To keep a cell healthy, proteins must go to the right places within a cell. To direct proteins to specific areas of the cell, they're marked with tags akin to zip codes on mail. These tags can direct one protein to the cell's nucleus, where it regulates gene expression, say, while another is sent to the hinterlands of the cell membrane, where it receives environmental signals. Protein targeting is crucial even as cells are building proteins—otherwise, for example, proteins won't fold into the proper shape.There's a pair of proteins that, across all organisms, plays a key role in protein targeting. Called Ffh and FtsY in bacteria, these proteins each have an active and inactive state; only when they're in their active state can they bind each other and deliver other proteins to their proper location. Both these proteins are GTPases, a class of proteins that work as molecular switches. However, among GTPases, Ffh and FtsY are unique. Other GTPases switch between active and inactive states by binding different forms of a small, energy-carrying molecule—either GTP or GDP. Such GTPases often require the help of other proteins to switch states. Ffh and FtsY, on the other hand, almost always have GTP bound. And when they interact, they change each other's state—allowing each other to convert GTP to GDP—without the help of other proteins. But researchers didn't know exactly how these proteins interacted or how they switched between their active and inactive forms.PLoS Biology, Shu-ou Shan and colleagues have found that when Ffh and FtsY bind and then activate each other, they likely go through an unusual, multi-step process in which the proteins change shapes, flexing so that different parts of the proteins become active at each step.Now, as reported in this issue of The researchers mutated 45 different sites in the gene that encodes the FtsY protein to produce a bevy of mutant proteins with different properties. The researchers chose mutations that produced amino acid substitutions at sites in the FtsY protein that have been preserved through evolution, and so are presumably crucial for the protein's function. These sites, it turns out, are all on the protein's surface where it interacts with Ffh.The mutant versions of FtsY varied in how well they bound to the normal version of Ffh and how quickly the two proteins activated each other. The researchers were able to sort the different mutations into four different classes based on the type of problem the proteins had: some bound Ffh very loosely, some bound Ffh well but only weakly turned on its GTPase activity, and so on. All of these mutations would presumably foul up the protein targeting system, so this explains why certain amino acids have been preserved through evolution.Both Ffh and FtsY change shapes as they bind, activate each other's GTPase activity, then cleave GTP and release each other, the researchers infer. They don't have direct evidence for these shape changes, but the postulated bends and twists during interaction are consistent with the build of the proteins. These shape changes, they speculate, could switch different parts of each protein between active and inactive states. By showing how this unique type of GTPase switch likely works, Shan and colleagues have helped explain how cells target proteins to specific areas—and perhaps have paved the way for others to find similar switches elsewhere within cells.


Over the past decade RNA interference (RNAi) has emerged as a natural mechanism for silencing gene expression. This ancient cellular antiviral response can be exploited to allow specific inhibition of the function of any chosen target gene. RNAi is proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, RNAi technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes.This review briefly describes the molecular principles underlying the biology of RNAi phenomenon and discuss the main technical issues regarding optimization of RNAi experimental design. In 1998 Fire and coll. coined the term RNA interference (RNAi) referring to the phenomenon of post-translational silencing of gene expression that occurs in response to the introduction of double-stranded RNA (dsRNA) into a cell [Introduction of long dsRNA into a mammalian cell triggers a vigorous nonspecific shutdown of transcription and translation, in part due to activation of dsRNA-dependent protein kinase-R (PKR) . ActivatRNAi is a highly conserved mechanism throughout taxonomical species . In addiBesides gene silencing, RNAi might be involved in other phenomena of gene regulation. DNA/RNA interactions are known to influence DNA methylation. It appears that RNAi can also function on this level by methylating cytosines as well as CpG sequences more classically associated with methylation. If the target sequence shares homology with a promoter, transcriptional silencing may occur via methylation. Moreover, RNA appears to interact with chromatin domains, which may ultimately direct DNA methylation. Studies of C. elegans have shown that RNAi can spread among cells through mechanisms that may not hinge upon siRNA . The sysSeveral strategies for inducing siRNA-mediated gene silencing have been developed, each of them presenting specific advantages and disadvantages Table .Synthesis, purification, and annealing of siRNAs by industrial chemical processes  is becomSuppression of gene expression by RNAi is generally a transient phenomenon . Gene exSeveral crucial considerations should be beard in mind while designing RNAi experiments. The below examples regard RNAi experiments performed with chemically synthesized siRNA.1. The first step is to design a suitable siRNA sequence. A growing number of libraries of validated siRNAs directed toward some frequently targeted genes are available. However, if the gene of interest has not been targeted using siRNA before, a novel siRNA must be developed. In mammalian cells RNAi is mediated by 21- to 23-nucleotide siRNAs containing symmetrical two nucleotide 3' overhangs. Given a siRNA sequence alone, it is not currently possible to predict the degree of gene knockdown produced by a particular siRNA. Nevertheless, several observations have been made that can be taken into account to increase the probability of producing an effective siRNA. The chief variable is the gene target site. Generally, it is recommended that a target site located at least 100–200 nucleotides from the AUG initiation codon is chosen. Targets within 50–100 nucleotides of the termination codon should instead be avoided. The 5' and 3' untranslated region (UTR) should also be avoided, since associated regulatory proteins might compromise RNAi. This is just a general recommendation, as some siRNAs targeting the 3' UTR have also been shown to induce RNAi . Numerou2. The structural characteristics of the siRNA molecules are another crucial aspect to be considered while designing RNAi experiments. SiRNA of 21 nucleotides with 3'-d(TT) or (UU) overhangs are considered the most effective . Despite3. To induce RNAi, siRNA must be transfected into the cells of interest. Several transfection reagents exist, the most commonly used being liposomal or amine-based. In some cases electroporation may be used, but cell toxicity can be high with this technique . Cell li4. Recently, experimental design features have been suggested to guarantee the rigor of RNAi experiments . Due to 5. The effect of RNAi should be quantified at both the mRNA and the protein level. The knockdown of a protein should be probably evaluated after mRNA reduction has been proved: in fact, a reduction in protein levels not accompanied by a decrease in mRNA might indicate that other mechanisms are at work, such as RNAi mediated by microRNA. Northern blot analysis is considered by many to be the gold standard. Real-time reverse transcriptase polymerase chain reaction, incorporating internal controls to quantify "housekeeping" gene transcript levels, can also be used . ProteinRNAi is now commonly used in biological and biomedical research to study the effect of blocking expression of a given gene. As the effect is rarely complete, it is generally termed a "knock-down" to distinguish it from the "knock-out" achieved by deletion of the gene. Although significant advances have been made as compared to previous methods, RNAi has its own limitations. Not every sequence works, most researchers reporting a success rate of about one in three. Moreover, although the effects are generally believed to be highly sequence-specific, some doubts remain as to whether or not some of the observed effects are "off target." Some residual activation of the interferon system has been reported, as well as degradation of closely related, but non-identical, mRNAs. Nevertheless, RNAi remains the most promising functional genomics tool recently developed. DNA microarray technology has now enabled the level of expression of every gene in the genome to be determined under any condition ,35. This


Phosphoinositides mediate one of the intracellular signal transduction pathways and produce a class of second messengers that are involved in the action of hormones and neurotransmitters on target cells. Thyroid hormones are well known regulators of lipid metabolism and modulators of signal transduction in cells. However, little is known about phosphoinositides cycle regulation by thyroid hormones. The present paper deals with phosphoinositides synthesis de novo and acylation in liver at different thyroid status of rats.Myo-[3H]inositol, [14C]CH3COONa, [14C]oleic and [3H]arachidonic acids were used to investigate the phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis. 1-methyl, 2-mercaptoimidazol-induced hypothyroidism was associated with the decrease of myo-[3H]inositol and [3H]arachidonic acids incorporation into liver phosphoinositides and total phospholipids, respectively. The thyroxine (L-T4) injection to hypothyroid animals increased the hormones contents in blood serum and PtdInsP2 synthesis de novo as well as [3H]arachidonic acids incorporation into the PtdIns and PtdInsP2. Under the hormone action, the [14C]oleic acid incorporation into PtdIns reduced in the liver of hypothyroid animals. A single injection of L-T4 to the euthyroid [14C]CH3COONa-pre-treated animals or addition of the hormone to a culture medium of hepatocytes was accompanied by the rapid prominent increase in the levels of the newly synthesized PtdIns and PtdInsP2 and in the mass of phosphatidic acid in the liver or the cells.The experiments were performed in either the rat liver or hepatocytes of 90- and 720-day-old rats. 4 injection to the animals was followed by the time-dependent increase of polyphosphoinositide synthesis in the liver. The both long-term and short-term hormone effects on the newly synthesized PtdInsP2 have been determined.The data obtained have demonstrated that thyroid hormones are of vital importance in the regulation of arachidonate-containing phosphoinositides metabolism in