864).The GenBank (


Plasma levels of tumor necrosis factor (TNF)-α and of C-reactive protein (CRP) are elevated in smokers. Previous studies failed to show an association between the G-308A polymorphism in the promoter region of the TNF-α gene and coronary artery disease (CAD). We investigated whether smoking would interact with the TNF-α G-308A polymorphism in determining plasma levels of TNF-α and CRP.Study participants with a complete data set in terms of smoking and the TNF-α G-308A polymorphism were 300 middle-aged male and female industrial employees. After excluding 24 irregular smokers, analyses were performed on 198 "non-smokers" (life-long non-smokers or subjects who quit smoking >6 months ago) as compared to 78 "regular smokers" (subjects currently smoking >3 cigarettes/day). All subjects had a fasting morning blood draw to measure plasma levels of TNF-α and CRP by high-sensitive enzyme-linked immunosorbent assays.1,250 = 5.67, p = .018) but not for genotype (F1,250 = 0.33, p = .57). The interaction-term between genotype and smoking status was not significant (F1,250 = 0.09, p = .76). The fully adjusted model with plasma TNF-α failed to show significant main effects for smoking and genotype, as well as for the smoking-status-genotype interaction.The cardiovascular risk factor adjusted analysis regressing log-transformed CRP levels against smoking status, genotype, and smoking-status-genotype interaction revealed a significant main effect for smoking status (FThe findings suggest that the TNF-α G-308A polymorphism does not mediate the effect of smoking on plasma CRP levels. It remains to be seen whether other genetic polymorphisms along the inflammatory pathway may modulate vascular risk in smokers. Coronary artery disease (CAD) is associated with chronic inflammatory processes in which blood-derived macrophages play a key role . Tumor nSmoking is one of the strongest cardiovascular risk factors for atherosclerotic diseases . Severalin vivo.Another candidate polymorphism that might mediate the cardiovascular risk with smoking, and that is at the initiation of the inflammatory cascade, is the G-308A polymorphism of the TNF-α gene promoter site. The TNF-α G-308A polymorphism is a single base pair polymorphism located at position 308 in the TNF-α gene that maps to human chromosome 6 (p21.1-p21.3) resulting in substitution of the nucleotide adenine (A) for guanine (G) . In the in vitro [While the TNF-α G-308A polymorphism has been associated with increased production of TNF-α in vitro ,18, previn vitro -25. Howein vitro -28.If alleles are randomly distributed between smokers and non-smokers, case-control studies not explicitly investigating the possible environmental-genetic interaction might fail to unravel the role of genetic polymorphisms in CAD. Therefore, we speculated that smoking would affect plasma TNF-α and CRP levels depending on the TNF-α genotype. If so, the genotype might affect the rate of disease progression rather than the existence of atherosclerotic lesions.The study was conducted as part of a larger project in an airplane manufacturing plant in southern Germany. From a total of 1,760 employees, participation was offered to a representative sample of 647 men and women. Of those, 537  volunteered to participate. The Institutional Review Board approved the study protocol. All 537 subjects completed questionnaires on medical and psychosocial health status. A subsample of 332 subjects agreed to have a variety of biological measures assessed. Three hundred of these individuals had complete data on smoking status, smoking history and the TNF-α G-308A gene polymorphism.Based on self-reported smoking history, we categorized life-long non-smokers or those, who had quit smoking for at least 6 months as „non-smokers" (n = 198), smokers who were currently smoking >3 cigarettes per day as "regular smokers" (n = 78), and smokers reporting to consume up to 3 cigarettes/day or who had stopped smoking for less than 6 months as „irregular smokers" (n = 24). Because of the resulting small numbers of irregular smokers, the latter 24 individuals were excluded from further analyses. Of the remaining 276 individuals, we excluded those with a history suggestive of symptomatic atherosclerotic disease, individuals reporting intake of drugs or conditions that might affect CRP levels (including chronic inflammatory diseases such as active rheumatoid arthritis), and subjects for whom CRP data were missing because of occasional assay problems. Subjects reporting a positive history of elevated blood glucose were not excluded. This selection procedure resulted in a final dataset of 261 subjects.Data were collected on two occasions: First, subjects completed a medical questionnaire and examination. The medical assessment consisted of a 96-item questionnaire assessing the medical history and smoking behavior. The questionnaire was based on the Nurses Health Study  with queAfter completion of questionnaires, subjects had a 15-min rest period while