We report that spontaneous activation occurs in 34% of eggs, probably in response to mechanical stress during oviposition. Artificial activation, as evidenced by the slow block to polyspermy and the onset of zygote division, was elicited both by mechanical stimulation and calcium ionophore exposure in 64% and 83% of the cases, respectively. Finally, one in vitro fertilization protocol showed a 27% success rate, despite the fact that about one third of all unfertilized eggs obtained by hormone injection auto-activate. We expect these findings to aid in the conservation effort of Eleutherodactylus frogs, the largest vertebrate genus.Although much is known about the reproductive biology of pond-breeding frogs, there is comparatively little information about terrestrial-breeding anurans, a highly successful and diverse group. This study investigates the activation and  Import mothers ,11.Xenopus laevis and the North American leopard frog, Rana pipiens . Ind. IndXenons .Clearly, there are enormous advantages to being able to manipulate a species' reproduction in the laboratory. However, despite the multiplicity of studies concentrating on anurans, to date all model species are aquatic breeders. Yet amphibians have the largest diversity of breeding strategies among terrestrial vertebrates, and it is to be expected that species with different reproductive strategies will require different methods for their manipulation in the laboratory. Therefore, many species remain experimentally intractable. Notably, terrestrial-breeding frogs, a very large and diverse group of organisms, are largely inaccessible to reproductive investigations.Eleutherodactylus is characterized by terrestrial breeding and direct development without an aquatic larval stage. With more than seven hundred described species, this is the largest vertebrate genus ; to  biology ,31. Howe extinct ,33.Eleutherodactylus coqui are small tree frogs with internal fertilization and direct development [Eleutherodactylus species, E. coqui embryos develop directly into tiny froglets in terrestrial eggs, without a tadpole stage [E. coqui is arguably the best-studied terrestrial-breeding frog, we have focused on this particular species as a model for the development of reproductive techniques.elopment . This spelopment . As withle stage . Protocole stage ,38. A mele stage . It is kle stage . Corticale stage , but theEleutherodactylus coqui frogs were collected in Puerto Rico near El Verde Field Station in the Luquillo mountains and transported to Tulane University where they were housed and fed as previously described [Adult escribed . All aniNatural matings were performed by placing a gravid female and a calling male together as described . Mature,In vitro fertilization (IVF) experiments using sperm in solution were carried out by macerating the testes from a single frog in 500 μL of sperm dilution buffer , and adding this dropwise over the tops of the eggs or injecting it under the jelly coat using a tuberculin syringe . Alternatively, small pieces of macerated testes were placed directly on top of each egg without the use of buffer solution. Incubation of sperm with egg jelly was accomplished by vigorously vortexing the jelly from one egg in 100 μl of SDB. Sperm was then incubated for 10 min in the jelly/buffer supernatant.Sperm preparations were checked for morphology, movement and membrane integrity by fluorescent microscopy with Live/Dead sperm stain (propidium iodide and SYBR 14)  using an Olympus BH-2 microscope . Additio2 with 0.1 mM A23187 calcium ionophore. Scoring of activation was done by noting the appearance of the first cleavage furrow .E. coqui. This ionophore non-specifically activates the unfertilized eggs of a variety of species through stimulation of a calcium-dependent signalling cascade [Table  cascade . Ten hou cascade ; figure  cascade ). ActivaTo test whether the pseudocleavage response was an effect of our stimuli and not a reaction tied to other uncontrolled variables, we examined eight eggs that had remained undisturbed and that had not started pseudocleavage ten hours after laying. At this time, we poked them with fine forceps, and 6 of 8 (75%) began pseudocleaving six hours later table . This deE. coqui during natural matings because this species undergoes internal fertilization, and the eggs will have already been fertilized prior to deposition [E. coqui eggs are arrested or what signal takes them out of their arrest, we followed procedures used in Xenopus [An interesting observation was that one third of all eggs deposited in response to hormone treatment activated of their own accord. This may be due to the mechanical stress to which the eggs are exposed during oviposition. Clearly, mechanical stimuli are able to activate the eggs, and stress incurred in during transit from the ovisac may be sufficient to cause activation. This would presumably not affect position . In orde Xenopus . HoweverE. coqui eggs might be sensitive to aging, as has been reported under certain conditions for the externally fertilizing X. laevis [E. coqui has internal fertilization, we suspected that eggs laid unfertilized might degenerate rapidly, complicating the artificial manipulation of this species' reproduction. Consequently, we investigated the ability of eggs to be activated as a function of time. When we artificially activated eggs with the calcium ionophore at different time points and examined them six hours post treatment, the percentage that pseudocleaved was high even ten hours after being laid  – by simply mincing the testes and adding them directly over the eggs . We also examined the possibility that a component of the egg jelly coat may be important for sperm capacitance. To test this, we incubated sperm with SDB and jelly, or SDB alone, and injected this under the jelly coat of eggs. None of 12 eggs were fertilized by sperm incubated with SDB alone, while pre-incubation of sperm with an extract of the jelly coat in SDB resulted in one fertilization out of 10 eggs (10%). This is considerably less than the 27% efficiency following direct placement of minced testes over the eggs, but suggests that interactions between sperm and the jelly coat may play a role in sperm capacitance and subsequent fertilization.Sperm concentration may play a role in fertilization efficiency as the use of diluted sperm resulted in decreased fertilization. In support of this possibility, polyspermy has been observed in this species and is apparently not deleterious to fertilization and development . A seconis sperm . However36 = 3.2 × 10-5, or less than one in ten thousand. This shows that fifteen minutes after being poked the eggs have established a block to polyspermy, one of the defining functional characteristics of activation. Hence, although the first visible indication of activation –the formation of the first cleavage furrow- will not be seen for six hours, we can conclude that the egg is undergoing the normal activation processes within minutes of being stimulated.To test the functional response of the eggs, we attempted to fertilize artificially activated eggs. As was explained above, we were able to achieve an IVF success rate of 27%. However, if we poked the eggs fifteen minutes prior to direct fertilization, none 0/36) developed (see table 6 developE. coqui, we have observed that a large proportion of eggs that are laid unfertilized auto-activate. We showed that E. coqui eggs are easily activated by mechanical stimuli, leading to a need for careful manipulation of unfertilized eggs in all reproduction studies. Further, the cleavage pattern seen in mechanically activated eggs is similar to that of both auto-activated and chemically activated eggs, suggesting that mechanical stress, probably incurred in during oviposition, is responsible for the auto-activation mentioned above (table E. coqui, this phenomenon may relate to internal fertilization, and it would be interesting to investigate auto-activation of unfertilized eggs in closely related, externally fertilizing species such as E. antillensis [E. coqui eggs, however, remain intact and can be manipulated, showing signs of activation both at the morphological level, through the initiation of development, as well as the functional, through the slow block to polyspermy. By careful handling, we are now able to fertilize over half of the remaining, functionally viable eggs using a simple procedure. Finally, we have shown that E. coqui eggs do not degenerate rapidly and are capable of undergoing activation up to ten hours after deposition, thus creating a window of time for carrying out experimental procedures. Our results demonstrate efficient IVF in an internally fertilizing, terrestrial-breeding frog and help lay the foundation for future research and conservation possibilities in this unusually large genus of amphibians.In an effort to conserve declining populations of animals, the development of protocols for the artificial manipulation of reproduction is of great interest. In the case of the neotropical frog ve table . Facile illensis . Some E.ET carried out the experiments and prepared the manuscript. SFM carried out preliminary experiments and supervised the manuscript. The study was conceived jointly. Both authors approved the final manuscript.