the liver. The drug-induced malfunction of thyroid gland noticeably changed the phosphoinositides synthesis de novo. The L-T The phosphoinositides is a family of lipids which members play an essential role in the receptor-mediated intracellular signaling cascades, vesicle trafficking and cytoskeletal rearrangements [for review see ,2] and, 2) in the plasma [2 and other polyphosphoinositides production [The two pools of phosphoinositides are supposed to exist in the cells ,4. One oe plasma  and nucle plasma  membraneoduction ,11.The regulation of the key enzymes of the PtdIns synthesis pathway: the CDP-diacylglycerol (DAG) synthase and PtdIns synthase were examined . Express32P]phosphoinositides and inositol 1,4,5-trisphosphate were found to be significantly lower in the hypothyroid rat hearts [3H]inositol phosphate in the hypothalamic slices, whereas the thyroxine (L-T4) completely restored the hypothalamic [3H]inositol phosphate formation [3H]inositol pre-labeled tissue [Thyroid hormones are of vital importance in maintaining the initial level of phospholipids in cell membranes and fatty acids composition of the lipids. On the other hand, there is a little literature regarding the hormone regulation of phosphoinositides exchange. The levels of [t hearts . The efft hearts . The hypormation . The resd tissue . Thus, i4-altered thyroid status of animals on the phosphoinositides synthesis in the liver. Thus, it was determined that the hypothyroidism decreased considerably the levels of the newly synthesized lipids in the liver but thyroid hormones increased the synthesis of the polyphosphoinositides de novo and the arachidonic acid incorporation into the PtdIns and PtdInsP2. A single injection of the L-T4 to the euthyroid rats lead to the rapid and transient decrease of the newly synthesized PtdInsP2 followed by the increase of the PtdIns and PtdInsP2 levels in the liver.In the paper, we examined the influence of the drug- and L-T4-treated rats and intact animals after the single hormone injection has been studied.The present paper considers the influence of thyroid functional status on the phosphoinositides synthesis in the liver. To determine the role of thyroid hormones in the regulation of phosphoinositides synthesis de novo and lipids fatty acid remodeling, the euthyroid, MMI (1-methyl, 2-mercaptoimidazol)- and MMI+L-T4 induced hydrolysis of PtdInsP2 and inositol phosphates and DAG formation at the early stages of hormone action on the [14C]oleic and [14C]linoleic acid pre-labeled hepatocytes of adult 90-day-old rats [4-mediated PLC activation was accompanied by the protein kinase C (PKC) translocation to membranes [2-specific PLC activation in the L-T4-stimulated cells was a short-lived event. The hormone-stimulated rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was followed by its conversion into the biologically inactive inositol 1,4 -bisphosphate and inositol 1-phosphate [It has been reported that the L-Told rats ,21. The embranes  and PKC embranes  and acylembranes . The Ptd4 to the 90-day-old rats leads to the rapid and sustained increase in the L-T4 and triiodothyronine (L-T3) levels in blood serum , the PtdInsP2 is now recognized as an important plasma membrane signal that activates the PLD [for review see [2 is a membrane-associated cofactor of PLD [2-specific PLC by the L-T4 in the rat hepatocytes of adult 90-day-old rats was followed by the prominent PLD activation [2 – neomycin. Neomycin does not directly interact with PLC and PLD but interacts with the endogenous membrane PtdInsP2. Inclusion of the PtdInsP2 in mixed phosphatidylcholine/phosphatidylethanolamine liposomes [2 synthesis and the resting cellular levels of PtdInsP2 are insufficient for enzyme stimulation. The role of PKC has been determined in the regulation of PtdInsP2synthesis in the liver [4 to stimulate the PtdInsP2 synthesis in the liver cells of the 720-day-old rats Pview see ] and thur of PLD ,27. The tivation . The PLDiposomes  and stimiposomes . The reshe liver  and othehe liver . The PKChe liver ] and phohe liver  stimulathe liver  correlatats Fig. . Howevermyo-[3H]inositol incorporation into the PtdIns, PtdInsP and PtdInsP2 in the liver of the drug- and L-T4 -treated animals. The MMI administration to the rats was found to be accompanied by the decreased myo-[3H]inositol incorporation into the PtdIns, PtdInsP and PtdInsP2 in the liver  stimulate the phosphatidic acid and PtdInsP2 accumulation and did not change the PtdIns content in the liver cells, it could be assumed that in such case the L-T4 increases PtdInsP2 synthesis via phosphoinositide 5-kinase activation rather than the lipid synthesis de novo.Thyroid hormones stimulate lipogenesis in the liver by inducing the enzymes in the lipogenic pathway. The acyl-CoA-glycerol-3-phosphate acyltransferase, which is known to catalyze a rate-limiting step for the synthesis of phosphatidic acid in the rat liver, is dependent on the thyroid gland function . The L-Tatocytes . The resatocytes . It can 5-kinase  and thus3 administration to the euthyroid rats increases the saturated fatty acids and arachidonate/linoleate ratio of PtdIns in the liver cell mitochondria [It is well documented that the efficiency of polyphoshoinositide derived second messanger DAG in signaling pathways is closely dependent on the degree of its unsaturation. The unsaturated fatty acids are incorporated into the sn-2 position of the phospholipids by the deacylation-reacylation reactions. Some investigations demonstrated the changes of the fatty acid composition of membraneous lipids at the hypo- and hyperthyroid state of the rats [for review see ]. There chondria .3H]arachidonic acid incorporation into the liver total phospholipids  and decreased the content of the oleate-labeled PtdIns in the liver slices (Table 3D). The incorporation of the [14C]oleic acid into the PtdInsP2 did not differ between the liver slices of control, drug- and hormone-treated animals. As can be seen from the Table 4 and T3 levels in the blood serum, but did not remove completely the hormones from organism. The T3 content in the serum of the drug-treated animals was relatively high as compared with control animals. However, the MMI reduced the T4 level by 77%. The T4 administration to the MMI-treated rats increased significantly T3 and T4 contents in the serum. It seems possible that the 14C/3H fatty acids incorporation into the phosphoinositides was rather regulated by the T3 than T4, although the both hormones are participated in the regulation of the other phospholipids acylation in the rat liver.The MMI did not change markedly the s Figure . The L-T2 synthesis, which are the predominant substrates of PLC in the numerous signaling pathways. The both long-term and short-term effects of hormone on lipid synthesis have been determined  involved in the signal transduction in the stimulated cells, the abnormalities of the phosphoinositides synthesis at different pathological states of thyroid gland could disturb other hormones signaling in the target cells.The present data have demonstrated that the phosphoinositides synthesis de novo and arachidonic acid incorporation into phospholipids are strongly dependent on the thyroid status of organism. The marked enrichment of animal cell phosphoinositides in arachidonate and the results obtained suggest an important role of the thyroid hormones in the regulation of polyunsaturated PtdIns and PtdInsPd Figure . The L-Tynthesis ,37. Consmyo-[3H]-inositol (58 mCi/mmol), [14C]oleic acid (58 mCi/mmol) and [3H]arachidonic acid (60 Ci/mmol) – Amersham Corp. and [14C]CH3COONa (25 mCi/mmol) – BPO Isotop (Russia); silica gel from Woelm (Germany). Phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate lipid standards were obtained from Sigma (USA). T4 and 1-methyl, 2-mercaptoimidazol were from Zdorov'e Trudyaschikhsya (Kharkov, Ukraine). T4 and T3 radioimmunoassay kits were from Minsk (Belarussia). Other chemicals used were of chemically pure grade.4 (200 μg/100 g weight) 48 h prior to killing. Besides, T4 (200 μg/100 g weight) was injected to the normal rats 15 and 60 min prior to killing. Control rats received 0.9% NaCl of the same volume. The animals were starved overnight prior to experiment. The thyroid state of rats was monitored by radioimmunological determination of the T4 and T3 in blood serum.90- and 720-day-old male Wistar rats, which had a free access to food and water and were kept at 24°C on a cycle of 12 h light/12 h darkness were used for experiments. The MMI was injected intraperitoneally (1 mg/100 g weight) in 0.9% NaCl to the experimental animals every day during 16 days-experiment. In some cases, the MMI-treated rats were injected intraperitoneally by T14C]CH3COONa was intraperitoneally injected to rats four times every 30 minutes for 2 hours. The liver was perfused with 0.9% NaCl, then removed and washed in Krebs-Henseleit buffer, pH 7.4, containing 2 mM CaCl2 and 0.2% BSA. The pre-labeled liver was used to obtain 10% homogenates and to analyze the14C-phosphoinositides. Besides, the slices of unlabeled liver were labeled by incubation in the Eagle medium containing 10% fetal calf serum, 100 units/l streptomycin, 100 units/l penicillin, 13 mg/ml gentamycin and 0.1 μCi/ml of mio- [3H]inositol or 2.5 μCi/ml of [14C]oleic acid or 2.5 μCi/ml [3H]arachidonic acid for 1–2 h in 95% O2/5% CO2 atmosphere at 37°C. The lipids were extracted and analyzed as described below.The 1 mCi of [7/ml) were incubated in the Eagle medium containing 10% fetal calf serum, 100 units/liter streptomycin, 100 units/liter penicillin, 13 mg/ml gentamycin and in the presence of 100 nM NaOH (control) or L-T4(10 nM) for 30 min in 95% O2/5% CO2 atmosphere at 37°C. Before lipid extraction, the cells were washed twice with a Krebs-Henseleit buffer pH 7.4, containing 2 mM CaCl2, 25 mM HEPES, 0.1% BSA. The lipids were extracted and analyzed as described below.The hepatocytes were isolated from the liver by the method described in . Prepara2 at 37°C. The lipids were redissolved in chloroform/methanol (1:2, v/v) and applied on TLC plates. For a total phospholipids isolation, the solvent system: hexane/diethyl ether/acetic acid (80:20:2, v/v) was used, for PtdIns, PtdInsP and PtdInsP2 – chloroform/methanol/NH4OH (50:40:10, v/v). For phosphatidic acid separation the two-dimensional TLC was used. The TLC plates were developed in chloroform/methanol/ NH4OH (60:35:5, v/v) (first direction) and after – in the second direction in chloroform/methanol/acetic acid/water (80:60:7.4:1.2, v/v). The phospholipids masses were determined as described in [14C/3H]lipids were scraped and transferred to the scintillation vials. Radioactivity was measured by a scintillation counter.The phospholipids were extracted according to Folch et al. , the phoribed in . The gelNAB conceived of the study and participated in its design, coordination, and manuscript preparation. OAK participated in data collection and performed the statistical analysis.