sitting. Thereafter, systolic and diastolic blood pressure (BP) were measured twice within 5 min by sphygmomanometry, and the average of the two readings was computed. The waist-to-hip ratio was calculated based on waist circumference (as measured at its narrowest point between the ribs and iliac crest) and hip circumference . Blood samples after overnight fasting were collected within one day to two weeks after having obtained medical data. Blood sampling was scheduled two hours after awakening to minimize circadian variation in variables of interest.Venous blood was obtained using cooled (4°C) citrate tubes for the TNF-α assay. Plasma was snap-frozen after centrifugation until further processing. We chose high-sensitive enzyme-linked immunosorbent assays (ELISA) to measure plasma concentrations of TNF-α (Quantikine HS, R&D Systems Europe, Abington, United Kingdom) and of CRP . In contrast to standard CRP assays, the high-sensitivity assay for CRP allows stratification of subjects with CRP levels below the range used for infectious disease workup . MoreoveTo determine the TNF-α -308 G/A gene polymorphism, we extracted genomic DNA from the leukocyte-containing pellets remaining after centrifugation of coagulated blood using the QIAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany). The TNF-α G-308A polymorphism was assessed by fluorescent real-time polymerase chain reaction with melting curve analysis on a LightCycler (Roche Diagnostics, Rotkreuz, Switzerland) using the TNF-α G-308A ToolSet for LightCycler (Genes-4U, Neftenbach, Switzerland) containing specific primers and fluorescent mutation detection oligonucleotide probes, in conjunction with the Roche Light Cycler Hyb Probe Master Mix (Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturer's protocols. For statistical analyses, we used the following groups: a) the GG variant, and b) the rarer AA and GA genotypes combined.Descriptive data are presented as means ± SD or as median and interquartile range for severely skewed data. To approximate a normal distribution, we log transformed TNF-α and CRP values. General linear models were employed to elucidate the proportion of variance explained of log-transformed plasma TNF-α and CRP values (dependent variables). Independent variables were smoking status (regular smokers vs. non-smokers), gene variant, and an interaction term between smoking status and gene variant. Following this crude analysis, we entered possible covariates  into the equation. Results were considered statistically significant at the p ≤ .05 level; all tests were 2-tailed. To minimize possible type II errors when assessing interaction terms, we considered interaction terms when the F-statistic on the interaction term had a p-value < 0.2 . All regTable After excluding irregular smokers, we found the GG wild type polymorphism in 203 subjects (74%); GA heterozygote were 70 participants (25%) and AA homozygote were 3 participants (1%). Calculated allele frequencies amounted to 0.86 for the G allele and to 0.14 for the A allele. Regular smokers were slightly though not significantly more frequent amongst subjects with the GG genotype than amongst individuals carrying the GA or AA genotype . Table 1,250 = 5.67, p = .018), but not for genotype (F1,250 = 0.33, p = .57). The interaction-term between genotype and smoking status failed to gain significance (F1,250 = 0.09, p = .76), indicating that the effect of smoking on plasma levels of CRP is not affected by the TNF-α -308G/A polymorphism . Of the considered covariates, gender (F1,250 = 12.5, p = .0005), age (F1,250 = 4.55, p = .03), and HDL-C (F1,250 = 3.4, p = .065) were associated with CRP levels, but not BP or LDL-C. Men, older individuals, and those with lower HDL-C had higher plasma levels of CRP.The adjusted analysis regressing log-transformed CRP levels against smoking status, genotype, and smoking-status-genotype interaction revealed a main effect for smoking status (F1,246 = 1.08, p = .30) and for smoking (F1,246 = 0.33, p = .56), as well as for the smoking-status-genotype interaction. However, the adjusted model revealed a significant main effect for HDL-C (F1,246 = 20.7, p < .0001), suggesting that individuals with higher plasma levels of TNF-α had lower HDL-C. Post-hoc analyses revealed no interaction between smoking status and HDL-C or genotype and HDL-C.The fully adjusted model with plasma levels of TNF-α also failed to show significant main effects for genotype (FGenes, health-behavior and the psychosocial environment interact to determine whether or not, and, if at all, how rapidly silent atherosclerosis will progress to the clinical manifestation of CAD ,35. The While plasma levels of the proinflammatory cytokine TNF-α are regulated by several polymorphisms of the TNF-α gene , it is tvitro upon stimulation with lipopolysaccharide than cells from individuals being homozygous for the G allele [Interestingly, blood cells from individuals who carry the A allele of the TNF-α G-308A gene polymorphism express more TNF-α in G