Analysis of DNA microarray data usually begins with a normalization step where intensities of different arrays are adjusted to the same scale so that the intensity levels from different arrays can be compared with one other. Both simple total array intensity-based as well as more complex "local intensity level" dependent normalization methods have been developed, some of which are widely used. Much less developed methods for microarray data analysis include those that bypass the normalization step and therefore yield results that are not confounded by potential normalization errors.Instead of focusing on the raw intensity levels, we developed a new method for microarray data analysis that maps each gene's expression intensity level to a high dimensional space of SEDs (Signs of Expression Difference), the signs of the expression intensity difference between a given gene and every other gene on the array. Since SED are unchanged under any monotonic transformation of intensity levels, the SED based method is normalization free. When tested on a multi-class tumor classification problem, simple Naive Bayes and Nearest Neighbor methods using the SED approach gave results comparable with normalized intensity-based algorithms. Furthermore, a high percentage of classifiers based on a single gene's SED gave good classification results, suggesting that SED does capture essential information from the intensity levels.The results of testing this new method on multi-class tumor classification problems suggests that the SED-based, normalization-free method of microarray data analysis is feasible and promising. DNA microarray technology is now playing an increasingly important role in biomedical research. Microarray technology gives one the opportunity to measure gene expression levels of thousands to tens of thousands of genes simultaneously, in order to study the differential gene expression pattern between different developmental stages, diseases states and samples treated with drugs or other compounds. Before comparing data from different arrays to address these biological questions, however, a "much more mundane but indispensable " normalization step  is curreBecause of the slight difference in RNA quantities, imaging settings and other variables, even in very controlled experiments the intensity levels from different arrays are of different scales and need to be normalized before they can be compared with each other. Various normalization methods have been developed and some are widely used. The simplest method is total intensity based normalization ; this apFor most current applications, these normalization methods seem to be adequate. However, the residual left by a less than perfect normalization procedure is another source of non-biological variation that is usually non-desirable, especially when the differences in expression levels are expected to be small . In addietc. We decided to use multi-class tumor classification as a test case to illustrate the power of our approach. We compared our results for a multi-class tumor classification problem with more conventional approaches published by Ramaswamy et al. , we will use (sij) to compare gene expression differences between arrays. Since we expect measurement variations within an array will be less than those between arrays and we take the signs of relative expression differences to get SED, we expect the SED will be less "noisy". However, the value of any single sij may still vary between technical and biological replicates. One would expect more sij would change values randomly if the technical replicates was done on arrays that were fabricated in a different run than arrays from same run, for example. Biological variations are expected to be even more frequent. However, we hypothesize that we can perform statistical analysis on the SED, which contains tens of thousands of sij for a single gene, and minimize the impact of such noises.Instead of directly using the intensity levels x, and its derivatives such as the mean μ, the standard deviation σ and the signal to noise ratio (S2N) between two sample groups A and B, , since the NB method is sensitive to the extreme values of sp, and samples can be over-predicated without thresholds.Because of the uneven and relatively small sample sizes for each tumor class (mostly 8 but up to 24), extra care was taken in computing spIn addition, several alternatives were tested to demonstrate that our results were reasonably robust and not sensitive to the particular choices we made:i.e. only the first 8 samples were used to calculate spij with no significant change of results observed;1) To examine the influence of the sample size, in a separate analysis the sample size of ME, LE, CNS class was artificially reduced to 8, ij depends on the sample size n for each tumor class, we have applied a "sample replacement" strategy in addition to the usual "take-one-sample-out" approach for cross-validation, i.e. when one sample is taken out as the test sample, another sample from the same class is duplicated to take its place to keep the sample size constant. Essentially the same results were obtained. Results reported are from the sample replacement runs.2) Since spij between all 14 classes was calculated as:In Feature Filtering, the variance of sp2 = /14 - /14)2     (4)σwith the test sample taken out, and used as the criterion for feature exclusion.Feature filter was done as in the Naive Bayes Method.The analysis was done on a computer (Pentium M 1.5 GHz) operating under Microsoft XP. Both Naïve Bayes and Nearest Neighbor Classifiers are implemented in Java. Since SED can be easily calculated from the raw intensities only the later are kept in memory and SED are computed from the intensities on an as-needed bases. Memory needed to analysis the 144 samples is less than 64 MB.The most computationally intensive algorithm that we tried is the Nearest Neighbor method without any feature selections and it takes about 10 sec to calculate SED score for one pair of tumor samples with about 16000 genes.hw14@columbia.edu.A Java program named SED (including source code) to perform nearest neighbor analysis of microarray samples is freely available by contacting author at H.W. conceived of the SED study and performed implementations. H.H. refined the approach and provided additional statistical insight on SED. Both authors read and approved the final manuscript.