With the genome assembly archive, it is possible to examine the raw data that underlies the DNA sequence in any sequenced genome Scientists have dedicated considerable effort to decoding the genomes of an ever-growing list of species, ranging from small viruses, whose genomes may be just a few thousand nucleotides in length, to large mammalian genomes, three billion nucleotides and larger. Many aspects of life science research have benefited by the accumulation of these data, but decoded genomes could be even more valuable if important information about the genome sequence, currently being lost, were preserved. Occasionally, questions arise about a specific position in the sequence—or a variant in the sequence is observed in a new sample. At times like these, it would be helpful to be able to go back to the experimental evidence that underlies the genome sequence at that position, to see if there is any ambiguity or uncertainty about the sequence. As things stand, that's almost impossible. To understand why this is the case, it is necessary to know a bit more about how a genome sequence is put together.Haemophilus influenzae in 1995 , which are then sequenced. The sequences are assembled to generate a consensus sequence that, if all steps work perfectly, matches the original DNA molecule. Since the sequencing of  in 1995 , most baWithout really being aware of it, the bioinformaticians who assemble genomes have for years been discarding the valuable information on how all of the individual sequence fragments align to the assembled chromosomes. This loss has gone largely unremarked because the scientific community has focused its attention primarily on the end product: the final genome sequence itself. It is only natural to regard the genome sequence, which is the basis for gene discovery and for functional understanding of the biology of the organism, as the primary result of a WGS project. In reality, though, a WGS project is an experiment in which large numbers of sequencing reactions are run, followed by a combination of computational work and additional sequencing to complete the genome. Three years ago, the Trace Archive  was developed to store the raw sequence data and to facilitate dissemination of this data, but currently there is no database that captures the alignment of these reads to the published genome sequence.Many scientists would be surprised to hear that genome assemblies are unavailable. One might infer that the assembly of a genome could be reconstructed from the genome sequence and the associated traces. However, aligning the traces to the genome will generally not reproduce the assembly, both because many of the traces will have alternate possible alignments and because, in some cases, parts of the assembly are manually refined based on additional experimental data. Furthermore, only a small number of large-scale centers have the computing hardware, software, and bioinformatics expertise to allow them to assemble a large genome.http://www.ncbi.nlm.nih.gov/projects/assembly). The archive has been developed to store both an archival record of how a particular assembly was constructed and the alignments of any set of traces to a reference genome. Assemblies contained in this archive will be available in the GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html), DDBJ (http://www.ddbj.nig.ac.jp/), and EMBL (http://www.ebi.ac.uk/embl) databases, and all underlying traces are required to be deposited in the Trace Archive.To bridge this gap, we have developed the Assembly Archive (Bacillus anthracis (the causative agent of anthrax), which have been sequenced as part of an effort to understand the detailed variation of that species. This includes the completed reference genome of the Ames strain, sequenced from a sample kept frozen since 1981, when it was originally isolated in West Texas . For the first time, the evidence behind each polymorphism in these assembled genomes will be directly accessible to the scientific community.The Assembly Archive's first entries are a set of seven closely related strains of B. anthracis bacterium (the bacterium sent through the United States mail in late 2001) in order to determine if there were any differences between it and a reference laboratory sample  are common causes of base-calling errors, because the peaks in the underlying trace data sometimes merge together. The sequence shown includes several monomer runs; several of the underlying traces are shown as well. For example, the run of six As at the far left of the figure is supported by several reads in which all six peaks are distinct, as well as other reads in which the six nucleotides appear as one broad peak. By examining data such as these, one can easily verify (or disprove) putative SNPs in this genome.Human polymorphism studies e.g.,  are a trIn contrast with prokaryotic genomes, the human genome  is diploid, and as a result many SNPs can be discovered within a single assembly, which contains the chromosomes representing the two parent organisms. SNPs can also be found through population studies in which the same locus is sampled from multiple individuals. In either case, the evidence for a SNP begins with the alignment of two different genomes. Despite the clear need for it, the original evidence for the genome itself—the assembly—is not available, and is not linked to the evidence in the Trace Archive. If it were available, many of the polymorphisms already reported could be validated, and many more SNPs might be discovered. Assemblies will also allow centers to better coordinate their gap-closing and finishing efforts, as has been recently noted .We hope that the availability of the Assembly Archive will encourage human genome sequencers, and sequencers of other genomes, to begin depositing their assemblies into this public resource, where it can be shared by all.


O(n6)time and O(n4) space algorithm by Rivas and Eddy is currently the best available program.The general problem of RNA secondary structure prediction under the widely used thermodynamic model is known to be NP-complete when the structures considered include arbitrary pseudoknots. For restricted classes of pseudoknots, several polynomial time algorithms have been designed, where the O(n4) time and O(n2) space to predict the energetically optimal structure of an RNA sequence, possible containing such pseudoknots. Evaluation against a large collection of known pseudoknotted structures shows the adequacy of the canonization approach and our algorithm.We introduce the class of canonical simple recursive pseudoknots and present an algorithm that requires RNA pseudoknots of medium size can now be predicted reliably as well as efficiently by the new algorithm. Pseudoknots have been shown to be functionally relevant in many RNA mediated processes. Examples are the self-splicing group I introns , ribosomn into unknotted structures requires O(n3) time and O(n2) space, finding the best structure including arbitrary pseudoknots has been proved to be NP-complete [Well established algorithms for the prediction of RNA secondary structures (MFOLD , RNAfoldcomplete ,8. In faO(n4) time and O(n3) space algorithm for base pair maximization has been given in [O(n3) time algorithm based on maximum weight matching in [The first route is to consider pseudoknots in full generality, but resort to an even more simplistic energy model. An given in , and an ching in  and 10]O(n4) timO(n6) time and O(n4) space [pknotsRE for later reference. Further improvements have been shown to be possible for yet more restricted classes, e.g. the non-recursive simple pseudoknots considered by Lyngsø and Pedersen [O(n5) time and O(n4) space, but to our knowledge, no implementations are available. Recently, an O(n4) time and O(n3) space algorithm based on the technique of [The second route is the one followed here: We retain the established thermodynamic model, but restrict to a more tractable subclass of pseudoknots. For some quite general classes of pseudoknots, polynomial time algorithms have been designed: Rivas and Eddy achieve 4) space . This alPedersen  with O(nnique of , that usnique of . While iThe new contributions reported here are the following:pknotsRG for folding RNA secondary structures including pseudoknots under the MFE model which requires O(n4) time and O(n2) space.• We present an algorithm pknotsRG.• The algorithm considers the class of simple recursive pseudoknots, further restricted by three rules of canonization. Each simple recursive pseudoknot has a canonical representative that is recognized by • While this class is more restricted than the one of the Rivas/Eddy algorithm, practical evaluation shows that our algorithm finds the same pseudoknots, while the length range of tractable sequences is increased significantly.• We provide an evaluation of the class of pseudoknots introduced here against known examples from the literature.RNAfold and, where feasible, with pknotsRE.• We perform a rigorous evaluation of our algorithm on 212 sequences from PseudoBase  plus 7 opknotsRE by Rivas and Eddy. It recognizes pseudoknots that can be nested and can have unlimited chains of helices involved in crosswise interactions. The drawback of this powerful, but computationally expensive algorithm is the following paradox: Pseudoknots with complex helix interactions naturally require longer primary sequence than simpler ones. The high runtime complexity of O(n6), however, as well as the space consumption of O(n4) restricts the use of this algorithm to a maximal sequence length of around 150 nucleotides. Most of the pseudoknots predicted belong to a much simpler structural class and do not exhibit chains of crosswise interactions.It is not easy to relate the classes of pseudoknots recognized by the different algorithms mentioned above. We refer the reader to the review by Lyngsø and Pedersen , which cO(n4) and space complexity O(n2). The runtime improvement, compared to pknotsRE, results from an idea of canonization, while the space improvement results from disallowing chained pseudoknots. These improvements extend the range of tractable sequences to a length up to 800 nucleotides, and we can locate pseudoknots up to this size in even longer sequences.The algorithm developed here achieves time complexity simple pseudoknot is a crosswise interaction of two helices, as shown in Figure recursive pseudoknots, we allow the unpaired strands u, v, w in a simple pseudoknot to fold internally in an arbitrary way, including simple recursive pseudoknots. Let us call this class sr-PK. More complex knotted structures like triple crossing helices or kissing hairpins, as shown in Figure Following the terminology of , a simplbefore it is written in detail. We consider elements of RNA structure as sequence motifs of different types: hairpins, bulges, multiloops, etc. The following notation is taken from the algebraic dynamic programming approach [Thermodynamic RNA folding is implemented via dynamic programming (DP). We start with a semi-formal discussion of how to estimate the efficiency of a DP algorithm for folding (or any kind of motif search) approach . By an em = f <<< a ~~~ b ~~~ c | | | g <<< c ~~~ am can be composed in two alternative ways: The first case, labelled by f, requires adjacent occurrences of motifs a, b and c. The second case, labelled by g, requires adjacent occurrences of motifs c and a. When motif m is to be scored, f and g are seen as the scoring functions that combine the local score contribution of each case with the scores of sub-motifs a, b, and c.we specify that the sequence motif m in an input sequence x of length n, say at sequence positions i through j? First we assume that all motifs can have arbitrary size between 0 and n. The algorithm must consider all boundary positions (i, j) for motif m, which requires O(n2) steps at least. In case g, it must consider all boundary positions k where motifs c meets a, such that the runtime for case g is in O(n3). In case f, there are two such moving boundaries k and l between the three sub-motifs, so we obtain O(n4) overall for motif m.What is the computational effort of locating motif a is a single base, for example, the exponent of n decreases by 1 in both cases. Furthermore, if motif b is (say) a loop of maximal size 40, then one factor of n is reduced to a constant factor and overall asymptotic runtime is now O(n2). Sometimes a motif description can be restructured to improve efficiency by reducing the number of moving boundaries. Whether or not this is