allele . DespiteG allele -25. TherIn spite of two recent studies, which found an interaction between smoking and polymorphisms of molecules participating in the inflammatory response ,15, the It must be noted that our findings are preliminary, and, they do not allow us to reject the overall hypothesis of a smoking-gene interaction modifying inflammatory processes contributing to atherosclerosis initiation and progression ,15. For in vitro studies [The lack of a difference in plasma TNF-α levels between subjects with the A allele as compared to those homozygous for the G allele stands in contrast to previous  studies ,18. Howe studies  may playOur study suggests that both plasma TNF-α and CRP levels are not regulated by an interaction between smoking and the G-308A polymorphisms of the TNF-α gene promoter site. We thus remain far from adopting a clinical practice that would counsel smokers to quit smoking based on a particular gene polymorphism. Nonetheless, our results do not refute the overall hypothesis that genetic polymorphisms along the inflammatory pathway may account for the differential effect of tobacco consumption on the cardiovascular risk in individuals who smoke.The authors declare that they have no competing interests.MG participated in the design of the study and drafted the first version of the manuscript.JF participated in the design of the study, in data acquiring, performed the statistical analyses, and critically revised the manuscript.FM carried out and supervised the molecular genetic studies.RvK participated in the design of the study and wrote the final version of the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Biologists have long known that the African great apes (including the chimpanzee, bonobo, and gorilla) are our closest relatives, evolutionarily speaking. The recent release of the chimp draft genome sequence confirms this relationship at the nucleotide level, showing that human and chimp DNA is roughly 99% identical. Given the genetic similarity between human and nonhuman primates, the next big challenge is to identify those changes in the human genotype (the genetic complement of an organism) that generated the complex phenotype  that distinguishes humans from the great apes. For example, modern humans have larger brains and a larger cerebral cortex than both nonhuman primates and their forebears, the early hominids. Elucidating the molecular mechanisms that account for this expansion will provide insight into brain evolution.ASPM gene cause microencephaly, a rare incurable disorder characterized by an abnormally small cerebral cortex. Since the microencephalic brain is about the same size as the early hominid brain, researchers hypothesized that ASPM—whose normal function is unclear—may have been a target of natural selection in the expansion of the primate cerebral cortex. Last year, researchers showed that selective pressure on the ASPM gene correlated with increased human brain size over the past few million years, when humans and chimps diverged from their common ancestor. Now, Vladimir Larionov and colleagues report that the selective pressure began even earlier—as far back as 7–8 million years ago, when gorillas, chimps, and humans shared a common ancestor.One way to figure out which genes are involved in a physiological process is to analyze mutations in the genotype that generate an abnormal phenotype. Such efforts are easier in the relatively rare instance that one gene affects a single trait. Mutations in the ASPM gene, including promoter and intronic (noncoding) sequences, from chimpanzees, gorillas, orangutans, and rhesus macaques. They sequenced these YACs to determine the complete genomic sequence of the ASPM gene from each species. Next, they characterized sequence changes among these species, based on whether the resulting substitutions in amino acids produced changes in the ASPM protein, to determine how fast the protein was evolving. Larionov and colleagues found that different parts of the protein evolved at different rates, with the rapidly evolving sequences under positive selection  and the slowly evolving sequences under “purifying” selection (significant disruptions were jettisoned). Positive selection on genes is one important way to drive evolutionary change.The researchers used a newly developed technology  to extract specialized cloning agents in yeast  containing the entire ASPM gene, Larionov and colleagues show that the increase in human brain size—which began some 2–2.5 million years ago—happened millions of years after the gene underwent accelerated selective pressure. The ASPM gene, they conclude, likely plays a significant role in brain evolution. The next big challenge will be identifying the forces that preferentially acted on the human genotype to kick-start the process of brain expansion, forces that promise to shed light on what makes us human. New genomic technologies like TAR-cloning will likely accelerate this process.By reconstructing the evolutionary history of the