Plasmodium genus that are under consideration as candidates for inclusion in transmission-blocking vaccines. Previous research using transgenic rodent malaria parasites lacking P25 and P28 has demonstrated that these proteins have multiple partially redundant functions during parasite infection of the mosquito vector, including an undefined role in ookinete traversal of the mosquito midgut epithelium, and it has been suggested that, unlike wild-type parasites, Dko P25/P28 parasites migrate across the midgut epithelium via an intercellular, rather than intracellular, route.P25 and P28 are related ookinete surface proteins highly conserved throughout the via an intracellular route similar to wild-type parasites and subsequently develops into oocysts.This paper presents an alternative interpretation for the previous observations of Dko P25/P28 parasites, based upon a recently published model of the route of ookinete invasion across the midgut epithelium. This model claims ookinete invasion is intracellular, with entry occurring through the lateral apical plasma membrane of midgut epithelial cells, and is associated with significant invagination of the midgut epithelium localised at the site of parasite penetration. Following this model, it is hypothesized that: (1) a sub-population of Dko P25/P28 ookinetes invaginate, but do not penetrate, the apical surface of the midgut epithelium and thus remain within the midgut lumen; and (2) another sub-population of Dko P25/P28 parasites successfully enters and migrates across the midgut epithelium These hypotheses are tested by showing how they can account for previously published observations and incorporate them into a coherent and consistent explanatory framework. Based upon these hypotheses, several quantitative predictions are made, which can be experimentally tested, about the relationship between the densities of invading Dko P25/P28 ookinetes in different regions of the midgut epithelium and the number of oocyst stage parasites to which these mutant ookinetes give rise.The recently published model of ookinete invasion implies that Dko P25/P28 parasites are greatly, although not completely, impaired in their ability to enter the midgut epithelium. Therefore, P25 and/or P28 have a novel, previously unrecognized, function in mediating ookinete entry into midgut epithelial cells, suggesting that one mode of action of transmission-blocking antibodies to these ookinete surface proteins is to inhibit this function. P25 and P28 are related major ookinete surface proteins under consideration as candidates for inclusion in transmission-blocking vaccines -4. ConsePlasmodium genus [P25 and P28 are structurally similar proteins, highly conserved throughout the um genus ,31,35-43um genus , putativum genus ,28,29, tum genus ,17-24. Pum genus -21,44,45Plasmodium berghei rodent malaria parasites lacking P25 and P28 demonstrated that these proteins have multiple and partially redundant functions during parasite infection of the mosquito vector [P. berghei parasites exhibit greatly reduced levels of oocyst infection compared to wild-type or Sko P25/P28 parasites, ookinetes lacking both P25 and P28 are still able to cross the midgut epithelium and establish oocyst infections [P. berghei ookinetes migrate intracellularly through the midgut epithelium causing significant damage to invaded midgut epithelial cells [via a solely intercellular route [Previous research using transgenic o vector ,27. Althal cells -48, whical cells -48, inclal cells ,45, protal cells ,45,48 anal cells ,49 and Sal cells . Furtheral cells ,45. Dko al cells ,45,48. Car route  suggestsin vivo support this model [P. berghei ookinetes [The route of ookinete migration across the midgut epithelium of the mosquito vector has long been controversial . The majis model . Accordiis model ,51,52. Sis model  and Figuis model ). Ookineokinetes ,55. Subsokinetes ,52,56, mP. berghei parasite infection of the mosquito vector are proposed. First, some Dko P25/P28 ookinetes invaginate, but are unable to penetrate, the apical surface of the midgut epithelium. Second, other Dko P25/P28 parasites are able to successfully enter and migrate across the midgut epithelium via an intracellular route, in a manner similar to wild-type parasites.Following the model of ookinete invasion of the midgut epithelium outlined above . If Dko P25/P28 ookinetes do migrate across the midgut epithelium via a solely intercellular route there is no known reason why these parasites should not also be melanized in the basal region of the midgut epithelium. Consequently, if solely intercellular migration does occur melanized parasites should be found that are not associated with any morphologically abnormal midgut epithelial cells.First, all melanized Dko P25/P28 parasites in the refractory via a solely intercellular route greatly exceeds the number of intracellular ookinetes [via a solely intercellular route, the number of oocysts should greatly exceed the number of ookinetes migrating via an intracellular route .Second, there should be a quantitative relationship between the density of Dko P25/P28 ookinetes associated with morphologically abnormal midgut epithelial cells and the number of oocysts that subsequently develop on the basal surface of the midgut epithelium. Specifically, the number of oocyst stage parasites should be equal to or less than the number of Dko P25/P28 ookinetes associated with morphologically abnormal midgut epithelial cells, as only ookinetes migrating intracellularly are predicted to become oocysts. The Dko P25/P28 ookinetes located between morphologically normal midgut epithelial cells are not expected to transform into oocysts, as these parasites do not enter, and hence cross, the midgut epithelium. The number of ookinetes apparently migrating okinetes . ConsequP. berghei parasites implies that there are two sub-populations of Dko P25/P28 ookinetes, neither of which migrate across the midgut epithelium via a solely intercellular route (Figure An. gambiae and do not give rise to oocyst parasite stages. These parasites remain surrounded by morphologically normal midgut epithelial cells, which do not exhibit the morphological abnormalities associated with parasites invading intracellularly [An. gambiae [The re-interpretation presented here of previously published work on Dko P25/P28 llularly . A seconllularly , GNBP [2llularly , NOS andllularly , undergo gambiae , and for gambiae . Accordi gambiae . HoweverP. berghei ookinetes is unknown. One explanation is that loss of P25 and/or P28 impedes, but does not entirely prevent, penetration of the apical plasma membrane of midgut epithelial cells. Consequently, the entry of Dko P25/P28 ookinetes into the midgut epithelium may be protracted, prolonging the period of exposure to the hostile environment of the midgut lumen, which results in the death of most parasites before completion of midgut epithelial cell penetration. This interpretation is consistent with the observation of lysed Dko P25/P28 parasites on the luminal side of the midgut epithelium [The reason for the existence of the two distinct sub-populations of Dko P25/P28 ithelium .In summary, the unified model of the route of ookinete invasion across the mosquito midgut epithelium suggests a novel, previously unrecognized, function for P25 and/or P28 in mediating ookinete entry into the midgut epithelium. Specifically, the interpretation presented implies that the loss of P25 and/or P28 greatly impairs, but does not entirely abolish, ookinete entry into midgut epithelial cells and probably has relatively little effect on the ability of ookinetes to traverse through the cytoplasm of midgut epithelial cells. A role for P28 in parasite entry into the midgut epithelium is suggested by the deposition of this molecule at the site of ookinete penetration into midgut epithelial cells ,45. ThisCTRP = circumsporozoite and thrombospondin-related anonymous protein-related protein; Dko P25/P28 = double knockout of P25 and P28; GNBP = gram-negative binding protein; MAOP = membrane-attack ookinete protein; NOS = nitric oxide synthase; Sko P25/P28 = single knockout of either P25 or P28; SRPN10 = serine protease inhibitor 10.LAB wrote the manuscript and prepared the figures. LRC revised the manuscript. Both authors read and approved the final version of the manuscript.