possible does not depend on the motif structure, but on the scoring scheme! This is a somewhat surprising observation from [This can be improved if there is an upper bound on the size of some motif involved. If motif ion from , where sa and c have the same length, then the boundaries k and l in case f are related by k - i = j - l. When iterating over k, we can use l := j - k + i (rather than k ≤ l ≤ j)and save another factor of n.In the sequel, we shall exploit another source of efficiency improvement. If the lengths of two sub-motifs are coupled somehow, say When the search space of a combinatorial problem seems to be too complex to be evaluated efficiently, heuristics are employed. Canonization restricts the search space in a well-defined way, arguing that all the relevant solutions in the full search space have a representative that is canonical, and hence, nothing relevant is overlooked. One such technique is the purging of structures that have isolated basepairs. Here the plausibility argument refers to the underlying energy model, where base pairings without stacking have little or no stabilizing effect. This canonization does not affect efficiency, but it achieves a significant reduction of the search space (figures in ), which canonized simple recursive pseudoknots, csr-PK. Using the notation introduced above, the motif definition of a simple recursive pseudoknot is given byWe shall introduce three canonization rules that reduce class sr-PK to the class of knot = knt <<< a ~~~ u ~~~ b ~~~ v ~~~ a' ~~~ w ~~~ b'i, e, k, g, f, l, h, j as shown in Figure with boundaries at sequence positions a forms a helix with a', and b with b'. Segments u, v, and w can have arbitrary structures, including pseudoknots. Naively implemented, we can expect a DP algorithm of time complexity O(n8) according to our efficiency estimation technique introduced above. We now apply canonization. Note that it only applies to helices forming pseudoknots; other helices are unaffected. We first present the technical aspects; the discussion of these restrictions is deferred to the next section.Segment a| = |a'| and |b| = |b'|. (b) Both helices must not have bulges.(a) Both strands in a helix must have the same length, i.e. |Note that (b) is a stronger restriction and trivially implies (a). Under the regime of Rule 1 we may conclude:f = l - (e - i)h = j - (g - k)O(n6).We are left with 6 out of 8 boundaries that vary independently, and runtime is down to a, a' and b, b' facing each other must have maximal extent, or in other words, compartment v must be as short as possible under the rules of base pairing.The helices a and a' is fixed once i and l are chosen. The maximal helix length stacklen(i, l) can be precomputed and stored in an O(n2) table. The same observation holds with respect to the other helix, and we fixWe observe that the maximal length of e = i + stacklen(i, l)g = k + stacklen(k, j).i, k, l, j –, and can hope to obtain an algorithm with runtime O(n4). Scores of pseudoknots found between i and j are stored in table knot(i, j), and hence the space requirements are O(n2), which is the same asymptotic space efficiency as in the folding of unknotted structures.Thus, we are left with only four independently moving boundaries – v, or in other words, the length of v would become negative. This case is addressed byA subtlety arises when both helices, chosen maximally, compete for the same bases of If two maximal helices would overlap, their boundary is fixed at an arbitrary point between them.m and m' be the helix lengths so determined. We finally obtainLet e = i + mg = k + m'ak denote a terminal symbol of k times the letter a. The grammar uses a single nonterminal symbol S and its productions areThe language of pseudoknots in class csr-PK can be defined by a simple context free grammar over an infinite terminal alphabet. Let k, l ≥ 1. For example, the simple pseudoknot of Figure for arbitrary .. [[[......{{..]]]]..........}}.This grammar is useful to judge how different an experimentally determined structure is from class csr-PK. It is not useful for programming, since it is ambiguous and does not distinguish the fine grained level of detail required in the energy model.A careful discussion is required to show that each simple recursive pseudoknot, if not canonical by itself, has (a) a canonical representative of (b) similar free energy.i' and j'. It is not canonical if one of the two helices contains bulges. However, there must be at least one pair of shorter helices without bulges at i, j with i' ≤ i and j ≤ j', which serves as a canonical representative, albeit with somewhat higher free energy.Rule 1 (b) affects the length of helices that are considered in forming the pseudoknot. Let there be a pseudoknot between i, k, l, j there is a canonical one with maximal helices, whose free energy is at least as low – except for the following case: The maximal helices compete with the internal structure of u, v and w. It may be possible to contrive a structure where shortening (say) helix a – a' by one base pair allows to create two pairs with new partner bases in u and v, resulting in a structure which has slightly lower energy. Still, the free energy of the canonical pseudoknot must be very similar.Rule 2 is justified by the fact that the energy model strongly favours helix extension. Clearly, for each family of pseudoknots delineated by a' or the b – b' helix.Finally, Rule 3 requires a decision where to draw the border between two helices facing each other and competing for the same bases. An arbitrary decision here can only slightly affect free energy, as the same base pairs are stacked either on the a – E(s) denote the free energy computed for structure s. Summing up, we have shown that for each simple recursive pseudoknot K, there is a canonical one C in the search space. While we cannot prove that E(C) ≤ E(K), we have argued that this is likely, and if not, the energies will at least be close. Still, there might be another, energetically optimal canonical structure S (knotted or not) such that E(K) <E(S) <E(C). In this case, if only the "best" structure S is reported, neither K nor its canonical representative C is observed. Let Finally, let us add that the implementation described below is actually slightly more general that the "pure" csr-PK model described above: We do allow a single nucleotide bulge in either helix of a pseudoknot, which complicates the program, but does not affect asymptotic efficiency.To evaluate how well the class csr-PK covers known pseudoknots, we considered 212 pseudoknot structures from PseudoBase. The observations are shown in Table We find 172 simple recursive pseudoknots, and 40 of more general shapes. We find that 135 out of the 172 pseudoknots lie in csr-PK, i.e. they are their own canonical representatives. 11 more fall into the relaxed csr-PK, where we allow a single nucleotide bulge in Canonization Rule 1. Thus, we cover 146 out of 212 (68%). 26 simple recursive pseudoknot do not fall in class csr-PK, since they contain isolated basepairs, non canonical basepairs or one of the helices has not maximal extent.a – b – c – d – c' – a' – d' – b', where helix d – d' is very short – only two base pairs. Deleting it, helix c – c' is no longer interacting with other helices, and the pseudoknot falls within class csr-PK.Considering the remaining 20% complex pseudoknots, note that often pseudoknots in more general classes also have a good representative in csr-PK. For example, the pseudoknot of Hepatitis delta virus Figure  consistsk best structures. Secondly, we shall provide three variants of our program:There are many reasons why "the" MFE structure may only be part of what we want to know about a molecule's foldings. To deal with the problem when the optimal (knotted) structure is non-canonical, and its canonical representative is dominated by an unrelated structure, we provide two means: First of all, our algorithm is non-ambiguous, the prerequisite for a non-redundant enumeration of near-optimal structures . We can pknotsRG-mfe computes the mfe structure (or the k best), pseudoknotted or not.pknotsRG-enf picks out from the folding space the energetically best structure that contains at least one pseudoknot.pknotsRG-loc computes the energetically best pseudoknot that can be formed locally, i. e. somewhere in the sequence. "Best" is defined here as minimal free energy per base, to avoid a built-in bias towards large pseudoknots.The best local pseudoknot motif is included by adding two cases:bestPK = skipleft <<< base ~~~ bestPK ||| bestPKlbestPKl = skipright<<< bestPKl ~~~ base ||| knotO(n2) and preserve the non-ambiguity of the algorithm. If desired, an enumeration of near-optimal "local" pseudoknots is also feasible.These clauses have time complexity RNAfold and in an older version with pknotsRE), and the results in this section may improve if this model is further improved in the future.We first consider the predictive accuracy achieved by our approach. We have already evaluated the class csr-PK against the known pseudoknots, and we know that our algorithm correctly implements this class in its search space. What is really tested in the following is the adequacy of the current thermodynamic model . Let BP be the number of basepairs in the reference structure from the database or literature. We define the sensitivity as (TP/BP), selectivity as (TP/TP+FP).We compare our results to the output of RNAfold. Both, the sensitivity and the selectivity increase. Compared to pknotsRE our results are slightly better, probably because we are using the newer and subtler energy model. For example, for the sequence of hepatitis delta virus, our algorithm predicts all helices except for the very short helix 5 . For all programs the overall prediction accuracy for tRNAs is not very high. tRNAs are a known hard case for structure prediction because they contain many modified bases.We also folded 14 randomly selected human tRNAs  take about 2 hours (see Table O(cn3) with a rather large constant c. This enables us to fold sequence of length 1000 in approximately 12 hours. We can further observe that the space requirements scale quadratically with the input size, as expected.Clearly, we are able to fold sequences that are longer than equences 00 bp takpknotsRE is not strictly wasted: It is spent on assuring that the optimal folding of the input RNA sequence does not contain pseudoknots with chained interacting helices of lower free energy than the reported structure. pknotsRG does not consider such structures and hence cannot make this assertion.For a fair comparison, the reader should keep in mind that the extra time spent by In the following, we discuss extensions of the implemented model and their expected computational costCanonization Rule 1 can be relaxed further to allow larger bulges inside the helices forming a pseudoknot. As long as their number (and hence the length difference of the two arms of a helix) is bounded by a constant, asymptotic efficiency is not affected.Two examples of non-simple pseudoknots are shown in Figure kiss = kss <<< a~~~u~~~b~~~v~~~a'~~~w~~~c~~~x~~~b'~~~y~~~c'triple = trp <<< a~~~u~~~b~~~v~~~c~~~w~~~a'~~~x~~~b'~~~y~~~c'O(n6), bringing runtime back to the efficiency class of the Rivas/Eddy algorithm. But note that the space requirements remain O(n2). This is due to the fact that we now consider three interacting helices, but not arbitrary chains.Canonization can be applied as above, with Rule 3 becoming more sophisticated for the triple interaction case. This would yield an algorithm of runtime in vivo as in vitro must be understood as a hierarchical process, where small structures in close vicinity form first, and then combine to larger ones [pknotsRG gives us the possibility to test this using a fairly large structure containing pseudoknots that have been proved experimentally. We considered the sequence of the group I intron from Tetrahymena thermophila (419 NT) (V01416).RNA folding ger ones . The folThe MFE-structure found was quite different from the "true" structure taken from the literature. We hand-coded the experimental structure and evaluated its stability in our energy model. The result was striking: the experimental structure  was significantly far from the possible minimum of free energy . So far in fact that it seems infeasible to detect the structure by scanning the space of near-optimal structures. This could be interpreted as the energy model being incorrect, but since it works well for short sequences, we suggest that this is an indication that the kinetics of folding already have a strong influence with this size of sequence, at least when pseudoknots are involved.While we have achieved a considerable speedup for predicting small pseudoknotted structures, it seems that minimum free energy approach is not meaningful with the largest structures which it now can handle algorithmically. However, the situation changes when we are looking for particular structural motifs (see below).pknotsRG-mfe, based on the MFE-model, for finding the best RNA structure including the pseudoknot class csr-PK. This requires O(n4) time and O(n2) space. The algorithm variant pknotsRG-enf returns the energetically best structure that contains a pseudoknot (interesting when the MFE structure is unknotted), while pknotsRG-loc reports the best pseudoknot  somewhere in a sequence. We achieve a high prediction accuracy for moderate length sequences, whereas long sequences, at least when pseudoknots are involved, seem to have a folding scheme that cannot be modelled with minimum free energy folding.We presented an algorithm pknotsRG is based on a simpler grammar model than the crossed interaction grammars [pknotsRE, as well as the communicating grammars underlying the recent approach by Cai [pknotsRG can serve as a template for a new class of programs we call thermodynamic matchers.Algorithm grammars  underlyih by Cai . It requh by Cai . Furtherthermodynamic matchers, which are RNA folding programs, based on the established MFE model, but specialized to the particular structural motif at hand. Such a matcher returns the optimal way to fold a sequence into the motif structure, together with the free energy of this folding. Comparing this energy to the MFE of an unrestricted folding can give us a hint with respect to the significance of such a match.Many functionally important RNAs like RNase P or group-I-introns have known structures that include pseudoknots. The search for such motifs using combinatorial matchers like RNAmotif  is hampepknotsRG was implemented using the method of algebraic dynamic programming (ADP) [Using the ideas presented so far, our folding algorithm can be implemented in any language suitable for dynamic programming, say FORTRAN or C. However, we are interested in a reusable implementation that can be integrated without change in specialized folding programs called thermodynamic matchers. Therefore ng (ADP) ,23.knt, skipleft, skipright) that build the trees as scoring functions. The grammar needs to be annotated with respect to tabulation and the application of the objective function (in our case, minimization).In ADP, the search space of a DP problem is defined on a declarative level, specified by clauses like the ones we have already seen above. Together they form a tree grammar, defining a tree language whose elements are all the candidates in the search space. In our case, the candidates are RNA structures represented as trees. The typical DP recurrences are implicit in this description. Scoring is achieved by interpreting the operators . A significant, but constant factor of speedup can be gained by explicitly formulating the recurrences and implementing them in a lower level language. Automating this process is part of our current work.We start from an ADP algorithm for folding RNA secondary structures (excluding pseudoknots) provided by Dirk Evers . We showO(n3) time and O(n2) space. A size constraint of 30 is used to bound loop length in internal loops. Closed substructures are defined such as to avoid lonely base pairs. While all this is easily expressed within the standard ADP framework, our new algorithm requires extensions which are now explained.The shown code abstracts from efficiency annotation and the treatment of dangling bases. The complete algorithm is found on the ADP WWW pages . It is bi, j) denotes input sequence positions inpi+1.. .inpj. [...] denotes lists, and <- denotes enumerating a list of alternative values.The implementation strictly follows the outline given in the methods section, except that a considerable amount of detail related to the energy model has to be taken care of. While ADP bans the use of subscripts, our canonization ideas require to explicitly manipulate subscripts. We show the concrete pseudoknot code, but explain only the essential points. A subscript pair (knot (i, j) = [pk energy a u b v a' w b' | k <-[i+2 .. j-1], l<-[k+1 .. j-2],k and l from the interval (i, j), and put together the results from a, u, b, v, a', w, b' under the scoring function pk. Each helix must have a minimum length of two bases. Due to stereochemical reasons one base in the front part and two bases in the back part are left explicitly unpaired; these bases should bridge the stacks. This consideration is taken over from pknotsRE. The next definitions implement canonization rules 1, 2 and 3. They determine the helix lengths, finally computed into the variables m and m'. If either of them is smaller than 2, a pseudoknot is not possible at this particular location.These line chooses cut shortens the helix b – b' as much as necessary in case of overlapping helices. The next lines define the pseudoknot components a through b', plus the local energy contribution. To avoid an extra factor of n in time complexity, the energies of maximal length helices are also precomputed in table stackenergy. If the helix b – b' must be chosen shorter than maximal to avoid overlap, a correction term has to be subtracted. This explains the negative term in the energy computation.The function front. For a full implementation of the algorithm see Left to be defined are the interior structures front, middle, and back. For reasons of space, we only show the definition of b-helix, and if the remaining region is not empty, an arbitrary list of substructures (comps) is recognized. idd, frd and pul are the corresponding functions from the energy model.This case takes care of a potentially dangling base from the v is smaller or equal to 1, the pseudoknot helices stack coaxially on each other and we further add the appropriate stacking energy. In [Overall, the energy of a pseudoknot consists of stabilizing and destabilizing terms. Where possible, we use the values from the current thermodynamic energy model . As stabergy. In  a pseudoergy. In . Finallyergy. In . Of courk, l inside (i, j), computes m and m' using the precomputed maximal helix information, and passes these boundaries to the pseudoknot compartments. Methodically, this is a use of inherited attributes with the underlying tree grammar, and appears to be a novel technique in dynamic programming, at least in its grammar oriented tradition [The first clause (knot) chooses radition ,28-30.pknotsRG can be judged from the sizes of the tree grammars required, which are summarized in Table The relative effort of implementing the three variants of pknotsRG-mfe, pknotsRG-enf, and pknotsRG-loc are available as executables and source code on the Bielefeld Bioinformatics Server [The three variants of the algorithm s Server .RG had the initial idea for the algorithm. JR developed and evaluated the software. All authors read and approved the final manuscript.Source code of pknotsRG-mfeClick here for file


Researchers from the Royal Netherlands Army are studying the potential of isolated lumbar extensor training in low back pain in their working population. Currently, a randomized controlled trial is carried out in five military health centers in The Netherlands and Germany, in which a 10-week program of not more than 2 training sessions (10–15 minutes) per week is studied in soldiers with nonspecific low back pain for more than 4 weeks. The purpose of the study is to investigate the efficacy of this 'minimal intervention program', compared to usual care. Moreover, attempts are made to identify subgroups of different responders to the intervention.Besides a baseline measurement, follow-up data are gathered at two short-term intervals (5 and 10 weeks after randomization) and two long-term intervals (6 months and one year after the end of the intervention), respectively. At every test moment, participants fill out a compound questionnaire on a stand-alone PC, and they undergo an isometric back strength measurement on a lower back machine.Primary outcome measures in this study are: self-assessed degree of complaints and degree of handicap in daily activities due to back pain. In addition, our secondary measurements focus on: fear of movement/(re-) injury, mental and social health perception, individual back extension strength, and satisfaction of the patient with the treatment perceived. Finally, we assess a number of potential prognostic factors: demographic and job characteristics, overall health, the degree of physical activity, and the attitudes and beliefs of the physiotherapist towards chronic low back pain.Although a substantial number of trials have been conducted that included lumbar extension training in low back pain patients, hardly any study has emphasized a minimal intervention approach comparable to ours. For reasons of time efficiency and patient preferences, this minimal sports medicine approach of low back pain management is interesting for the population under study, and possibly for comparable working populations with physical demanding job activities. For the last ten years, clinical researchers from the Royal Netherlands Army (RNLA) have studied the potential of physical training modalities in preventing and alleviating nonspecific low back pain (LBP) in their working population.Many military and civilian job functions in the RNLA involve heavy manual material handling and, therefore, spine-loading activities. In general, the incidence of back problems is higher in physically demanding tasks than in sedentary activities . ConcordCurrently, there is strong evidence that exercise therapy is more effective than usual care . ExercisThe specific character of military tasks nowadays, e.g. (preparation for) crisis management operations abroad, interferes with the schedules of rehabilitation programs for back-injured soldiers which, in general, take several days per week over a considerate number of consecutive weeks. Therefore, the search for effective and (time-) efficient exercise therapy protocols has led us to a specific form of lumbar extension training. Each training session consists of no more than 5 to 10 minutes training of the isolated lumbar extensors on a special training device.For a number of years, we have experience with high-intensive, isolated training of the lumbar extensors in military personnel with nonspecific LBP, by using a special training device. In this a sports medicine approach is followed, partly according to established exercise protocols ,4, in whIn individual cases, we observed satisfying to sometimes excellent results in terms of pain relief and functional restoration, when giving a training stimulus of no more than 5 to 10 minutes (1 to 2 training sessions) per week. These findings, however, need to be confirmed in a randomized controlled trial.Four main reasons led to the choice of doing research on the efficacy of this sports medicine approach for our working population with LBP.First, recent systematic reviews indicate that exercise therapy is a successful approach for the restoration of chronic and recurrent LBP, at least in the short term ,3. HowevMoreover, controversy remains regarding the impact of a training stimulus, in terms of intensity, duration and frequency, on the reduction of LBP. Different explanations for this lack of specificity are given in the literature, such as non-specific, more centrally induced training effects, e.g. a shift in pain perception , or largFrom this perspective, back strength and endurance training in CLBP patients with the use of training devices is an interesting concept for our military population. RNLA personnel are, from their very first initial military education, used to participate in physical exercise programs, including progressive resistance training on exercise machines. The RNLA is well equipped with an extensive line of modern fitness devices on all major military locations throughout the country, including state-of-the-art lower back machines. Moreover, protocolized treatment sessions with our training device take no longer than 5 to 10 minutes once or twice a week from both patient and provider, compared to (on an average) 30 minutes in regular treatment sessions. We expect this time efficiency to be highly appreciated by our personnel, who work in a typical