Prediction of structure and function for uncharacterized protein families by identification of evolutionary links to characterized families and known structures is one of the cornerstones of genomics. Theoretical assignment of three-dimensional folds and prediction of protein function even at a very general level can facilitate the experimental determination of the molecular mechanism of action and the role that members of a given protein family fulfill in the cell. Here, we predict the three-dimensional fold and study the phylogenomic distribution of members of a large family of uncharacterized proteins classified in the Clusters of Orthologous Groups database as COG4636.Gloeobacter violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. Thus, members of COG4636 appear to be a new type of selfish genetic elements, which may fulfill an important role in the genome dynamics of Cyanobacteria and other species they invaded. Our analyses provide a platform for experimental determination of the molecular and cellular function of members of this large protein family.Using protein fold-recognition we found that members of COG4636 are remotely related to Holliday junction resolvases and other nucleases from the PD-(D/E)XK superfamily. Structure modeling and sequence analyses suggest that most members of COG4636 exhibit a new, unusual variant of the putative active site, in which the catalytic Lys residue migrated in the sequence, but retained similar spatial position with respect to other functionally important residues. Sequence analyses revealed that members of COG4636 and their homologs are found mainly in Cyanobacteria, but also in other bacterial phyla. They undergo horizontal transfer and extensive proliferation in the colonized genomes; for instance in Thermus thermophilus Hb8, to be published). Our analysis of the Tt1808 structure reveals that we correctly predicted all functionally important features of the COG4636 family, including the membership in the PD-(D/E)xK superfamily of nucleases, the three-dimensional fold, the putative catalytic residues, and the unusual configuration of the active site.After submission of this manuscript, a crystal structure of one of the COG4636 members was released in the Protein Data Bank (code 1wdj; Idaka, M., Wada, T., Murayama, K., Terada, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S.: Crystal structure of Tt1808 from  The PD-(D/E)XK domain is ubiquitously found in enzymes involved in metabolism of nucleic acids, mostly in nucleases with diverse biological functions. The first structurally characterized members of the PD-(D/E)XK superfamily were restriction enzymes (REases) . CrysD...Xn...(D/E)XK (where X is any amino acid). The Lys residue serves to position a water molecule for an in-line attack on the scissile phosphodiester bond, while the carboxylate residues coordinate a Mg2+ ion, which acts as a cofactor. Despite the wealth of structural and biochemical data, obtained mainly for REases (summarized in a collection of reviews: [All members of the PD-(D/E)XK superfamily share a common structural core, comprising a mixed β-sheet of 4 or 5 strands flanked on both sides by α-helices ,2,12. Threviews: ), there reviews: ,15. Morereviews: -19. In areviews: -22.2+ binding site common to its cousins that cleave phosphodiester bonds in DNA. Remarkably, on the opposite side of the common fold, EndA developed a different active site, whose geometric configuration is very similar to that of a His-Tyr-Lys triad in structurally unrelated RNase A [Saccharomyces cerevisiae exhibits perfect conservation of the restriction enzyme-like structure, but lacks any catalytic residues – it is postulated that it functions as a nucleic acid binding domain devoid of any catalytic activity [Crystallographic analyses have also revealed the PD-(D/E)XK fold in proteins that do not function as deoxyribonucleases at all and exhibit no conservation of the active site with the above-mentioned enzymes. The structure of the C-terminal catalytic domain of tRNA splicing endoribonuclease (RNase) EndA is identical to the minimal core of the PD-(D/E)XK fold , yet thi RNase A . Finallyactivity .The divergence exhibited by the members of the PD-(D/E)XK superfamily is remarkable. Even enzymes with very similar biological functions, such as REases that recognize and cleave the same substrate, can exhibit little or no significant sequence similarity. Thus, most of the afore-mentioned enzymes were considered unrelated until the corresponding crystal structures were solved. Only in a few cases the membership in the PD-(D/E)XK superfamily was successfully predicted using bioinformatics  before the actual structures were determined -29. The In the course of analyses of proteins with unknown structures, we came across a family of sequences grouped together in the Clusters of Orthologous Groups (COG) database  as COG46In order to test the hypothesis of the evolutionary connection between COG4636 and the PD-(D/E)XK superfamily we carried out the fold-recognition analysis, which allows to predict the three-dimensional fold of the target protein by matching its sequence with the available protein structures and assessing the sequence-structure compatibility using a combination of criteria, such as sequence similarity, match of secondary structure elements, compatibility of residue-residue contacts, etc. (review: ). SequenNostoc sp. PCC 7120, GI: 17231142) and used it as a platform to study the three-dimensional arrangement of conserved residues. A homology model of all3650 was constructed using the "FRankenstein's Monster" approach (see Methods and ref. [In order to identify the putative active site of newly predicted members of the PD-(D/E)XK superfamily, we modeled the structure of one of the COG4636+ members, whose sequence was close to the consensus calculated for the whole family , startiand ref. . The cooand ref.  and on-lThe model of all3650 reveals a typical PD-(D/E)XK nuclease-like spatial arrangement of one Lys ε-amino group (from the residue K127) and two carboxylate groups (from residues D83 and E111) Figure . The modG. violaceus, GI: 37520478) the Lys residue is present both at the "classical" and alternative position, thereby forming a "PD-EXK-K" variant of the active site. This arrangement resembles a putative evolutionary intermediate between the "classical" active site and the newly discovered rearranged variant. In another lineage of the COG4636+ family, an Asp residue appears in the position normally occupied by Lys in the C-terminal half-motif. Some of the members of this lineage  exhibit therefore the "PD-EXD-K" motif, but the majority  lack the Lys residue and exhibit only the "PD-EXD" variant. In another lineage  the Lys residue is replaced by Asn to form the "PD-EXX-N" variant of the predicted active site. The conserved His residue  is present in most members of the COG4636+ family, with the exception of a small lineage of closely related proteins  in which it is substituted by Gln, and a larger group of more diversified sequences, in which it is substituted by Thr or Ser. Most members of the latter group possess a Lys or Arg residue in the "catalytic" position and hence exhibit "PD-EXK-K" (see above) or "PD-EXR-K" variants of the active site. It will be very interesting to determine experimentally, which of those residues in different configurations are involved in catalysis, and which are only auxiliary. In particular, it would be interesting to find if both or either of the Lys residues present in the potential "intermediate" versions of the active site are required for catalysis.Inspection of the multiple sequence alignment reveals that only two carboxylates  are practically invariant in the COG4636+ family, while all the others undergo various substitutions . It is astonishing that members of COG4636+ represent over 2% of all protein-encoding genes of G. violaceus PCC 7421  originate from Cyanobacteria; of these, 84% were found in just 6 genomes , other 30 total  and depoD. hafniense DCB-2 (Firmicutes), 7 sequences from C. aurantiacus , and 6 from S. coelicolor (Actinobacteria) each form a separate species branch on the phylogenetic tree, while 14 sequences from T. thermophilus HB27 (Deinococcus-Thermus