military culture of "running into extremes": relatively quiet (maintenance) periods on the military base are interspersed with extremely busy periods shortly before and during out-of-area operations. For several target groups, longstanding and time-consuming rehabilitation programs are out of the question. For instance, recruits who drop out of their initial training because of (back) injuries, need to return as quick as possible to prevent a stagnation in their military career. For soldiers standing by for military operations, everything revolves around the mission when being commanded to be prepared within the next weeks.A second reason for our approach is that the majority of studies on LBP management consist of multimodal interventions, which include physical, behavioral, educational and/or ergonomic elements. To obtain a better view on the (relative) efficacy of either of these concepts, unimodal intervention programs like ours need to be evaluated ,8. BesidThird, the choice for a particular intervention approach depends in many cases on the stage and severity of the back problem, the extent to which psychosocial aspects are involved, and the needs and preferences of the patient. For instance, behavioral therapy is mainly focused on issues that are prevalent in chronic patients, such as low feelings of self-control or fear of movement/(re-)injury. Since the population of the present study, military employees of the RNLA, is a working population with mostly short-term, intermittent and moderately severe LBP, we chose to apply a more physical approach. As we have seen in our previous research (see the next paragraph), this links well with the health perceptions of our target population, in which perceived health problems were not severe and much more focused on physical than on mental aspects.Fourth, the efficacy of isolated extension training in chronic back patients has been studied by several other research groups as well . Althougst trial) or 8 weeks .Especially in recent years, we have scientifically studied the potential of our sports medicine approach. In two previous trials we compared the efficacy of a high-intensive, progressive resistance training program of the isolated lumbar extensors, with a low-intensive, non-progressive program of the same extend, in a group of workers with nonspecific LBP. Total intervention time of both 'minimal intervention programs' was limited to 14 sessions of 5 to 10 minutes, over a period of 12 weeks  directly after the 'minimal intervention' treatment, as well as a higher compliance to the treatment and a higher willingness to participate in physical exercise on the longer term. In the present multicenter study, we aim at identifying relevant subgroups of patients that show higher success rate due to this training approach.In a randomized, single-blinded multicenter trial, we evaluate the efficacy of progressive, isolated resistance training of the lumbar extensor muscles, compared to the usual care. The trial started in April 2002; data will be collected till the end of 2005. The source population (n = 23,000) consists of military employees of the RNLA. Our in- and exclusion criteria are listed in table Recruitment of participants takes place during regular office hours of the military general practitioner. A brief outline of the study design is presented in Figure Almost every military location in the Netherlands has a health center, in which general practitioners, dentists and physiotherapists give primary care to military personnel of the RNLA. For the present study we selected five military health centers on the basis of: (1) representing a major part of the total military population; (2) holding at least two full-time physiotherapists, and (3) the willingness to unconditionally participate in the project.The selected military health centers include the following locations:• Amersfoort, in the middle-east of the Netherlands: approximately 15% younger soldiers on stand-by for military operations abroad (18–25 years), 25% military instructors (35+ years), 10% staff personnel (40+ years), and 46% civilian workers from supporting units (40+ years);• Den Haag, in the west of the Netherlands: approximately 90% older staff personnel (40+ years) at office work;• Oirschot, in the south of the Netherlands: approximately 75% younger soldiers in their initial military education or on stand-by for military operations abroad (18–25 years), 25% military instructors and staff personnel (35+ years);• Schaarsbergen, in the south-east of the Netherlands: approximately 65% younger soldiers in their initial military education or on stand-by for military operations abroad (18–25 years), and 30% staff personnel (35+ years);• Seedorf, in the north-west of Germany (part of the 1 German/Netherlands Corps): approximately 65% younger soldiers on stand-by for military operations abroad (18–25 years), 20% staff personnel (35+ years), and 10% civilian workers from supporting units (40+ years).All general practitioners at each of the selected study centers identify potential subjects from among their clinic's patients, according to the aforementioned criteria. Each subject is submitted to regular history taking and physical examination by the physician; checklists with standard elements for LBP have been provided to all physicians. If eligible, patients are informed about the study. All relevant information from the intake is written down in a referral; a visit to one of the physiotherapists of the center is planned within the next days.At the first visit to the physiotherapist, patients receive further information about the trial. A pre-assessment of the isometric back strength is taken. A written explanation of one of the questionnaires, in which patients have to choose the three most disabled daily activities due to back pain, is given them to take home, which allows them to make a well-considered choice at the next visit. Written informed consent is obtained from all patients who are willing and eligible to participate. At the second visit, participants undergo a baseline measurement consisting of a compound questionnaire and an isometric back strength test. At the third visit, patients are randomized into either the back strengthening group or usual care group. Directly after randomization, the first treatment session starts. Patients are allowed to withdraw from the study at any time, although the importance of full compliance of every participant is emphasized.Randomization is done by means of a computer-generated table of random numbers per study center, using a block size of ten. Prestratification is applied for the duration of the back complaints, with a cut-off point of 12 weeks, suspecting duration of complaints to influence the individual response to the exercise program. One intervention group receives a back-strengthening program; the other group receives a usual care program.The randomization is concealed, which means that the treating physiotherapist obtains the allocated treatment by means of a computer software program, by entering the name and military registration number of the patient.The study protocol was approved by the Medical Ethics Committee of the Netherlands Central Military Hospital.Subjects allocated to the back-strengthening program (BS) undergo a 10-week, progressive resistance-training program of the isolated lumbar extensor muscle groups. The program includes 14 training sessions (2 days per week) and 3 isometric back strength tests (in week 1, 5, and 10). The initial training load is set at approximately 35% of the maximal isometric back extension strength of the participant, measured at baseline. The goal of every training session is to perform 15 to 20 repetitions (reps) on the lower back machine, equivalent to approximately 50% and 70% of the one-repetition maximum (1 RM) respectively. If the subject is able to perform a higher number of reps, 2 1/2 kg weight is added in the next training session. Vice versa, if the participant is unable to perform the minimal number of reps, the subsequent training load is lowered with 2 1/2 kg. This training protocol is partly based on existing protocols , and parTraining sessions are carried out on a Total Trunk Rehab . This lower back machine is equipped with a knee-lock system and a thigh-restraining belt to immobilize both hips and thighs, allowing the participant only to move the isolated lower back.All training sessions are conducted as much as possible by the same physiotherapist. The physiotherapist pays special attention to the execution of the training in terms of pace and movement. The flexion and extension of the lower back has to be executed in the full range of motion of the participant, and movements have to be slow and controlled . During this movement, emphasis is put on the hollowing and flattening of the lumbar lordosis. Every training session is preceded by a 5-minute all-body warming-up on an arm/leg ergometer . The weight load used and the number of reps completed during each training session are recorded.Subjects allocated to the usual care program (UC) receive regular physical therapy for their lower back for at most 10 weeks, or earlier when the patient indicates to be free of complaints. Based on the physiotherapist's judgment, this could include 'hands-on' treatment  and/or 'hands-off' treatment . In the RNLA, active therapy forms are favored. To increase the contrast between both intervention programs, physiotherapists are not allowed to use the lower back machine in their usual care. Patients are not allowed to undergo co-treatment beside the interventions programs during the treatment period, nor exercise on equipment that mimics the specific components of the lower back machine.Therapeutic activities in every therapy session as well as the number of sessions are written down on a form.In this study we have chosen primary outcome measures that are most relevant to the patient and clinician: self-assessed degree of complaints and degree of handicap in daily activities due to LBP. In addition, our secondary measurements focus on several other LBP-related areas, like kinesiophobia or mental health perception.We have included some potential prognostic factors into our measurements, i.e. characteristics of the patient that possibly influence the effect of the intervention: job characteristics, overall health, physical activity, patient satisfaction with the allocated treatment, and attitudes and beliefs of the physiotherapist. This trial is mainly focused on the efficacy of our minimal intervention strategy, and not on unraveling the physiological or psychological working mechanisms of isolated lumbar extension training on LBP. Nevertheless, by including potential prognostic variables, starting points for further research into the 'black box' of this type of intervention could be identified.The following demographic variables are registered during the intake: age, time since first episode of LBP, pain radiation, treatment history, and work absenteeism due to LBP in the last year.Moreover, the status of the patient before treatment, in terms of job aspects, overall health and the degree of physical activity, is assessed using a compound questionnaire. Several job characteristics, i.e. content, relation with superior and colleagues, conflicts, and physical aspects of the job, are measured using subscales of a validated Dutch version of the Job Content Questionnaire . PhysicaOverall health is assessed with one item from the MOS 36-item Short Form Health Survey : "What dThe degree of physical activity is measured with the Short Questionnaire to Assess Health Enhancing Physical Activity , a validTo assess the attitudes and beliefs of the participating physiotherapists about the relationship between low back pain and function before the start of the study, we use the Pain Attitudes and Beliefs Scale for Physiotherapists . These aGlobal perceived effect  is measuPatient-specific functional status  is measuLow-back specific functional status is measured by the validated Dutch version of the Roland Disability Questionnaire ,24, a wiFear of movement or re-injury is measured by the Tampa Scale for Kinesiophobia , a 17-itMental health is measured by the Dutch translation of the 12-item General Health Questionnaire ,30, asseSocial health is measured by a subscale of the Impact on Participation and Autonomy Questionnaire , focusinOverall work status is measured by a 4-level item .Individual back extension strength progression was evaluated using repeated isometric measurements on the lower back training and testing machine. A detailed description of these measurements can be find elsewhere .Patient satisfaction is measured at the end of the treatment program by two 3-level items and one 5-level item, in which the degree of satisfaction with the allocated treatment is assessed: (a) "Were you satisfied with the allocated treatment at the start of the program?" (b) "Has your opinion about the treatment changed during the program?" (c) "How satisfied are you now about the treatment that was given to you?"Beside a baseline measurement, follow-up data are gathered at two short-term intervals and two long-term intervals. Short-term follow-up measurements are at 5 and 10 weeks after randomization. Long-term follow-up measurements are at 6 months and one year after the end of the intervention, respectively. At every test moment, participants fill in a compound questionnaire on a stand-alone PC, and they undergo an isometric back strength measurement on the lower back machine. The content of the questionnaire varies per test moment: parts of the questionnaire referring to potential prognostic variables  are only displayed at baseline, and at 6 and 12 months of follow-up. Figure Double blinding or placebo control is virtually impossible in trials that involve treatment modalities like the ones used in this study, since both patient and provider are inevitably aware of the content of the treatment.Because our physiotherapists are aware of which treatment they provide, there is always the possibility that they may inadvertently convey different expectations to the patients in each treatment program. This could enhance non-specific effects in either of the two groups. Therefore, as mentioned, beliefs and attitude of the physiotherapists towards back treatment in general are evaluated at baseline, as well as at the end of the study period.Another limitation of trials comparing a relatively new treatment to the usual care is the risk of a potential nocebo effect; i.e. patients might feel disappointed after being allocated to the standard therapy. To minimize this effect, both programs are introduced to the patient as potentially equally effective treatments in restoring back function, with the relative efficacy of both programs as the main focus of the study. Moreover, at baseline patients are informed about the opportunity they have to continue with a treatment modality of their choice (e.g. our training device) after finishing the treatment period. Patient satisfaction with the allocated treatment is measured directly after the treatment period.Other efforts to achieve a certain level of blinding within patients, physiotherapists, and data researchers, are:• low back strength training and measurement are done as much as possible by two different physiotherapists;• an independent data manager collects data from all study locations, and recodes patients and locations to unique codes before handing the database to the researchers.We attempt to enroll 200 patients at the four military health centers, i.e. 100 patients per treatment group. According to power calculations (α = 0.05 and 1-β = 80%), this sample size is sufficient to detect a 20% difference in our primary outcome measure Global Perceived Effect, between the BS and UC program. In our beliefs, a 20% difference reflects a clinical relevant change in health status.Statistical analysis will be performed according to the intention-to-treat principle; i.e. patients will be analyzed in the treatment group to which they were randomly allocated. In addition, a per-protocol analysis will be performed, in which only patients with no major protocol deviations will be analyzed. Comparing these analyses with the results of the intention-to-treat analysis will indicate, to what extend protocol deviations and lack of compliance might have biased the results.In our analyses, we will compare the size of the effect, if any, of isolated back strengthening and of usual care for the low back on our primary and secondary outcome variables. Further analysis will determine whether several potential prognostic variables will influence the magnitude of the treatment responses, and if subsets of patients can be distinguished that can be indicated as good-/bad-responders to our specific back training. Demographic and clinical characteristics, as well as baseline outcome measures will be summarized by descriptive statistics. Longitudinal multilevel analyses will be used to examine differences in all continuous outcome measures at 5 and 10 weeks after randomization, and 6 and 12 months of follow-up.In order to obtain full commitment from the participating military health centers, as well as to make sure that every center uses the study protocols in the same way, we organize several feedback and feedforward sessions with all participating physiotherapists. In these sessions, we explain different aspects of the trial design, give instructions on the test and training protocols, and answer remaining questions.After these sessions, we install the required equipment (test/training machine, soft- and hardware) at the four locations. Each center has a practice period of about 8 weeks to become familiar with the instruments, logistics and protocols, by means of ad hoc training and test sessions with non-participating (regular) back patients. Our researchers join these sessions and, if necessary, give correcting feedback during and after a test or training.After this practice period, each location officially starts with the study. Once every two weeks, our researchers visit the centers to monitor progress in recruitment and data handling, and to observe the execution of test and training sessions.This article describes the rationale and design of a multicenter randomized controlled trial in which the efficacy of a specific type of lumbar extension training and usual care are compared in patients with nonspecific LBP longer than 4 weeks. A substantial number of trials have been conducted that included lumbar extension training in low back pain patients ,12,32,33Our population at risk can be seen as a selected population of mainly male employees, who work in a dynamic organization with a strong culture of physical fitness. We realize that this selection might limit the external validity of the outcomes of this study. However, results of the trial may be extrapolated to other working populations with a more-than-average degree of physical straining job activities, e.g. policemen and firemen on duty or construction workers.Besides, despite the "fit-and-healthy" image that soldiers have in our society, a large health survey among a cross-section of male military personnel of 30–40 years showed no favorable scores on several cardiovascular and fitness parameters in comparison to other populations of Dutch men ,35. In tMoreover, for reasons of homogeneity it might be even an advantage to only have a study population of working men with (probably) moderate low back trouble in this study.We hope with this trial to give greater insight to caregivers within and outside the RNLA on treatment options for workers in the sub-acute or chronic phase of their LBP. If, for instance, our minimal intervention approach is equally or more effective than the usual care, medical decision makers may consider implementing this treatment modality in the daily practice of the physiotherapist, by weighing the costs  and benefits (e.g. reduction of treatment time).RNLA = Royal Netherlands ArmyRCT = randomized controlled trialLBP = low back painBS = Back Strength interventionUC = Usual Care interventionrep = repetition on back extension machineThe authors declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.We are reporting here about new Sting Millennium Suite (SMS) version which is fully accessible , web based software for molecular structure and sequence/structure/function analysis. The new SMS client version is now operational also on Linux boxes and it works with non-public pdb formatted files (structures not deposited at the RCSB/PDB), eliminating earlier requirement for the registration if SMS components were to be used with user's local files. At the same time the new SMS offers some important additions and improvements such as link to ProTherm as well as significant re-engineering of SMS component ConSSeq. Also, we have added 3 new SMS mirror sites to existing network of global SMS servers: Argentina, Japan and Spain.Sting Millennium Suite) is web-based publicly available software developed to aid researches in their quest for translating information about the structures of macromolecules into knowledge. SMS allows to a user to interactively analyze molecular structures, cross-referencing visualized information with a correlated one, available across the internet. SMS is already used as a didactic tool by some universities. SMS analysis is now possible on Linux OS boxes and with no requirement for registration when using local files.SMS is already established software package and many key data base and software servers worldwide, do offer either a link to, or host the SMS. SMS ( A need to integrate, visualize and mine large amount of protein structure data is accelerating. In order to accommodate visualization of data originating from several sources and make analysis of protein structure and structural parameters easier, we developed Sting Millennium Suite (SMS). SMS is a web-based suite of programs and databases providing visualization and a complex analysis of molecular sequence and structure for the data deposited at the Protein Data Bank (PDB) .Using SMS it is possible to analyze: sequence to structure relationships, quality of the structure, nature and volume of atomic contacts of intra and inter chain type, relative conservation of amino acids at the specific sequence position based on multiple sequence alignment, indications of Folding Essential Residue (FER) based on relationship of the residue conservation to the intra-chain contacts, Cα – Cα and Cβ – Cβ distance geometry etc.. Specific emphasis in SMS is given to Interface Forming Residues (IFR) – amino acids that define interactive portion of the protein surfaces. SMS may simultaneously display and analyze previously superimposed structures.Parsing of data from relevant Data Bases ) to conveniently visualize large amount of physical-chemical, structural and biological information about the proteins with known structure. Variety of one-click-away renderings and color schemes helps to visualize bonding interactions and locations of residues of interest, as well as to localize patterns of evolution/conservation. The interactions which occur in the protein or between protein and its inhibitor/substrate, can be analyzed in great details with SMS.IFR Graphical Contacts). Both Graphical and IFR Contacts are fully integrated with SMS so that information about any particular amino acid is highlighted in simultaneous fashion across sequence, structure and contacts window.SMS offers to the user a graphical presentation of inter-atomic contacts established between amino acids in form of the fan. The base point of the fan is the selected amino acid. From the base point a user can detect number of colored lines connecting to other residues (presented by single letter code). Colors of the fan lines follow SMS code of contacts. A specific HTML table displays residue name and number, its pair in contact establishing, type of the contact, distance between contacting atoms and accessibility and entropy of two contacting residues. Such contacts are divided in number of classes: hydrogen bonds, hydrogen bonds with intermediary water molecules, hydrophobic contacts, aromatic ring stacking contacts, electrostatic (attractive and repulsive) contacts and finally disulphide bridges. A special table is built for those interactions across the interface  multiple alignment (relative sequence entropy) and histograms representing the atomic contacts , as well as IFR residue identification and IFR contacts. In addition, comparison of the Secondary Structure annotated by PDB, by DSSP [The  by DSSP  and by S by DSSP  is preseSTINGpaint it is possible to paint amino acids within multiple alignment of sequences according to two optional color schemes: STING's scheme and William Taylor [With m Taylor  color scConSSeq presents a sequence for a given PDB file and a consensus sequence (as found in the HSSP). A consensus sequence is obtained from the sequence alignment of the sequence-wise homologous proteins. Above those two sequences, ConSSeq shows a graphic bars colored by scale of colors according to the sequence conservation. The height of graphic bars is reflecting relative entropy. ConSSeq also offers information about residues present in other homologous sequences, with their respective frequency. For fast inspection of data, this program also generates a sequence logo. Complete interactivity with both sequence and chime-structure frame/window of the SMS is now operational, offering much better conditions for the thorough analysis of structure and sequence  interdependence.The Java Cα-Cα [Cβ-Cβ] Distance Plot is a diagram where the distances between the α [β] carbon of one residue and all α [β] carbon atoms of other residues, within a single chain of the PDB file, are represented by colored squares in a symmetrical plot.The All the above mentioned modules and some others available from SMS, can be accessed either from the STING Millennium's sequence window or entering through the independent entry web page. An extensive list of links is available to increment a volume of information on a protein under the study.In this new SMS release we introduced ProTherm  link, exThe Sting Millennium and some of the SMS components are now capable of importing local files in PDB format.SMS is organized in two logical layers: SMS server and SMS client. The server side is responsible for updating regularly all relevant public domain databases used by SMS. At the same time, SMS server is also responsible for calculation of a number of macromolecular properties for each PDB structure. The SMS client side provides to a user a friendly graphical interface and communicates to the SMS server, sending user's requests and receiving SMS responses.