lineage) form three separate branches. Several monophyletic groups of closely related sequences are also observed in G. violaceus . There is also one branch comprising 6 closely related sequences in C. watsonii, GI numbers: 45527153, 45527776, 45524526, 45527777, 45527775, 45527774). Other statistically significant branches, however, comprise members from different species, suggesting that they were either formed prior to speciation or that their members were transmitted horizontally between different genomes of already existing species.In order to reconstruct the evolutionary history of the COG4636+ family, we calculated the phylogenetic tree, based on the same reliable section of the multiple sequence alignment that was used for protein structure prediction (see Methods). Unfortunately, in all trees obtained with different methods and parameters, the majority of deep branches received very low bootstrap support (data not shown), hence the relationships within the whole family must be regarded as unresolved. We were able, however, to identify a number of branches with bootstrap support >90%. Many of such branches comprise members from one species only. This situation is characteristic for sequences found in a few non-Cyanobacterial species; for instance 8 sequences from C. watsonii WH8501, all from the above-mentioned branch of 6 closely related sequences (the other two relatives are located elsewhere on the chromosome). We also found a few tandem duplications: 9 in C. watsonii WH8501 and 5 in G. violaceus PCC7421, 5 in Nostoc sp. PCC6803, 2 in N. punctiforme PCC73102, 2 in A. variabilis ATCC 29413, 2 in Synechocystis sp. PCC6803, 2 in T. thermophilus HB27, 1 in T. erythraeum IMS101 and 1 in M. magnetotatcticum MS-1. In general, however, tandem duplications are rare and the distribution of COG4636+ family members along the chromosomes of Cyanobacteria with completed genomes seems completely erratic  in the range of 3000 bp upstream and downstream, we weren't able to identify any recurrent type of proteins, either with respect to the molecular or cellular function or the predicted three-dimensional fold (data not shown). Also no preference for occurrence of COG4636+ family members within or near any apparent mobile genetic elements (putative prophages etc.) was observed. Thus, insertion of the genes encoding putative COG4636+ nucleases seems virtually random. The only notable exception is a neighborhood of another member of COG4636+, suggesting tandem duplication. We identified one instance of 4 consecutively arranged genes in the genome of Our results suggest that functionally uncharacterized proteins grouped together in COG4636 are a branch of the PD-(D/E)XK superfamily, which has not been identified to date due to a presence of an unusual variant of the active site, which lacks the conserved Lys residue at the typical position in the primary sequence. That the catalytic Lys can migrate in the framework of the active site of PD-(D/E)XK nucleases has been suggested earlier, based on the sequence analysis of another nuclease domain found in site-specific, non-long terminal repeat retrotransposable elements , but to C. watsonii WH 8501, which was isolated from tropical waters of the Western Atlantic and Pacific oceans. It is remarkable that members of COG4636+ are almost absent from the genomes of Synechococcus and Prochlorococcus species thriving in the Sargasso sea, as well as in the environmental samples isolated from that region. On the other hand, in G. violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. This phylogenetic distribution resembles that of mobile genetic elements such as introns or insertion sequences (reviews: [T. thermophilus – three independent successful colonizations).Phylogenomic analyses show that putative nucleases grouped in the COG4636+ family are exceptionally abundant in genomes of certain Cyanobacteria, but absent in others. They are typically abundant in the sequenced genomes of freshwater species, but scarce in the genomes of marine species, with the exception of reviews: ,42) and Anabaena PCC 7120 [We hypothesize that the mechanism by which these putative nucleases induce their proliferation in a genome is similar to that displayed by homing nucleases and restriction enzymes , namely PCC 7120 , and theAnabaena sp. PCC 7120 [Microcystis sp. [C. watsonii WH 8501). Summarizing, it is plausible that members of COG4636+ fulfill an important role in the genome dynamics of Cyanobacteria and other species they colonize. We hope that our predictive study will facilitate experimental determination of the molecular and cellular function of members of this intriguing protein family.One possibility is that COG4636+ members serve as a part of the restriction barrier, similarly to the unrelated NucA family of extracellular nucleases found in Cyanobacteria, e.g. PCC 7120  and Micrstis sp. . They costis sp. . From thstis sp. . ConversSearches of the non-redundant (nr) database were carried out at the NCBI using PSI-BLAST  with def. The "coded_by" field from each GenPept file was used to identify the corresponding DNA sequence, which were also downloaded into the database. The sequence in the range of 3000 bp upstream or downstream from the region encoding a COG4636+ member were scanned for the presence of annotated Open Reading Frames (ORFs). Initially, the functional categorization of these ORFs was carried out based on the automatic assignment into the PFAM and COG families. In the absence of any recurrent function, the annotations of all ORFs were carefully re-analyzed visually and in uncertain cases, additional searches against the CDD database were carried out [The Eutils module from the Biopython package was used as an interface to access remotely the NCBI databases . The Genried out . The dis[Secondary structure prediction and tertiary fold-recognition was carried out via the GeneSilico meta-server gateway at . Seconda, PROFsec, PROF [6, SABLE [, JNET [6, JUFO [6, and SAM. Solvent and JPRE. The fol, SAM-T02, 3DPSSM , BIOINBG, FUGUE [, mGENTHR, and SPA. Fold-re.Fold-recognition alignments to the structures of selected templates were used as a starting point for homology modeling using the "FRankenstein's Monster" approach , comprisThermus thermophilus Hb8 To be Published). Our analysis of the Tt1808 structure and its comparison with the model of all3650 confirms our predictions. Tt1808 does indeed exhibit the PD-(D/E)xK fold: the DALI [After submission of this manuscript, a crystal structure of one of the COG4636+ members was released in the Protein Data Bank ; bp, base pair(s); nt, nucleotide; e, expectation; REase, restriction endonuclease; ORF, product of an open reading frame,MF carried out all sequence analyses and structure predictions using fold-recognition methods and ROSETTA. JMB built the homology model, analyzed spatial vs. sequential conservation of the putative active site, and wrote the manuscript. Both authors have read and accepted the final version of the manuscript.The additional data file all3650.pdb contains the coordinates of the original all3650 model (obtained before the Tt1808 structure was published) in the PDB format.Click here for file


Plasmodium falciparum was based on the amplification of MSP1, MSP2 and GLURP genes. This method enabled confirmation of the same P. falciparum genotype in all three patients as well as in a fourth one (index case). The presence of nosocomial malaria was confirmed and it was concluded that the most likely source of transmission was through multi-dose preparations of heparin applied to heparin locks.Three cases of nosocomial malaria are reported from patients of the Internal Medicine Ward of a tertiary University teaching hospital in Medellin, Colombia. Epidemiological research, based on entomological captures, medical records review and interviews of nursery staff about patient care practices potentially involving contact with blood, were carried out. Molecular characterization of  Plasmodium falciparum-infected patient.The city of Medellin is located in the north-western region of Colombia and, although malaria is endemic in the country, absence of anophelines within the city makes vector transmission of the infection impossible. However, this city, located within the Andes Mountains  at 1,588 m above sea level (a.s.l.), is surrounded by highly endemic regions and imported cases are often referred to the University hospital for treatment. Here, three cases of nosocomial malaria, which occurred in hospitalized patients in the Internal Medicine Ward of Hospital A, are described. These patients were being treated for diverse conditions and they shared the hospital facilities during different periods. One of these patients was in the Emergency Ward at the same time as a P. falciparum genomic DNA was amplified using specific primers for polymorphic streches of Merozoite Surface Antigen-1 (MSP-1), Merozoite Surface Antigen-2 (MSP-2) and Glutamate Rich Protein (GLURP), and the profile observed among the different isolates was compared.In order to confirm an association between all the cases, P. falciparum asexual forms . A post-mortem confirmed as cause of death complicated malaria. Dengue and yellow fever IgM antibodies were absent. The patient remained in the emergency ward for 18 hours prior to his death.A 23-year-old male woodcutter, resident in Vigia del Fuerte , was referred to the hospital on 11 April 2001, diagnosed with yellow fever. Vigia del Fuerte is a highly endemic malaria region located in the Atrato River basin with a mean Annual Parasite Index (API) of 72.6 during the past five years. The patient presented 15 days with fever, headache, chills, jaundice, arthralgia, melena, dark urine and bilious vomiting. He was given a heparin lock for administration of I.V. treatment. The patient deteriorated throughout the night and died before thick smear results were available. Peripheral blood thin and thick smears confirmed the presence of th April 2001, and remained there for 24 hours before being transferred to the Internal Medicine Ward with diagnosis of systemic lupus erythematosus (SLE). In addition to symptoms and signs of SLE, he presented fever on 23 April, and received treatment with fenitoin, dipirone, ampicillin, ranitidine, ceftriazone, methilprednisolone, oxacillin, amikacin and enoxaparine (via an heparin lock). As part of the treatment for his condition, he was administered chloroquine 150 mg daily from 24th April 2001. He was discharged on 19th May 2001. Then, on 6th June 2001, he was hospitalized with a diagnosis of a febrile syndrome. P. falciparum malaria was confirmed on 20th June 2001 after observation of the parasite in a blood cell count. A thick smear revealed the presence of 14,440 asexual forms per μl in peripheral blood. The patient was administered quinine and sulphadoxine-pyrimethamine with good treatment response.A 19 year-old male, unemployed, resident in Angelopolis , a non-malarious area, located 1,900 m a.s.l., attended the emergency service of the hospital on 10th May with diagnosis of status asthmaticus. He was administered aminophyllin, hydrocortisone, ranitidine, enoxaparine (via a heparin lock) and was discharged on 15th May 2001. He returned to the hospital on 5th June 2001 complaining of fever, chills and adynamia and was admitted with diagnosis of toxic hepatitis due to the presence of dark urine, jaundice and hepatomegaly. On 6th June 2001, diagnosis of complicated malaria was confirmed by observation of the parasite in a blood cell count. Later, the presence of 87,500 trophozoites of P. falciparum per μl was confirmed by a thick smear. He received treatment with quinine, sulphadoxine-pyrimethamine and was discharged on June 23rd 2001A 46 year-old male, unemployed, resident of Santa Barbara , a non-malarious area located 1,800 m a.s.l., was hospitalized in the Internal Medicine Ward on 8th May 2001 with superior vena cava syndrome (100% obstruction) and was applied a heparin lock for administration of I.V treatment. The patient was diagnosed with a mediastinal mass, compatible with lymphoma, and received treatment with dexametasone (I.V.) and radiotherapy. On June 9th 2001, he evidenced fever and on 9th June 2001, he was diagnosed as complicated malaria by a cell blood count. A thick smear confirmed the presence of 166,440 asexual forms of P. falciparum per μl. He received treatment with quinine, sulphadoxine-pyrimethamine with good treatment response. However, on 1st July 2001, he died as a consequence of multiple non-malaria related complications.A 41 year-old male, flower-grower, resident of El Carmen del Viboral , a non-malarious area located 2,150 m a.s.l.., was admitted directly to the Internal Medicine Ward of the hospital on 7Anopheles was carried out by expert personnel from the Secretariat of Health, both within the ward and in the surrounding gardens, around the time of malaria diagnosis of patients 2 and 4. These confirmed the absence of the vector in the hospital area.Search for P. falciparum genomic DNA was extracted from whole blood collected onto filter paper or from paraffin embebbed brain tissue (for the index case). Paraffin was removed from post-morten material using xylene, followed by 2 washes with 100% ethanol. DNA was isolated by proteinase K digestion, followed by four rounds of phenol-chloroform extraction. Purified DNA was stored at -20°C until amplification.The Region 2 of MSP-1 and the central region of MSP-2 were amplified by a nested Polymerase Chain Reaction (PCR); the region RII of GLURP was amplified by a semi-nested PCR [P. falciparum infection by matching strains in all 4 patients  gel , stained with ethidium bromide and visualized under ultraviolet light. Size analysis of the amplified fragments revealed identical pattern distribution for all the examined markers assayed, confirming the presence of P. falciparum malaria infection in three patients without history of malaria or travel to malaria-endemic regions is described. The first of these patients (Patient 2) shared the Emergency Ward with a fatal case of falciparum malaria. The incubation period for this nosocomial infection was 12 days, however, since he was administered high doses of chloroquine, fever receded for about two weeks. Resistance of P. falciparum to chloroquine is highly prevalent in the Antioquia region [The presence of  Patient  shared tPatient 2 shared the Internal Medicine facilities with patients 3 and 4 at least for one week during May. Patient 3 evidenced an incubation period of 22 days, while in Patient 4 this was 26 days.Since patients 3 and 4 had no contact with the index case, further analysis of the incubation periods allowed us to conclude that transmission occurred from index case to patient 2 and the latter was the source of infection for patients 3 and 4. This means that at least 2 separate episodes of contamination were involved in transmission to the different patients (see figure Transmission of malaria via heparin locks has been reported previously by other authors, the source of contamination was a multidose heparin container . AlthougP. falciparum to confirm nosocomial transmission of malaria [P. falciparum observed in the Antioquia region [P. falciparum among unrelated parasites may be possible, but unlikely, since examination of these 3 different genes has been routinely used to discriminate unrelated parasites [Other researchers have reported on the use of molecular typing of  malaria . Genotypa region , the prearasites . MoreoveThese observations are of major relevance to sanitary and health authorities, since they confirm the importance of biosafety during minor procedures, such as application of heparin to peripheral locks. In addition, it highlights the need to rule-out malaria infections in all febrile patients sharing hospital facilities with malaria patients as well as in those not responding as expected to antimicrobial therapy.GL was involved in the review of clinical records, staff interviews and preparation of the manuscript. OJ coordinated the epidemiological and entomological researches. FL coordinated the microscopy diagnosis and following of the patients. AM participated on the epidemiological research. RE carried out the molecular analysis of the samples. BS, the Grupo Malaria leader, participated in the treatment and following of the patients. MA attended the patients, coordinated the clinical and molecular aspects of the research and prepared the manuscript.


An obituary commemorates the life and works of Jacov Tal. At the time of his passing, Jacov was the Head of Virology at Ben-Gurion University Medical School, Israel. In brief, four of us, who befriended Jacov in different capacities, write our remembrances. It is appropriate to recall the words of a colleague who on another occasion upon the passing of a giant in American science remarked, "Well, ghosts can't make men do anything!" Thus, the true reflection of a person is not what (s)he through his/her prestige, wealth, and position makes others do in life, but how (s)he is remembered by others in death. Jacov started as a young retrovirologist in J. M. Bishop and H. Varmus' laboratory; and it is fitting that he is remembered by friends in Retrovirology.A friend's passing elicits a set of emotions. Collecting and sharing remembrances are steps of closure in bidding farewell. Here, we honor and remember Jacov Tal Fig.  who pass"I remember Jacov as my actual mentor in my first year as a graduate student in the laboratory of Uri Littauer that Jacov had joined a year earlier. Jacov took similar care of other new comers interested in nucleic acids and molecular biology including Hiroshi Inouye, Inder Verma and Jacque Beckman, even to the point of neglecting his own work. Ibelieve that Jacov's selfless involvement in public affairs and later decision to become a virologist date back to a childhood experience; namely, only a ruling elite and diplomatic corpse in a country that Jacov's parents were stationed in, knew about the polio epidemic, considered then an ideological insult.Jacov Tal studied at the Hebrew University in Jerusalem where he obtained the Bachelor degree in Biochemistry and Microbiology in 1964 and the Master degree in Biochemistry in 1966. He continued his studies at the Weizmann Institute of Science where he obtained his PhD degree in Biochemistry in 1971. Jacov Tal did his postdoctoral training first with Hershel Raskas in Washington University where he investigated adenovirus gene expression and later with Harold Varmus and Michael Bishop at USCF where he studied the relation between the retroviral genome and the genome of its cellular host. After these formative years as a molecular virologist Jacov Tal joined the newly founded Ben Gurion University in the Israeli desert city Beer Sheva where he initiated and led the Medical School's Virology Department." (Gabriel Kaufmann)"Professor Jacov Tal will be best remembered by the scientific community for his extensive studies of the parvoviruses adeno-associated virus (AAV) and minute virus of mice (MVM). One of his significant achievements was the determination of parameters of site-specific integration of AAV, leading to development of potential vectors for gene therapy. Other important contributions were his insights into MVM's ability to kill cancerous cells, while leaving normal cells unaffected. The students of Professor Tal will also remember him for his dedication to training them to be rigorous and discerning scientists, and his concern for their well being. His colleagues will miss his sharp wit and analytic acumen." (Maureen Friedman)"Jacov Tal and I collaborated closely to his last days. Together, we found that during embryonic development MVM somehow senses a differentiation signal, and we suggested a relation between this observation and the MVM's anti tumor cell activity. I recall Jacov's 'freshman' enthusiasm in private, and his public posture as one who speaks up his mind without hesitation; standing up against any perceived injustice." (Claytus Davis)"I met Jacov towards the latter years of his career. In 1993, Jacov came to the US to do a sabbatical in Peter Chiang's laboratory. By and by, he drifted into my laboratory and actually spent the entire year working with me. Jacov by then, already a senior scientist for many years, not unexpectedly struggled heroically (and largely unsuccessfully) at the bench; and certainly it was not his bench-skills that impressed me. What did impress me was Jacov's common sense and his very human and generous attitudes. I recall an incident during Jacov's first week when he had not yet gotten to know all the members of my lab. At that time, there was a tall, curly-haired, darkly-handsome and academically gifted young man, working as a post-doc with me, who had graduated from Yale, obtained his MD degree from Duke, and received house staff and infectious diseases training from the University of Virginia. This person also has four siblings who are MDs. Jacov upon meeting this young man, whispered to me excitedly, 'Now here is a nice Jewish boy who is going to make the mother of a Jewish daughter very happy!' Surprise, surprise...that person turned out not to be Jewish, but a Catholic Lebanese-American of Arabic descent. Afterwards, a sheepish Jacov explained to me that it is very difficult to nearly impossible to tell between an Arab and a Jew in Israel; and as far as he was concerned, it made no difference whether Arab-American or Jewish-American. I was struck by his frankness and openness. In his typically thoughtful and 'dovish' ways, over the next many years, Jacov would email me from Israel his periodic 'roadmaps' for peace in the Middle East, accompanied by his incisive commentaries. I will deeply miss my friend's common sense advice and humor." (Kuan-Teh Jeang)


The objective is to present the design of randomised clinical trial (RCT) on the effectiveness of physical therapy added to general practitioners management compared to general practitioners management only in patients with an acute lumbosacral radicular syndrome .Patients in general practice diagnosed with an acute (less than 6 weeks) lumbosacral radicular syndrome and an age above 18 years are eligible for participation. The general practitioners treatment follows their clinical guideline. The physical therapy treatment will consist of patient education and exercise therapy. The primary outcome measure is patients reported global perceived effect. Secondary outcome measures are severity of complaints, functional status, health status, fear of movement, medical consumption, sickness absence, costs and treatment preference. The follow-up is 52 weeks.Treatment by general practitioners and physical therapists in this study will be transparent and not a complete "black box". The results of this trial will contribute to the decision of the general practitioner regarding referral to physical therapy in patients with an acute lumbosacral radicular syndrome. Publishing the design of the trial has several advantages. It may prevent publication bias . A studyThe lumbosacral radicular syndrome (LRS) is a complex of symptoms related to the lumbosacral nerve roots. The LRS is a disorder with radiating pain in the leg below the knee in one or more lumbar or sacral dermatomes, and can be accompanied by phenomena associated with nerve root tension or neurological deficits  -7. A proMost patients seeking medical care in the Netherlands will first visit a general practitioner (GP), who is regarded as the 'gatekeeper' of the health care system. The majority of health problems presented to GPs are treated by the GPs themselves and they are responsible for most referrals to (para) medical specialists. In 1996 the Dutch College of General Practitioners published their clinical guideline for LRS . There iPrimarily, treatment consists of adequate pain medication, giving information about the natural course of LRS, which in general is favourable, and stimulating to continue the normal daily activities of the patient. GPs in the Netherlands largely comply with the recommendations stated in the clinical guideline regarding management in patients with LRS . HoweverThe LRS trial aims to assess the effectiveness of PTs management added to GPs management compared to GPs management only in patients with acute LRS. We will use a multicentre, randomised clinical trial as study design. Figure Participating GPs in and around Rotterdam, the Netherlands, will invite patients with suspected acute LRS to participate in the trial. GPs will invite patients from May 2003 till November 2004 if they have radiating (pain) complaints in the leg below the knee; duration of the (pain)complaints is less than 6 weeks, the age is above 18 years and they present one of the following symptoms: more pain on coughing, sneezing or straining, decreased muscle strength in the leg, sensory deficits in the leg, decreased reflex activity in the leg or a positive straight leg raising test. Patients will receive a letter of information about the LRS trial from their GP. Patients' name and telephone number will be faxed to the research institute. Subsequently, a researcher (PL) will screen eligible patients by telephone and make an appointment to check inclusion and exclusion criteria, to complete the informed consent procedure and to perform the baseline measurement. Figure Randomisation will take place after baseline measurement by the research assistant. We use a concealed randomisation procedure using a computer generated randomisation list developed by an independent person. Patients' specific and unique trial number will be typed in a special developed database  and the random allocation will appear on screen. In order to prevent unequal treatment group sizes, block randomisation will be used with blocks of 10 patients . This meFor obvious reasons GPs and PTs are not blinded for treatment allocation. But they are not involved with treatment effect measurements. The patients cannot be blinded because of the ethical reasons as stated by the Medical Ethical Committee. The researcher is involved in the statistical analysis, but the analysis and interpretation of the findings will be audited and verified by an independent and not involved statistician. In this trial the primary outcome measurement and most of the secondary outcome measurements will be scored by the patients. Studies from Ostelo et al  and SchoAll patients will be treated by the GP according to their clinical guideline  are not allowed in the PT treatment. This PT treatment protocol was accomplished in a consensus meeting with participating PTs. The PT will report what kind of information/ advice and what type of exercise the patient receives in each session. Both GP and PT intervention will be restricted to a maximum of 9 treatments/ consultations in the first 6 weeks after randomisation.In the Netherlands, PTs are mainly taught the biomechanic model . This moIt is possible that patients may suffer from a fear of movement because of pain . Good adThis trial attempts to enrol 182 patients with LRS, 91 patients in both treatment groups. This sample size is regarded sufficient to detect a difference of 20% (with a α of 0.05 and a power of 80%) in the primary outcome (GPE) between the two treatment groups. A difference of 20% is considered to be clinically relevant .Figure The primary outcome measure is the Global Perceived Effect (GPE), measured on a 7 points scale ranging from 1 = completely recovered to 7 = vastly worsened. It is regarded a clinical relevant outcome measure and is regarded valid and responsive to measure the patients' perceived benefit -20.Pain severity of the leg and the back will be scored on a 11 points Visual Analogue Scale (VAS) ranging from 0 = no pain to 10 = unbearable pain. Reliability, validity and responsiveness of the VAS have been shown -23.The functional status will be measured with the Roland Morris Disability Questionnaire (RDQ) for sciatica . The scoHealth status will be measured by the 36-item short form (SF-36)  and the Fear of movement will be measured by the Tampa scale for kinesiophobia (TSK) ,39. The Costs will be calculated and include LRS related sickness absence from work, medical consumption , out-of-pocket expenses and paid help. Patients' treatment preference will be evaluated at baseline and at 4 follow-up measurements.Baseline comparability will be investigated by descriptive statistics to examine whether randomisation was successful. If necessary, adjustments for baseline variables will be performed in the analysis. Group differences and 95% confidence intervals will be calculated for all outcome measures. The statistical analysis will be performed according tot the intention-to-treat principle, analysing the patients in the treatment group to which they were randomly allocated. Between group differences will be calculated using the Student t-test for continuous variables or Chi-Square for dichotomous variables. In addition a per-protocol analysis will be performed, analysing only those patients with no serious protocol deviations. Comparing the results of the intention-to-treat and the per-protocol analysis will indicate if and to what extent protocol deviations might have biased the results. Multivariate regression analysis will be conducted to examine the influence of baseline variables on outcome.This article introduces a design of a RCT to evaluate the additional effectiveness of PTs management added to GPs management in patients with LRS. The study is designed in a way that GP and PT treatment is transparent (according a guideline and a consensus meeting) and not a complete "black box". The results of this trial will contribute to the decision of the GP regarding referral of patients with LRS to PT. The inclusion of patients will run until the end of the year 2004. The follow-up measurements will be completed in the end of the year 2005.The pre-publication history for this paper can be accessed here:


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 , and at Caesarean section, at elective  and intrapartum  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  [260. To eliminate any residual contaminating genomic DNA, all RNA samples were DNase-treated with the DNA-free™ DNA removal kit , 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 , 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 , 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 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  to remove plasmid DNA. The supernatant, containing purified cRNA, was pipetted onto pre-hydrated NucAway™ spin columns  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 , 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 , 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.