++ programming language, mostly for complex calculation of specific parameters.SMS interactive interface has been mostly implemented in the Java programming language, taking advantage of its object oriented design and graphical representation capabilities. Most important Java classes in SMS are dedicated to sequence and structure parameter presentation, depiction and interaction. Additional classes are used for efficient data handling utilities. As it is known, the object oriented software design is suitable especially because of its ease in code reusability and also because it provides interfaces for linking new software modules, resulting in systems easily expandable and built with extended capabilities. In addition, the Java programming language is very attractive to users for reasons of portability a key feature in today's versatile computing world. SMS also make extensive use of the CJava Plugin 1.3.1 and CHIME. Some restrictions apply, so a user is invited to consult details of SMS Requirements. Users can run the SMS program by selecting a previously deposited structure in the Protein Data Bank, or using local files with pdb format.SMS runs in the Netscape browser or in Microsoft Internet Explorer (for Microsoft Windows operating system) and in the Netscape/Mozila (for the Linux OS) and requires installation of SMS accepts the PDB format files from RCSB/PDB repository and also accepts local files of the same format, at the client end. A user is able to see structure of the local file in chime/SMS structure window as well as a sequence corresponding to this particular structure. The sequence itself is presented in the separate sequence window. Additionally, some other SMS components will work fine with user's local files: Graphical Contacts, IFR Graphical Contacts, SMS Ramachandran Plot, Scorpion, Formiga, Ca-Ca and Cb-CB contacts and Protein Dossier .Increase in availability of molecular structure data during the last decade, urged the development of computer applications for sequence/structure analysis and visualization. Consequently, numerous approaches have been made to the problem of sequence/structure visualization and analysis, resulting in diverse software packages: Protein Explorer, Cn3D, Swiss PdbViewer and ProCheck -14. EachSMS, as well as comparable software resources, come with intuitive user friendly GUIs, allowing for easy navigation through the vast amount of structural data.SMS main advantage is the clear presentation of sequence along with the structure in addition to number of visualizing tools for variety of structure describing parameters. In the input layer SMS uses data from public databases: PDB, HSSP, DSSP and SwissProt. Simultaneous display of computed features/parameters/descriptors along with available annotations from above Databanks provides a useful and reach environment, which may complement and in many cases substitute and exceed the already existing tools for sequence/structure/function analysis and visualization.Structure analysis is a difficult task due to the large amount of possible parameters/descriptors that can be calculated and associated with the sequence and corresponding structure. The way in which structure data and structure descriptors are stored and displayed, represents a major challenge when interactivity of a user with the data dispersed among many resources is addressed. Several structure viewers already exist, each one of them better suited to different needs and research interests. SMS offers an easy to use computer environment, designed to facilitate concomitant display of as many parameters as possible, coupled in a consistent fashion to each other. Experimental data and calculated information are all embodied in a clear display that offers instantly an intuitive aspect of a given structure and a large amount of biological information at hand. Inspection of the SMS displayed information can lead to valuable conclusions and cover a wide variety of biology issues concerning entire protein families.SMS has already been applied as a didactic tool for learning details about sequence/structure/function relationship in several universities. Future plans to extend the software platform include the ability to handle ever more descriptors/parameters of protein structure with the simultaneous display and analysis including data extracted from the statistical elaboration of common features among members of certain protein families/folds.In order to achieve such goal, we count with most generic yet very usable tools: Chime viewer and JAVA programming language. In addition, we count on growing interest of other research groups in participating in this project, contributing with their data and benefiting from the resulting unification of data format and data display. Issues such as the geometrical increase in the volume of the disk space and available CPU time for updating such a large data base should be taken into account.SMS is available free and can be accessed through the web. A user has to be careful with proper configuration of IT components  so that SMS can be used to its fullest potential. The detailed online manual/help/tutorial for viewing and analyzing displayed data is available and recommended for frequent consultation.Project Name: STING Millennium SuiteLab Home Page: Project Home Page: Operating System(s):Servers: Extensively tested on SGI IRIX 6.5, SUN Solaris7.0 and 8.0 and LINUX Red Hat 7.3, 8.0Clients: MS Windows XP, NT, 2000 with Netscape 7.0 and IE 6.0 SP1, platform with Java Runtime Environment (JRE) 1.3.1 installed and Linux Rad Hat with Mozila/Netscape 7.0 and CrossOver plugin.Chime 2.6 SP3/SP4 (depending on OS and browser used) plugin is essential for structure presentation.Programming Language: JAVA, C++, Fortran, JavaScriptOther requirements: Installation of JRE 1.3.1.License: Free for Academic use.SMS: Sting Millennium SuiteIFR: Interface Forming ResiduesFER: Folding Essential ResiduePDB: Protein Data BankRCSB: Research Collaboratory for Structural BioinformaticsGUI: Graphical User InterfaceRH created the Graphical User Interface and part of the data processing programming and general procedures for SMS mirror installation, RT worked on general GUI and statistics for SMS access, AJM worked on ConSSeq integration to SMS, JP worked on HTML design and SMS help pages, IO worked on SMS implementation on Linux OS, PK worked on general data interpretation and GUI suggestions, MY worked on mathematical algorithms for parameter calculation, AM carried out most of the data processing and programming, and GN coordinated the whole project, suggesting the general directions and innovating features of the application. All authors have read and accepted the final manuscript.


Lichen sclerosus is a chronic inflammatory disease with a predilection of the anogenital region. Because of the potential side effects of repeated local application of potent glucocorticosteroids, equally-effective, safer therapeutic options are required, especially in the treatment of children.We report on the efficacy of twice-daily application of pimecrolimus 1% cream in four prepubertal girls (range of age: 4 to 9 years) who suffered from anogenital lichen sclerosus. After three to four-month treatment, all patients had almost complete clinical remission including relief from itch, pain and inflammation. Only minor improvement was observed for the white sclerotic lesions. No significant side effects have been observed.Topical pimecrolimus appears to be an effective and safe treatment for children with anogenital lichen sclerosus. The clinical benefits observed in the four patient presented particularly include relief of pruritus, pain and inflammation. Vehicle-controlled studies on a larger number of patients are now warranted to substantiate our promising findings, and to investigate long-term efficacy and safety of topical pimecrolimus in anogenital lichen sclerosus. Lichen sclerosus (LS) is an inflammatory sclerotic skin disease of unknown origin. LS affects all age groups and occurs in about 15% in female children mostly involving the anogenital region ,3. The dWe describe four prepubertal girls between the age of 4 and 9 years who suffered from LS of the anogenital region Tab. . Because®, Novartis Pharma, Basel, Switzerland) was applied twice daily in a thin layer to the affected areas. Clinical examination and recording of patients symptoms was performed before, after six weeks, and after three and four months of therapy, respectively. On the six-week follow-up visit, substantial improvement of pruritus, dysuria, and painful defecation has been reported by the patients which had already occurred during the first weeks of treatment. Accordingly skin lesions had improved as well. The patients were then encouraged to continue the treatment twice daily for further six and eight weeks, respectively (Table Pimecrolimus 1% cream (ElidelPimecrolimus belongs to the ascomycin class of macrolactam immunosuppressives, acting by the inhibition of T-cell activation via the calcineurin pathway and inhibition of the release of numerous inflammatory cytokines, thereby preventing the cascade of immune and inflammatory signals. In contrast to GCS, there is no potential to induce skin atrophy . PimecroSuccessful treatment of genital LS with calcineurin inhibitors has been reported recently. Böhm et al.  used topOn the basis of our case observations, pimecrolimus ointment seems to be effective in LS. However, we cannot fully exclude that the benefit observed was due to emollient effect of the ointment. For example, it has recently been demonstrated in a vehicle-controlled study that the beneficial effect of testosterone ointment claimed in the past was probably due to the emollient effect only . NeverthIn conclusion, pimecrolimus 1% seems to be a safe and effective treatment modality in pre-pubertal children with anogenital LS. Our case observations provide further evidence for the beneficial effects of topical pimecrolimus on pruritus and inflammatory features. As the recurrence rate of active LS in prepuberty is relatively high and many of the patients have continuing symptoms after menarche as well, a long-term treatment regime not based on GCS is needed to avoid the well-known side effects linked to GCS. Vehicle-controlled studies on a larger number of patients are now warranted to substantiate our promising findings, and to investigate long-term efficacy and safety of topical pimecrolimus in anogenital LS.Lichen sclerosus: LS; glucocorticosteroids: GCSThe authors declare that they have no competing interests.SB conceived of this case study including its conduction and drafting of the manuscript. TG and AK participated in the literature search and critically revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: