Analysis of the gene expression program in yeast cells suggests that commitment to sporulation involves an active modulation of the gene expression program. Meiosis in budding yeast is coupled to the process of sporulation, where the four haploid nuclei are packaged into a gamete. This differentiation process is characterized by a point of transition, termed commitment, when it becomes independent of the environment. Not much is known about the mechanisms underlying commitment, but it is often assumed that positive feedback loops stabilize the underlying gene-expression cascade.We describe the gene-expression program of committed cells. Sporulating cells were transferred back to growth medium at different stages of the process, and their transcription response was characterized. Most sporulation-induced genes were immediately downregulated upon transfer, even in committed cells that continued to sporulate. Focusing on the metabolic-related transcription response, we observed that pre-committed cells, as well as mature spores, responded to the transfer to growth medium in essentially the same way that vegetative cells responded to glucose. In contrast, committed cells elicited a dramatically different response.Our results suggest that cells ensure commitment to sporulation not by stabilizing the process, but by modulating their gene-expression program in an active manner. This unique transcriptional program may optimize sporulation in an environment-specific manner. Meiosis is a specialized cell division by which haploid gametes are generated from diploid cells. The principal features of meiosis are common to all eukaryotic organisms and include a single round of DNA replication ('premeiotic' replication) followed by two consecutive nuclear divisions, meiosis I and meiosis II. In the first meiotic division homologous chromosomes segregate to opposite poles, whereas in the second division the two sister chromatids separate from each other. Meiosis is characterized by a high frequency of recombination events, occurring during a prolonged prophase that separates DNA replication from the first meiotic division. This genetic exchange between homologous chromosomes ensures that they segregate properly and that the offspring differ genetically from their parents and from each other.Saccharomyces cerevisiae, meiosis is coupled to the process of sporulation, in which the four haploid nuclei are packaged into spores  cells complete some meiotic processes, such as high-frequency recombination ,13, but RTG may occur only up to a certain stage in the sporulation process. Beyond this stage, cells will continue with sporulation events even if challenged with rich nutritional conditions. This stage of irreversibility was termed "commitment to meiosis" ; it occuSPO14 gene, which codes for phospholipase D and is required for late sporulation events, are able to return to growth even after completing meiosis I [The molecular mechanism underlying commitment is not understood. It was proposed that commitment to the meiotic process involves the separation of spindle pole bodies (SPBs) . Indeed,eiosis I . The rolTo better understand the process of commitment to meiosis, we examined the genome-wide transcription response triggered by the transfer of sporulating cells to rich growth medium at different stages of the process. At all stages, the transfer initiated large-scale changes in the gene-expression pattern. The majority of genes that were induced during sporulation were immediately repressed upon the transfer to growth medium, even in committed cells that continued to sporulate. At the same time, committed cells displayed a unique response to nutrients, which was dramatically different from that observed in all other cell types examined . This unique response consisted of metabolic-related genes, as well as genes involved in competing developmental processes such as pseudo-hyphal growth. Our findings suggest that commitment to meiosis is achieved not by stabilizing the transcription cascade, but rather by an active modulation of the gene-expression program, the detailed nature of which is only starting to be unraveled.To examine the transcription program associated with commitment to sporulation, we employed the RTG experimental paradigm ,18. The To define the time of commitment, we first followed meiotic landmarks events Figure . Previouet al. [We used DNA microarrays representing the full yeast genome to characterize the genome-wide expression profile at subsequent time points following the transfer of sporulating cells to rich growth medium . The number of repressed genes varied somewhat between early and late sporulating cells: around 1,200 genes were repressed over twofold in cells that were transferred early, whereas only around 480 genes were repressed when cells were transferred later in the sporulation process. Evidently, sporulating cells sense and respond to the growth medium at all stages of the sporulation process, even after they have become committed to its completion.IME1 . Several genes involved in spore-wall formation were also identified. Thus, many of the genes that were immediately repressed upon the addition of YPD have a well-established role in sporulation.Large-scale phenotypic studies have shown that only a portion of the genes that are induced during sporulation are also required for the process ,29. One IME2 and SMK1, were also repressed upon transfer to rich medium . In particular, NDT80, the master regulator of the mid-sporulation genes, was downregulated as well , and were also induced in YPD, at variable times that appeared to be linked to the progression of sporulation Figure . Second,DIT1 and SPS100). Surprisingly, however, most genes in this group are not in fact classified as sporulation genes, and do not have a recognized role in the process. Rather, most genes in this group are associated with the general environmental stress response (ESR), which is invoked in response to a variety of different environmental stresses [HSP12, GRE1, and SIP18), for the stress-induced transcription factor Xbp1, and for the Tor1 kinase. Accordingly, the promoter regions of most of those genes contained the stress-response element CCCCT. The induction of this gene group may reflect some stress signal associated with the progression of sporulation.About 60 genes display a 'process-linked' expression pattern . It is likely that repression of these genes assists in the inhibition of this alternative developmental route.The 'commitment-specific' gene group included around 65 genes that were induced by YPD specifically after commitment. In parallel, about 50 genes were repressed by YPD specifically after commitment , which are activated upon the addition of glucose ,39 Figu. In parts Figure . To gains Figure . Taken tDifferentiation processes proceed through a sequence of events involving coordinate modulations of morphology and gene expression. In many cases, once the process has passed a certain stage it becomes determined and loses its dependence on environmental signals. This transition is termed commitment. Mechanisms for achieving commitment can be classified into two broad classes. First, a passive mechanism might be imagined, whereby the process becomes effectively insulated from the external signals. Alternatively, commitment might require an active modulation of the signaling apparatus, which senses the external signal, but interprets it in a specific manner that optimizes the continuation of the process in changing conditions.Xenopus oocyte at least this positive feedback loop has been shown to be important for commitment [Differentiating cells could be insulated from the environment through the inhibition of essential sensory receptors, such as the receptors for glucose or nitrogen in the case of yeast sporulation. Effective insulation of the differentiation process could also be achieved through positive feedback loops that would render the process self-sustaining. Such positive feedbacks are ubiquitous in differentiation cascades. In the case of the mmitment . Also inOur gene-expression analysis ruled out the possibility that commitment to sporulation is achieved through a passive mechanism. Cells responded to nutrients at all stages of the sporulation process . In fact, within 5 minutes of the addition of rich medium, we observed a change in the expression pattern of over 1,000 genes (around 15% of the yeast genome). Importantly, this large-scale transcription response was dramatically different in committed versus non-committed cells, and was highly specific to the type of medium added. In particular, the response seen on addition of YPD (containing both glucose and nitrogen) was distinct from that observed on the addition of YPA  or on the addition of glucose alone. This indicates that the reduction in mRNA is a result of combinatorial regulation. The machinery of committed cells responds to the combination of glucose and nitrogen signals. This behavior suggests that active modulation of the response to external signals takes place. This modulation may play a central role in enabling the continuation of the sporulation process after the addition of nutrients.How do the cells achieve commitment to meiosis? The continuation of sporulation in rich media poses two complementary challenges. First, cells need to overcome mitogenic signals, which in vegetative cells facilitate the mitotic program. Second, cells need to ensure the continuation of the sporulation process itself. Our data suggest that these two aspects are goverened by complementary molecular mechanisms.BCY1, the gene coding for the negative regulatory unit of the PKAs. This inhibition is probably required in order to overcome mitogenic signals, to ensure the continuation of the meiotic program upon the addition of nutrients.The mitogenic effects of glucose are mediated to a large extent by the PKA pathway ,43. ThisThe addition of either YPD or glucose to committed cells failed to induce the typical set of glucose-responsive genes. In contrast, the addition of YPD  had a dramatic effect on the sporulation cascade itself. In fact, the vast majority of genes that are induced during sporulation in SPM were immediately repressed. This repression did not reflect the lack of functional requirement, as many of the repressed genes had a well defined role in the two meiotic divisions or in the formation of the spore wall. It may be that the proteins encoded by these genes are already synthesized at the time of commitment in sufficient amounts to complete the meiotic division. Alternatively, it is possible that the downregulation of at least some of the mid-sporulation genes is in fact required to prevent a shift back to the mitotic cell cycle upon the addition of nutrients, thus augmenting the cells' commitment. Further work is needed in order to better understand the nature of this downregulation and its potential contribution to the commitment process.SPO14 in both the formation of a spore membrane and the commitment to sporulation [Whereas the transcription program characterizing sporulation in sporulation medium (SPM) was practically eliminated in YPD, a group of around 60 late-sporulation genes was also induced in YPD, in a temporal manner that appears to be linked to the progression of the sporulation process. Surprisingly, the vast majority of these genes do not have a known role in sporulation. Rather, this group included a majority of stress-related genes, which are also induced in response to a variety of environmental stresses . The timrulation . This prIn conclusion, previous theoretical and experimental work has shown that bistability, generated through positive feedback loops, can render a process irreversible by effectively isolating it from external signals ,51-53. IpSPS4-3HA-NDT80 strain (GF18) was constructed by a one-step PCR-based replacement method [GAL1 promoter fragment in pFA6a-pGAL-3HA-KanMX6 with a 1 kb PCR fragment of the SPS4 promoter. Transformed haploids NKY1712 were mated with strain NE30. Integration to the correct site was verified by PCR. Diploids were selected on minimally supplemented yeast synthetic dextrose (SD) plates and were checked for sporulation efficiency on sporulation plates.All strains used for this study are of the SK1 genetic background and are listed in Additional data file 3. The t method  using th8 cells were frozen in liquid nitrogen. To monitor the meiotic divisions, 2 ml of cells were kept for staining with 4,6-diamidino-2-phenylindole (DAPI). To monitor recombination frequencies at the HIS4 locus, cells were plated on both YPD and -His plates, and colonies were counted after three days incubation at 30°C. All liquid cultures were grown at 30°C with constant shaking. Sporulation efficiency was determined by counting asci in the overnight SPM culture. For germination experiments, cells (DS1) were grown to saturation in YPD for 24 hours and plated on sporulation plates. Three-day-old asci were harvested and suspended in YPD at 30°C to initiate spore germination.The compositions of all media are given in Additional data file 3. Cells  were grown to saturation in YPD for 24 hours, diluted into YPA and grown overnight. The cells were then washed twice in sterile water and resuspended in SPM to initiate the sporulation process. At different stages of the sporulation process, cells were centrifuged and resuspended in rich medium. At each time point, 5 × 102HPO4, 2 mM KH2PO4), sonicated for 5 seconds and centrifuged. One microliter DAPI (at a concentration of 25 μg/ml) was added to the pellet and incubated in the dark for 10 minutes at room temperature. The number of nuclei per cell was counted using a fluorescence microscope. Around 300 cells were analyzed for each sample.Cells (2 ml) were centrifuged for 30 seconds, fixed in 0.25 mM Tris, 70% ethanol and kept at 4°C. Before staining, cells were washed in 1× PBS solution , RNase H Minus and 40 Units RNasin (Promega) was added to the RNA, and the reaction mixture was incubated for 2 hours at 45°C with 400 units of Superscript II . An additional 400 units of the enzyme was applied to the reaction mixture, which was incubated for another 2 hours. After incubation, RNA was hydrolyzed by adding 10 μl of 1 M NaOH and incubating at 65°C for 30 minutes. The reaction mixture was neutralized by titration with 1 M Tris-HCl pH 7.5, and DNA was precipitated with ethanol, resuspended in 10 μl 0.1 M sodium carbonate buffer pH 9.3. Cy3 and Cy5 (Amersham) were dissolved in 10 μl DMSO. Cy5 or Cy3 (1.25 μl) was added to the sample and incubated at 25°C for 1 hour. Labeled cDNA was purified by Strataprep PCR purification kit  according to the manufacturer's instructions. Dye incorporation was measured using a spectrophotometer. Reference RNA for all microarrays in this work was from YPA-grown cells, just before the transfer to SPM.Total RNA was extracted using the RNeasy Midi Kit  and reverse transcribed using M-MLV reverse transcriptase RNase H Minus . cDNA products were labeled with Cy3 and Cy5 by the indirect amino-allyl method  with minFor each hybridization, cDNA samples were labeled with Cy3 and Cy5 and combined with blockers: 5 μg herring sperm (Promega), 5 μg tRNA (Gibco) and 17.5 μg poly(A) (poly(A) oligonucleotides were synthesized at mixed lengths of 40, 50, and 60 adenine residues). The labeled cDNAs were concentrated to 40 μl using Microcon  and 40 μl of 2× hybridization solution  was added. Microarrays containing all yeast open reading frames (ORFs) were prehybridized by incubating at 42°C for 45 minutes in a solution containing 1% BSA, 25% formamide, 5× SSC and 0.1% SDS. The slides were washed in sterile water and dried by centrifugation . The labeled samples were boiled for 5 minutes, centrifuged for 1 minute, hybridized on the slide and placed in a hybridization chamber  for overnight incubation at 42°C. The slides were then washed for 5 minutes at 42°C with a solution containing 2× SSC and 0.1% SDS. An additional wash was performed at room temperature with a solution containing 0.1× SSC and 0.1% SDS, followed by three additional washes at room temperature in 0.1× SSC solution. For most experiments, we used arrays purchased from the University Health Network Microarray Center, Toronto, Canada, where PCR products are printed. For the YPA experiment (35 arrays) we used slides obtained from F. Holstege , where genes are represented by 70-base oligonucleotides . Control2 ratios, and normalized by subtracting the median. Values of replicate spots on the slides were averaged . The data discussed in this publication have been deposited in GEO [Images were obtained using ScanArray 4000 . Image analysis was performed using QuantArray version 3 software , with spots quantified using the adaptive method. Low-quality spots were discarded following detailed visual inspection. The data were then transformed into logd in GEO  and are d in GEO ,58  ,61. In eWe included in this analysis genes whose expression was altered by the addition of glucose to vegetative cells. To reduce noise, we filtered out genes whose expression was not altered by YPD in our experiment (transfer of sporulating cells to YPD). We ended with 936 genes . For the vegetative cell data, we calculated the average expression profile over the three given time points , and considered a given gene to be down- or upregulated if its average expression change exceeded twofold. For the sporulation data, we considered the YPD expression relative to that observed during sporulation, and calculated the average expression change observed at 20 and 40 minutes after the transfer. For spores that completed the sporulation process, we calculated the average expression response observed at 15 and 30 minutes following the addition of YPD. Correlations were calculated between these average profiles. The correlation matrix of the sporulation data was clustered    groups wIME2 and SMK1. Figure S3 shows a matrix of pairwise correlations that exemplifies the general metabolic response to glucose. Additional data file 2 ratios). Addtional file The following additional data files are included with this article: Additional data file A checklist containing minimum information about a microarray experiment. (MIAME)Click here for fileIME2 and SMK1. Figure S3 shows a matrix of pairwise correlations that exemplifies the general metabolic response to glucose.Figure S1 shows polarized expression during sporulation and RTG. Figure S2 shows the expression pattern of Click here for fileTable S1 includes a list of early sporulation genes. Table S2 includes a complete list of genes used in Figure Click here for file2 ratios)Normalized data of the present study (in logClick here for fileA matlab program that enables the expression data discussed in this article to be viewed. Also contains a help file: 'ViewModules help.pdf'Click here for file


Exhaled nitric oxide (NO) measurement has been shown to be a valuable tool in the management of patients with asthma. Up to now, most measurements have been done with stationary, chemiluminescence-based NO analysers, which are not suitable for the primary health care setting. A hand-held NO analyser which simplifies the measurement would be of value both in specialized and primary health care. In this study, the performance of a new electrochemical hand-held device for exhaled NO measurements (NIOX MINO) was compared with a standard stationary chemiluminescence unit (NIOX).A total of 71 subjects , both healthy controls and atopic patients with and without asthma were included. The mean of three approved exhalations (50 ml/s) in each device, and the first approved measurement in the hand-held device, were compared with regard to NO readings (Bland-Altman plots), measurement feasibility (success rate with 6 attempts) and repeatability (intrasubject SD).NO range of 8–147 parts per billion (ppb). When comparing the mean of three measurements (n = 61), the median of the intrasubject difference in exhaled NO for the two devices was -1.2 ppb; thus generally the hand-held device gave slightly higher readings. The Bland-Altman plot shows that the 95% limits of agreement were -9.8 and 8.0 ppb. The intrasubject median difference between the NIOX and the first approved measurement in the NIOX MINO was -2.0 ppb, and limits of agreement were -13.2 and 10.2 ppb. The median repeatability for NIOX and NIOX MINO were 1.1 and 1.2 ppb, respectively.Success rate was high (≥ 84%) in both devices for both adults and children. The subjects represented a FEThe hand-held device (NIOX MINO) and the stationary system (NIOX) are in clinically acceptable agreement both when the mean of three measurements and the first approved measurement (NIOX MINO) is used. The hand-held device shows good repeatability, and it can be used successfully on adults and most children. The new hand-held device will enable the introduction of exhaled NO measurements into the primary health care. Since the original reports of the presence of nitric oxide (NO) in exhaled breath of mammals including humans , and of Up to now, a single type of NO analyser has been used for nearly all measurement of exhaled NO: the chemiluminescence NO analyser . These iIn this study, the new hand-held device was compared to a chemiluminescence-based stationary device for exhaled NO measurements that has previously proven to provide repeatable results . Both thNO (fraction of expired NO) value of < 8 ppb were excluded (tested with the chemiluminescence-based instrument). At the time of the study, the detection limit for the hand-held device was considered to be 8 ppb. This has later been corrected by the manufacturer (see Table Subjects were consecutively recruited at two separate clinics (adult and pediatric) at Uppsala University Hospital. A total of 75 subjects were invited; 34 adults  and 41 children . The total age range was 6–60 years. Twentyone subjects were non-atopic healthy controls, 52 subjects were atopic and 39 subjects had a diagnosis of asthma. None of the subjects had used any of the NO instruments over the preceding 6 months and were thus considered unexperienced with NO measurements. Subjects with a FENO measurements in each of the two devices (NIOX® Nitric Oxide Monitoring System (NIOX) and NIOX MINO® Airway Inflammation Monitor (NIOX MINO); Aerocrine AB, Solna, Sweden). Oral pressure was measured and the subjects were instructed to keep the pressure between 12–18 cmH2O in both devices with the help of visual feedback (provided via a mirror in NIOX MINO). Exhalation flow rate was kept at 50 ± 5 ml/s with calibrated dynamic flow resistors in both devices. In the NIOX, the mean NO concentration over the last 3 s of exhalation is calculated and the NO plateau is evaluated by linear regression, whereas in the NIOX MINO, the last 3-s portion of exhaled air is led into the measurement chamber containing the sensor. Analysis takes 100 s before a result representing the NO concentration in this mixed gas portion is presented. Sampling technique in both devices complies with current international guidelines  when appropriate. For comparison between devices, intraclass correlation coefficients (ICCs) were calculated and presented as reliability coefficients, and Bland-Altman plots were constructed. Repeatability was calculated from intrasubject SD. Student's paired t-test was used to compare the mean number of attempts in the two devices. The success rate was calculated as the proportion of subjects succeeding in obtaining three valid FENO measurements < 8 ppb and were excluded from the study. They were all younger children (age ≤ 13 years). Of all subjects (n = 71) who made an attempt to use the NIOX or the NIOX MINO under guidance of clinical personnel, only a few failed to obtain three approved FENO measurements out of a maximum of 6 attempts , the mean number of attempts required to obtain three approved measurements was 3.8 ± 1.0 and 3.4 ± 0.8 for the NIOX and the NIOX MINO, respectively. The number of attempts was significantly lower for the NIOX MINO . The mean number of attempts required by successful patients to obtain one approved measurement in the home-use environment was 1.1 ± 0.3.Three adverse eventswere reported ; they were all considered mild and were deemed unlikely to be caused directly by the study devices.NO range of 8–147 ppb. The overall mean values for the NIOX and the NIOX MINO were 26.5 ± 24.2 ppb and 27.5 ± 23.2 ppb . The reliability coefficient was high (r = 0.97) when comparing the individual mean values in the two devices  ppb, again suggesting that FENO measurements with NIOX MINO were slightly higher than FENO measurements using NIOX. The 95% limits of agreement were -13.2 and 10.2 ppb.In addition, we find the same degree of agreement between the NIOX and the NIOX MINO when comparing the mean of three approved exhaled NO measurements in the NIOX and the first approved measurement in the NIOX MINO in the clinical setting Fig , 3B. Theth percentile for the distribution of the repeatability  in the NIOX was 3.3 ppb compared to 4.6 ppb in the NIOX MINO. The median repeatability for NIOX and NIOX MINO was 1.1  and 1.2  ppb, respectively. The real and estimated distribution of intrasubject SDs are shown in Fig Repeatability was similar in the NIOX and the NIOX MINO. The 95Exhaled NO has been studied extensively over the past decade and reports of the clinical utility of this method in the management of patients with asthma are now appearing in the literature ,9. HowevNO measurements using the established chemiluminescence-based NIOX and the NIOX MINO, which incorporates an electrochemical sensor, the results suggest clinically acceptable agreement between the two instruments. Measured FENO levels obtained using the NIOX MINO were on average slightly higher than those obtained with the NIOX, and there was a tendency that the intrasubject FENO difference increased with increasing FENO. We believe that the difference between the two instruments is acceptable, considering the different measurement technologies and calibration procedures used in the two devices, and the results are in conformity with the declared accuracy for both the NIOX and the NIOX MINO. From a clinical point of view, accuracy will be more important in a FENO range close to a cut-off between health and disease (20–35 ppb) than at higher FENO levels. The NIOX MINO showed good agreement (within 95% limits of agreement) with the NIOX up to approximately 60 ppb, which indicates that the new hand-held device will be able to give clinical guidance with similar accuracy as the conventional chemiluminescence-based unit.When we compare the mean of three valid FEIn general, the NIOX MINO and the NIOX had similar repeatability, except for one extreme observation with poor reproducibility in the NIOX MINO. However, this was seen in a subject with very high exhaled NO values (range 125–147 ppb in the NIOX MINO), and such variability at these high NO levels is of minor clinical importance. The repeatability agreed with the devices' technical specifications.Success rates in achieving the required number of acceptable measurements were at least 84% for both devices and for both subject groups. Since all subjects were considered unexperienced with NO measurements, this indicates that both measurement techniques are generally well accepted by the patients. However, younger children failed slightly more frequently than adults when attempting to use the NIOX MINO. Interestingly, the number of attempts needed for successful subjects to achieve three acceptable measurements was significantly lower in the NIOX MINO compared to the NIOX. This could at least partly be explained by the fact that some measurements in the NIOX may be discarded after a linear regression analysis of the NO plateau has been performed, even though the number of regression failures was not recorded in the present study. The linearized plateau must not deviate more than 10% from the horizontal axis according to current guidelines . In the Four subjects were excluded because of a low exhaled NO value (< 8 ppb). However, three of these subjects had a measurement above 5 ppb which is now the established lower detection limit of the NIOX MINO.During the simulated home use, subjects were given the opportunity to use the NIOX MINO unassisted by study staff (children were assisted by their parents as they likely would be at home). This was performed after the clinical session, which would imitate what would normally happen, namely that the patient would receive training in the clinic prior to taking home the device. All subjects that succeeded in the clinical setting also succeeded in the simulated home environment. Using the mean of three measurements was advised in earlier guidelines , but thiThe results show that there is clinically acceptable agreement between the stationary NIOX and the new hand-held NIOX MINO, when similar conditions were considered and examinations were made as consistently as possible. The repeatability of measurements done using the hand-held device was similar to the stationary device, and adults and most children were able to successfully use both instruments. In addition, subjects displayed ability to operate the new hand-held device in a simulated home-use environment. The new hand-held instrument will enable the introduction of exhaled NO measurements in the primary health care.KA is a co-founder and a minority shareholder of Aerocrine AB. LN is a minority shareholder of Aerocrine AB. CJ reports no conflict of interest.KA participated in the analysis of data and drafted the manuscript. CJ and LN participated in the design and coordination of the study. All authors read and approved the final manuscript.


It is suspected that additional folding information is present in protein coding nucleic acid sequences, but this is not represented by the known genetic code.All the information necessary for protein folding is supposed to be present in the amino acid sequence. It is still not possible to provide specific p < 0.0001, n = 81). This periodic FFE difference is not present in introns. It is therefore a specific physico-chemical characteristic of coding sequences and might contribute to unambiguous definition of codon boundaries during translation. The FFEs of the 1st and 3rd residues are additive, which suggests that these residues contain a significant number of complementary bases and that may contribute to selection for local RNA secondary structures in coding regions. This periodic, codon-related structure-formation of mRNAs indicates a connection between the structures of exons and the corresponding (translated) proteins. The folding energy dot plots of RNAs and the residue contact maps of the coded proteins are indeed similar. Residue contact statistics using 81 different protein structures confirmed that amino acids that are coded by partially reverse and complementary codons (Watson-Crick (WC) base pairs at the 1st and 3rd codon positions and translated in reverse orientation) are preferentially co-located in protein structures.Nucleic acid subsequences comprising the 1st and/or 3rd codon residues in mRNAs express significantly higher free folding energy (FFE) than the subsequence containing only the 2nd residues  interacting amino acids are coded by partially complementary codons, which strongly supports the connection between mRNA and the corresponding protein structures and indicates that there is protein folding information in nucleic acids that is not present in the genetic code. This might suggest an additional explanation of codon redundancy. The protein folding problem has been one of the grand challenges in computational molecular biology. The problem is to predict the native three-dimensional structure of a protein from its amino acid sequence. It is widely believed that the amino acid sequence contains all the information necessary to make up the correct three-dimensional structure, since protein folding is apparently thermodynamically determined; i.e., given a proper environment, a protein will fold up spontaneously. This is called Anfinsen's thermodynamic principle .The thermodynamic principle has been confirmed many times on many different kinds of proteins in vitro. Critics says that the in vivo chemical conditions are different from those in vitro, the correct folding is determined by interactions with other molecules  and protein folding is much more complex than re-naturation of denatured poly-amino acids. The fact that many naturally-occurring proteins fold reliably and quickly to their native state, despite the astronomical number of possible configurations, has come to be known as Levinthal's Paradox .Anfinsen's principle was formulated in the 1960s using purely chemical experiments and a lot of intuition. Today, many sequences and structures are available to establish a logical and understandable link between sequence, structure and function. But it is still not possible to predict the structure (or a range of possible structures) correctly from the sequence alone, ab initio and in silico .There are two potential, external sources of additional and specific protein folding information: (a) the chaperons The idea that the nucleotide sequence itself could modulate translation and hence affect co-translational folding and assembly of proteins has been investigated in a number of studies -7. StudiAnother observation concerning the code redundancy dilemma is that there is a widespread selection (preference) for local RNA secondary structure in protein coding regions . A givenG, of the RNA and the magnitude of change is proportional to the number of base pair formations. Therefore the free folding energy (FFE) is used to characterize the local complementarity of nucleic acids [Gshuffled - dGnative)/L × 100, where L is the length of the nucleic acid, i.e., free energy difference between native and shuffled (randomized) nucleic acids per 100 nucleotides. Higher positive values indicate stronger bias toward secondary structure in the native mRNA, and negative values indicate bias against secondary structure in the native mRNA.Single-stranded RNA molecules can form local secondary structures through the interactions of complementary segments. Watson-Crick (WC) base pair formation lowers the average free energy, dic acids . The fremfold [G values and the lowest dG was used to calculate the FFE. The mfold also provided the folding energy dot plots, which are very useful for visualizing the energetically most favored structures in a 2D matrix.We used a nucleic acid secondary structure predicting tool, the mfold  to obtaiA series of JAVA tools were used: SeqX to visualize the protein structures in 2D as amino acid residue contact maps ; SeqFormStructures were generally randomly selected regarding species and biological function (a few exceptions are mentioned in the Results). Care was taken to avoid very similar structures in the selections. A propensity for alpha helices was monitored during selection and structures with very high and very low alpha helix contents were also selected to ensure a wide range of structural representations.t-tests were used for statistical analysis of the results.Linear regression analyses and Student's st, 2nd and 3rd letters of the codons. Similar phase separation was made for intronic sequences immediately before and after the exon. There are, of course, no known codons in the intronic sequences, therefore we continued the same phase that we applied to the exon, assuming that this kind of selection is correct, and maintained the denotation of the phase even for non-coding regions. Subsequences corresponding to the 1st and 3rd codon letters in the coding regions had significantly higher FFEs than subsequences corresponding to the 2nd codon letters. No such difference was seen in non-coding regions  species and different  protein functions and therefore the results might be regarded as more generally valid. The propensity for different secondary structure elements was recorded (as annotated in different databases). The proportion of alpha helices ranged from 0 to 90% in the 81 proteins and showed a significant negative correlation to the proportion of beta sheets (not shown). The coding sequences were phase separated by SeqForm into three subsequences, each containing only the 1s Figure .st and 3rd codon residues than in the 2nd indicate the presence of a larger number of complementary bases at the right positions of these subsequences. However, this might be the case only because the first and last codon residues form simpler subsequences and contain longer repeats of the same nucleotide than the 2nd residues. This would not be surprising for the 3rd (wobble) base but would not be expected for the 1st residue, even though it is known that the central codon letters are the most important for distinguishing among amino acids . It is A Figure .There is a correlation between the protein structure and the FFEs associated with codon residues. This correlation is especially prominent when the FFE ratios are compared to the helix/sheet ratios Figure .The unique, codon-related FFE pattern and its correlation to alpha helix content suggested some similarity between protein structures and the possible structures of the coding sequences. This possibility was examined by visual comparison of 16 randomly selected protein residue contact maps and the energy dot plots of the corresponding RNAs. We could see similarities between the two different kinds of maps Figure . HoweverAnother similar, but still not quantitative, comparison of protein and coding structures was performed on four proteins that are known to have very similar 3D structures although their primary structures (sequences), and their mRNA sequences, are less than 30% similar. These four proteins exemplify the fact that the tertiary structures of proteins are much more conserved than the amino acid sequences. We asked whether this is also true for the RNA structures and sequences. We found that there are signs of conservation even in the RNA secondary structure (as indicated by the energy dot plots) and there are similarities between the protein and nucleic acid structures Figure . CompariMore direct statistical support might be obtained by analyzing and comparing residue co-locations in these structures. Assume that the structural unit of mRNA is a tri-nucleotide (codon) and the structural unit of the protein is the amino acid. The codon may form a secondary structure by interacting with other codons according to the WC base complementary rules, and contribute to the formation of a local double helix. The 5'-A1U2G3-3' sequence  forms a perfect double string with the 3'-U3A2C1-5' sequence . Suboptimal complexes are 5'-A1X2G3-3' partially complemented by 3'-U3X2C1-5' .I searched for some pattern in the codons of co-locating amino acids and analyzed the frequencies of the 8 possible patterns in the 64 nucleic acid triplets Figure . The codThese perfectly or partially complementary codon patterns, read in direct or opposite directions, defined 8 different ways in which amino acids can be paired on the basis of their codon complementarities: the 8 possible amino acid – amino acid (or protein-protein) interaction codes.st and 3rd codon residues are complementary but the 2nd is not. If this is the case, and there is a connection between nucleic acid and protein 3D structures, one might expect that the 4 amino acids coded by 5'-A1X2G3-3' codons will preferentially co-locate with 4 other amino acids coded by 3'-U3X2C1-5' codons. We have constructed 8 different complementary codon combinations and found that the codons of co-locating amino acids are often complementary at the 1st and 3rd positions and follow the D-1X3/RC-3X1 formula but not the 7 other formulae  are preferentially co-located in protein structures.Our experiments with FFE indicate that local nucleic acid structures are formed under this suboptimal condition, i.e., when the 1e Figure . This meIt is well known that coding and non-coding DNA sequences (exons/introns) are different and this difference is somehow related to the asymmetry of the codons, i.e. that the third codon letter (wobble) is less important in defining the meaning of the codon than the first and second letters. Many Markov models have been formulated to find this asymmetry and predict coding sequences (genes) de novo. These in silico methods work rather well but not perfectly and some scientists remain unconvinced that codon asymmetry explains the exon-intron differences satisfactorily.Another codon-related problem is that the well-known, non-overlapping, triplet codon translation process is extremely phase-dependent and there is theoretically no tolerance for any phase shift. There are famous examples of single nucleotide deletions that destroy the meaningful translation of a sequence and are incompatible with life. However, considering the magnitude and complexity of the eukaryotic proteome, the precision of translation is astonishingly good. Such physical precision is not possible without a massive and consistent physico-chemical underpinning. Therefore, discovery of the existence of secondary structure bias (folding energy differences) in coding regions of many organisms  was veryst or only the 3rd codon letters showed significantly higher FFE than those consisting only of the 2nd letters. This FFE difference was not present in the intronic sequences preceding and following the exons, but it was present in exons from different species. This is an interesting observation because these phenomena might not only distinguish between exons and introns on a physico-chemical basis, but might also clearly define the tri-nucleotide codons and thus the phase of the translation. This codon-related phase-specific variation in FFE may explain why mRNAs have greater negative free folding energies than shuffled or codon choice randomized sequences [Our experiments with free folding energy (FFE) confirmed that this bias exists. In addition, there is a very consistent and very significant pattern of FFE distribution along the nucleotide sequence. Comparing the FFEs of phase-selected subsequences, those subsequences comprising only the 1equences .st and 3rd codon positions were additive, while the 2nd letter did not contribute to the total FFE; the total FFE of the entire (intact) nucleic acid was the same as that of subsequences containing only the 1st and 3rd codon letters (2nd deleted). This indicates that local RNA secondary structure bias is caused by complementarity of the 1st and 3rd codon residues in local sequences. This partial, local complementarity is more optimal in reverse orientation of the local sequences, as expected with loop formations.Free folding energy in nucleic acids is always associated with WC base pair formation. A higher FFE indicates more WC pairs (presence of complementarity) and a lower FFE indicates fewer WC pairs (less complementarity). The FFEs in the 1st, 2nd and 3rd codon bases separately are useful for showing that the 2nd codon position does not have the same significance in the codons as the other two positions [FFEs are obtained by considering free folding energies of substrings that do not represent the real molecule: forcing nucleotides to be consecutive is an extreme methodological approach to measuring the structure features of coding sequences. However, this bioinformatical method has been successfully used by others ,21. In aositions . Intronist and 3rd codon residues. This connection between the RNA secondary structure and codons immediately directs attention toward the question of protein folding and its long-suspected connection to RNA folding [It is known that single-stranded RNA molecules can form local secondary structures through the interactions of complementary segments. The novel observation here is that these interactions preferentially involve the 1 folding ,23.Only about one-third (20/64) of the genetic code is used for protein coding, i.e., there is a great excess of information in the mRNA. At the same time, the information carried by amino acids seems to be insufficient (as stated by some scientists) to complete unambiguous protein folding. Therefore, it is believed that the third codon residue (wobble base) contains information additional to that already present in the genetic code. A specialized database, the ISSD , was estWe found a significant correlation between FFE ratios and the helix/sheet contents of protein structures. It was possible to make direct visual comparison of mRNA structures  and protein structures (as 2D residue contact maps). This method suggests similarity between nucleic acid and protein structures.It is known that some complex protein structures are very similar even if there is less than 30% sequence similarity. It was interesting to see whether the same principle might apply to nucleic acids, and structural similarity might exist even when the sequence similarity is low. Furthermore, significant similarity between nucleic acid and protein structures might exist even without translational connection. Structure seems to be more preserved, even in nucleic acids, than sequence. However, although the matrix comparisons are suggestive, they remain semi-quantitative. Better support was necessary.st and 3rd positions and are translated in reverse orientation result in preferentially co-located (interacting) amino acids in the 3D protein structure. Other complementary residue combinations or translation in the same (not reverse) direction  did not result in any preferentially co-locating subset of amino acid pairs.A working hypotheses grew out of these observations, namely that (a) partial, local reverse-complementarity exists in nucleic acids that form the nucleic acid structure; (b) there is some degree of similarity between the folding of nucleic acids and proteins; (c) protein structure determines the amino acid co-locations; (4) in consequence, amino acids coded by interacting  codons might show preferential co-locations in the protein structures. And it seems to be the case: codons that contain complementary bases at the 1st and 3rd (but not the 2nd) codon positions.Construction of residue contact maps for protein structures and statistical evaluation of residue co-locations is a frequently used method for visualization and analysis of spatial connections among amino acids -27. The nd codon residue. Experimental in vitro confirmation is required to validate this recent theoretical and in silico prediction.The idea that sequence complementarity might explain the nature of specific protein-protein interactions is not new and was suggested as long ago as 1981 ,35,36. IAvailability: : SeqX, SeqForm.


Tobacco use, particularly smoking, is the most preventable cause of death in the United States. More than 400,000 premature deaths are associated with its use and the health care costs are in the billions. All health care provider groups should be concerned with patients who continue to smoke and use tobacco. The US Preventive Services Taskforce and Health People 2010 guidelines encourage providers to counsel smokers on cessation. Current studies, though limited regarding chiropractic advising practices indicate a low engagement rate when it comes to providing cessation information.To test a campaign regarding initial impact aimed at increasing chiropractic interns advising on cessation and delivery of information to smokers on cessation.Chiropractic interns do engage patients on smoking status and can be encouraged to provide more cessation messages and information to patients. The initial impact assessment of this campaign increased the provision of information to patients by about 25%. The prevalence of smoking among chiropractic patients, particularly at teaching clinics may be lower than the national averages.Chiropractic interns can and should be encouraged to advise smokers about cessation. A systematic method of intake information on smoking status is needed and a standardized education protocol for chiropractic colleges is needed. Chiropractic colleges should assess the adequacy of their advising roles and implement changes to increase cessation messages to their patients as soon as possible. Trichotimized variables were collapsed into 2 categories.There were a total of 538 usable surveys completed by patients in the Dallas clinic. A majority of patients completed a survey as all patients meeting the inclusion criteria were offered a survey and only 5 patients who met the criteria refused to complete one. Reasons for refusal included being in too much pain or aversion to surveys. The pre-intervention survey was completed by 342 individuals and 212 completed the post-intervention survey. The pre-intervention group 6 surveys were unusable either due to patient age or because a non-smoker completed the additional smoker-oriented questions on the survey by mistake. In the post-intervention group 6 surveys were considered unusable for the same reasons and an additional 4 were excluded from the study analysis because respondents identified themselves as past-smokers; however, they also completed the survey section designated for current smokers. Of the usable surveys, 475 (88.3%) patients did not smoke. The mean age of patients in the sample was 44.4 years with a rage of 18 to 84 years. Males, (n = 283) had a mean age of 45 (SD = 13.82) and females, (n = 255) had a mean age of 43.8 (SD = 15.19). Data were considered fairly normal in distribution but had bimodal peaks noted at the 25–30 age range and 50–55. There was no statistical difference in the mean age of smokers (43 years) and non-smokers (45 years). There were a total of 63 smokers (11.7%) with 41 being male (65% of smokers) and 22 females (35%). Of the 41 male smokers, 24 were in the pre-intervention group and 17 were in the post group. While females made up only 35% of smokers in the study population, there were only 13 in the pre-intervention group (21% of smokers in the study) and 9 in the post-intervention group (14.3% of smokers in the study). Additional demographic characteristics on patients in the survey are included in Table There were 179 interns and 68 total surveys returned. One survey was unusable as the intern was not seeing patients that semester, for a total of 67 usable surveys (38% participation rate). The mean age of students in the intern survey was 29.3 years (range 22–48 years). Forty (60%) of the interns were males and 27 (40%) were females. Of the interns in the study, only 4 (6%) were current smokers and 17 (25%) were former smokers, 46 (69%) having never smoked. The current smokers (n = 4) were all males.2 testing; there was a significant difference in the number of male versus female smokers in the study in aggregate. There were 242 males who did not smoke (85.5%) and 233 females (91.4%) Forty-one males reported smoking (14.5%) and 22 females (8.6%). This difference was significant at p < .035 indicating almost twice as many males smoking as females. Gender breakdown in pre versus post grouping was not significantly different. There were 173 males in the pre group and 163 females and there were 110 males in the post-group with 92 females (p = .504).Regarding patient surveys and whether patients would report differences in smoking status and subsequent intern engagement based on demographics, the following results were found after analysis on χ2 statistics for these analyses and includes the χ2 for smoking and gender as well.Regarding patients' reports of being asked about smoking status, receiving advice to quit if they smoked, and being asked if they wanted to try to quit, no significant difference in rates were noted among those in the pre-intervention groups when compared to post-intervention participants based on patient demographics. Table 2 testing. Among pre-intervention smokers (n = 37) only 2 (5.4%) said they had been given specific information on cessation. Among those smoking patients in the post-intervention group (n = 26), 8 (30.7%) said they were given information on cessation. This was significant at the .007 level with an odds-ratio of 7.8. Although numbers are small, this represents an increase in over 25% within one month and those in the post-intervention group were about 8 times more likely to have been given information on cessation.Regarding smokers who reported being given information on cessation there was a significant difference in rates among smokers in the pre-intervention and post-intervention groups on χWhen smokers were asked if they had been advised to quit on more than one occasion, about 50% in both pre and post-intervention groups answered in the affirmative.p = .014, OR = 8.196, 95% CI 1.54, 43.5).In logistic regression modeling only the pre/post grouping was significant when age, gender and pre/post group tested. Patients in the post-intervention group were about 8 times more likely to report being given information than the pre-intervention patient group , 9 said they didn't have time, 4 said it was not their place to advise smoking patients and only 3 said they didn't feel they had the skills.The purpose of this study was to determine if an education campaign, delivered to chiropractic interns, could have an initial impact on intern's advising roles with patients regarding assessment of smoking status and smoking cessation information. We believe the results generally support the idea that interns can be trained to assess smoking behaviors and deliver information to those patients on the importance of cessation. Intern demographics may, or may not, affect the rates of the advising roles. There are certain limitations of this study. First, it is an impact study to measure short-term changes in intern behavior, not an outcome study. Another limitation was that both patients and interns were essentially a convenience sample. Although patients were technically a consecutive sample, neither sample may be representative of the populations they are suggested to represent.Among patient participants all were patients at an outpatient teaching clinic at Parker College of Chiropractic in Dallas, Texas. Even though they ranged in age from 18 to 84 (mean = 44) they differed in smoking status from current US prevalence statistics. According to the CDC, as of 2004, 20.9% of the US citizens were current smokers.  In our sInterns in the sample (n = 67) seem typical regarding age and gender categories but when it comes to smoking prevalence, they do not smoke at a rate near the current US prevalence. Among all interns completing the survey, only 4 (6%) were current smokers and there were no current female smokers in the sample. The number of eligible interns who declined participation may have influenced this prevalence rate and there could have been reporting bias reflected in the sample as well. A majority of interns did not complete a survey.Regarding patient responses on whether they were asked about smoking status on their last visit, many wrote on their survey that they had never smoked and although their intern did not ask on the last visit, they did ask about smoking status on the patient's first visit and therefore, the patient felt there was no reason that the intern would have asked this again. Interns, by the same token, often wrote on their survey that they would have participated in the campaign but had no smoking patients in their patient-base. With only 11.7% of patients in the sample reporting smoking, this seems reasonable and therefore, rates of participation were likely affected by this fact.Regarding patients who reported being advised to quit on more than one occasion, about 50% said they had been told to in both the pre and post-intervention groups. This is slightly better than what has been reported on engagement by primary care physicians ,7. HowevGenerally, numbers were small in this sample when it came to those who smoked – both interns and patients. A larger sample of patients and interns, perhaps across the country and repeating this campaign at several campuses could yield more pertinent information on this topic. We suggest this be considered.The analysis of this data indicates that in a short period of time some impact can be made on intern behaviors regarding cuing patients to stop smoking. If cessation information is made easily available to interns, we believe they will provide it to their patients. Although not all results were encouraging and the number of smokers in the study population was small, information was given to smoking patients at a higher rate after the education intervention. This is the area where the ball is typically dropped. Most smoking patients have not been given specific information on cessation by their doctor ,6,7,18. This campaign was inexpensive and was well-received at Parker College of Chiropractic. It has now been integrated into the curriculum as part of the wellness class required in trimester 5 prior to interns seeing patients in student or outpatient clinics. It has been popular and generated a request for a smoke-free campus which will take effect in January 2007 with full faculty and board support.During the month-long campaign, 4 patients and 1 intern self-reported quit attempts to the principal investigator. If every chiropractic college and teaching clinic would integrate smoking cessation advising into the curriculum and clinic competencies of their colleges, we have no doubt that a significant effect would be seen in the amount of information provided to patients.Chiropractic has been long seen as a holistic profession that says it emphasizes wellness and better health for patients. We see no reason advising smoking patients on cessation should not be a part of routine clinical practice of chiropractic and feel it should be made a requirement in all chiropractic colleges.CAM-complementary and alternative providerCDC-United States Centers for Disease Control and Prevention5-A's-United States Surgeon General's 5-A's for advising smokers of cessationn-numberp-p-valueSD-standard deviationUS-United States2-chi squareχThe author(s) declare that they have no competing interests.ME developed the general study design, collected, manipulated and analyzed the data, and prepared the manuscript. CH assisted in the study design, developed the patient survey and assisted in development and revision of the manuscript. SS assisted in the study design and in the development and revision of the manuscript. All authors read and approved the final manuscript.


Data suggest that antibody responses to malaria parasites merozoite antigens are generally short-lived and this has implications for serological studies and malaria vaccine designs. However, precise data on the kinetics of these responses is lacking.Plasmodium falciparum merozoite antigens  among Kenyan children were monitored using ELISA for 12 weeks after an acute episode of malaria and their half-lives estimated using an exponential decay model.IgG1 and IgG3 responses to five recombinant The responses peaked mainly at week 1 and then decayed rapidly to very low levels within 6 weeks. Estimation of the half-lives of 40 IgG1 responses yielded a mean half-life of 9.8 days (95% CI: 7.6 – 12.0) while for 16 IgG3 responses it was 6.1 days (95% CI: 3.7 – 8.4), periods that are shorter than those normally described for the catabolic half-life of these antibody subclasses.This study indicates antibodies against merozoite antigens have very short half-lives and this has to be taken into account when designing serological studies and vaccines based on the antigens. An effective malaria vaccine is urgently needed, but to date it remains elusive. A common way of trying to establish if a given malaria parasite antigen is a good candidate for a malaria vaccine is by determining if an with protection against subsequent infections of malaria. However, a number of studies suggest that naturally acquired responses to malaria merozoite antigens are short-lived. Among the majority of people living in endemic areas, levels of antibodies to merozoite antigens appear to vary with the levels of malaria transmission i.e. they are highest during periods of intense transmission and lowest or undetectable at the end of periods of low transmission -3. FurthPlasmodium falciparum merozoite antigens among Kenyan children recovering from a clinical infection of malaria and the data used to estimate the half-life of the responses. The results of the study indicated that both IgG1 and IgG3 antibodies to merozoite antigens have very short half-lives.The implication of these observations is important as they suggests that during serological surveys, individuals who can nonetheless mount a rapid secondary antibody response to malaria antigens upon re-infection are likely to be classified as antibody negative depending on how recent their last malaria infection was. Conversely, individuals who are positive at the survey may be negative by the time they encounter the next infection. If indeed the antibodies responses are very brief, then data from longitudinal studies with long intervals between sampling days will not reflect well the dynamics of the responses. Unfortunately, estimates of the half-lives of antibody responses to malaria that can help guide the design of such studies are lacking. In this study, a closely spaced sampling schedule was used to monitor the kinetics of antibody responses to five recombinant g for 5 min to obtain plasma, which was stored at 20°C. The children were examined by a clinician and a thick malaria film prepared during the follow-up visits or any other time during the study when they were unwell. Malaria treatment (SP) was given for parasitaemia in the presence of fever (axillary temperature ≥ 37.5°C). Seven children from whom weeks 1 and 2 samples could not be obtained were considered lost to follow up, so the cohort for analysis comprised 41 children.This study was carried out at Kilifi District Hospital (KDH) on the Kenyan coast. Ethical clearance for the study was given by the Kenya Medical Research Institute ethics review board. Forty eight children admitted to the pediatric ward of KDH with a primary diagnosis of malaria, but who did not fulfill the World Health Organization criteria for severe malaria , were reP. falciparum apical merozoite antigen 1(AMA-1), the 11 kDa carboxyl portion of merozoite surface antigen 1 (MSP-119), region II of the 175 kDa erythrocytes binding antigen (EBA-175 RII), and two recombinant proteins representing the two major allelic types of MSP-2 was assessed in plasma samples from 41 children . Levels of IgM reactivity against the two allelic types of MSP-2 were also assessed. The AMA-1 antigen and EBA-175 region II were kind gifts from Sheetij Dutta and Arnoldo Barbosa  and have been previously described t are concentrations at time 0 and t respectively, k is a rate constant and plateau is the concentration at the end of the decay and half-life of the decay (t1/2) is calculated as follows:Where [C]Association between kinetics half-life of the responses and age of responder or peak level of antibodies at the beginning of the decay was assessed by linear regression using Stata ver. 8 , while differences between half-lives of response to the various test antigens was assessed using one-way ANOVA. In addition, ANOVA was used to test if the mean level of responses to the test antigens differed between individuals. The profiles were then grouped together depending on whether or not they had peaked before the child presented to hospital, and the mean half-lives of the groups compared using a student t-test.19 in subjects 23, 36, 38, 40, 41,47, 48 and 56, Figure There was considerable variation in the children's overall pattern of responses to the five test antigens but for each antigen, the children generally responded in one of three ways. Either, (1) they already had moderate to high levels of antibodies to the antigen by the time they presented to hospital , but not always . Only in 10 of the 43 individual antigen-specific boosting instances were IgG1 responses boosted to similar or higher levels than the initial peak . There was no association between half-life and age , peak level of antibody , or antigen specificity . The half-lives of the responses did not differ significantly between different individuals . The mean half-life of the IgG3 responses analysed was lower  than that of IgG1 but not significantly . As with IgG1, there was no association between half-life and age , peak levels of antibody , or antigen specificities   (8.9 days [95% CI: 5.5–10.8]) but the difference was not significant (P = 0.17). The mean half-life of IgG3 responses that peaked before presentation to hospital (n = 5) was 7.3 days (95% CI: 5.0 9.5), while that of the responses that peak post presentation (n = 10) was 8.1 days (95% CI: 4.8 – 11.5), a small difference that was not significant (P = 0.685)The profiles were grouped depending on whether or not they had peaked by the time the child was presented to hospital and compared their mean half-lives. IgG1 profiles which peaked before presentation to hospital (n = 8) had a longer mean half-life (13.1 days [95% CI: 8.2 – 21.4]) than those that peaked after presentation .Data from 11 children where responses to two or more antigens were analysed for half-life was used to determine the correlation between the half-life of responses to different antigens within the same child. A high level of correlation between responses to different antigens by the same child was observed  was similar to that of antibodies injected into healthy adults (8 days) [Estimating the half-life of antibody responses to malaria antigens among residents of malaria endemic areas is difficult as the kinetics of the antibodies are continually altered by reinfection. In this study, eventual boosting of the initial responses was evident in many of the children even in the absence of patent re-parasitization. As such, half-lives could only be estimated for those responses that showed no obvious boosting over at least four time points. However, it is still possible that the half-life estimates obtained may still have been subtly affected by sub-patent of re-parasitization. In addition the half-life of the some of the profiles that were not analysed because they did not meet the inclusion criteria may have been different from those estimates obtained but it was not possible to clarify this. As such, a future study is being planned that will include a group of children who are kept parasite-free over study period by chemoprophylaxis. Nevertheless, this study shows that among Kenyan children recovering from clinical malaria, antibody responses to (8 days) , that ofP. vivax merozoite surface protein 1 (MSP-119) during a clinical episode of malaria became sero-negative within two months of treatment [P. falciparum MSP1 and rhoptry associated protein 1(RAP-1) in Sudanese individuals often last for less than three months after treatment for clinical malaria [Such short half-lives could explain observations from previous longitudinal studies that reported a rapid decline of antibodies to malaria antigens within a few months of malaria treatment. For instance, 44% of Brazilian individuals who made antibodies to the C-terminal fragment of reatment . Similar malaria ,14.Data on the half-lives of natural responses to other infectious agents such as bacteria are generally lacking and it is not possible to say if the short-lives observed here are unique to malaria or common to acute infection by different pathogens. Future studies will have to incorporate patients with other conditions for comparison. On the other hand, antibodies responses to viral infections and vaccines such as measles, appear to last for many years ,16 althoAlthough the half-life of IgG1 is usually cited as 21 days , early sSince in natural responses, the kinetics of antibodies is a balance between production and decay the profiles observed in this study suggest that production of antibody against merozoite antigens is not sustained following an acute episode of malaria. Antibody production can be sustained through re-stimulation of memory B cells by persistent antigens ,26 or byInterestingly, in many instances, re-parasitization after the episode did not boost the initial responses or only did so mildly. While polymorphism in the new infection might explain some of the failure, the fact that boosting occurred in many instances but only to a lower level than the initial responses suggests that there may be a period of suppression of antibody responses following a clinical episode of malaria. Reduced reactivity to viral vaccines during malaria episodes and recovery of reactivity following malaria treatment has been reported among Nigerian children .This study shows that following an acute episode of malaria in children living in an endemic area, antibody responses to malaria merozoite often have very short half-lives; less than those reported for normal antibody clearance. This has implication for studies monitoring responses to these antigens and indicates the need for closely spaced sampling within the first few weeks of an episode. Further studies on the fate of antibody producing cells following a malaria episode are required in order to elucidate the mechanism underlying this brevity. In addition, it is important to establish whether the brevity of the responses is due to malaria disease processes or whether it is inherent to the antigens. Would vaccines based on the antigens also induce short-lived responses in healthy children, and would this compromise their use, or would memory B cell populations be adequate for a sufficient antibody response upon infection?The author(s) declare that they have no competing interests.SMK carried out longitudinal sampling, the ELISA and prepared the manuscript DJC developed the ELISA and helped in the manuscript preparation DEL participated in the preparation of the antigens used in the study KM helped in designing the study and setting up of the longitudinal framework


Positive end-expiratory pressure (PEEP) is used in mechanically ventilated patients to increase pulmonary volume and improve gas exchange. However, in clinical practice and with respect to adult, ventilator-dependent patients, little is known about the short-term effects of PEEP on breathing patterns.2O, and then again at 5 cmH2O for 30 s each, using the automatic tube compensation mode.In 30 tracheally intubated, spontaneously breathing patients, we sequentially applied PEEP to the trachea at 0, 5 and 10 cmHP < 0.0001). For respiratory rate, a 1 cmH2O change in PEEP in either direction resulted in a change in rate of 0.4 breaths/min. The effects were exclusively due to changes in expiratory time. Effects began to manifest during the first breath and became fully established in the second breath for each PEEP level. Identical responses were found when PEEP levels were applied for 10 or 60 s. Post hoc analysis revealed a similar but stronger response in patients with impaired respiratory system compliance.Increases in PEEP were strongly associated with drops in minute ventilation (P < 0.0001) and respiratory rate (In tracheally intubated, spontaneously breathing adult patients, the level of PEEP significantly influences the resting short-term breathing pattern by selectively affecting expiratory time. These findings are best explained by the Hering–Breuer inflation/deflation reflex. Pulmonary stretch receptors affect the resting respiratory pattern by vagal afferents. Increased stretch receptor activity shortens the duration of inspiration in animals , human nE]), whereas tidal volume (VT) and inspiratory time were not significantly affected.Our aim in the present study was to investigate whether these effects also become apparent during standard clinical conditions. To this end, we repeatedly applied a pattern of different levels of PEEP in a heterogeneous population of tracheally intubated patients during unsupported spontaneous breathing. Using this approach we were able to demonstrate that increased PEEP was strongly associated with prolongation of the expiratory time  required to maintain their arterial oxygen tension and arterial oxygen saturation above 10 kPa (75 mmHg) and 92%, respectively, had to be 50% or less. Furthermore, the patients had to be able to maintain blood gas values within normal ranges, to exhibit no severe coughing or repeated swallowing, or obvious signs of discomfort, and to have stable cardiovascular and respiratory conditions . Of these patients, 19 (63%) could be successfully extubated within 1–24 hours after termination of the study. The remaining 11 patients (37 %) had to be kept on the ventilator for a mean of 3 ± 2 days for prolonged weaning from mechanical ventilation for various reasons.Thirty patients were investigated during weaning from mechanical ventilation. The characteristics of the patients and their underlying diseases are summarized in Table 2O. If necessary, FiO2 was adjusted to maintain oxygenation within comfortable ranges . After 20-min period to stabilize the temperature and humidity of the system and to allow the patient to become accustomed to the ventilator, the ventilatory mode was switched from conventional PSV to the automatic tube compensation mode [The study protocol was approved by the ethics committee of our institution, and informed consent was obtained either from the patient (in the case of elective surgery) or from the patient's next of kin. After study inclusion criteria had been fulfilled, patients were connected to a modified study ventilator , in whicion mode ,17. We uion mode .The study ventilator was driven by an external control unit, which – in accordance with the study protocol – automatically changes the PEEP level in synchrony with the patient's breathing pattern (i.e. upward steps in PEEP during inspiration and downward steps during expiration). Even though this helps to minimize interference with the patient's breathing pattern, the duration of expiration of the first breath after PEEP adjustment was shortened and prolonged, respectively, by upward steps and downward steps in PEEP. Consequently, these breaths had to be rejected from our analyses.2O, and the 5 cmH2O again. Each level of PEEP was applied for 30 s  and 60 s (n = 18).Instead of airway pressure, the tracheal pressure was the target for PEEP adjustment. To this end, the tracheal pressure was continuously calculated  and repe0 s Fig. . To mini2 Analyzer 930; Siemens-Elema, Solna, Sweden) that was previously calibrated with a carbon dioxide concentration of 8.4%. Measured signals were digitized with 12-bit precision and stored at a rate of 100 Hz in a personal computer for further analyses. The mechanics of the respiratory system  were calculated from tracings originating from a preceding period of controlled mechanical ventilation [The flow (V') was measured using a heated Fleisch No. 2 pneumotachograph , which was placed at the proximal end of the ETT. Airway pressure was measured between the pneumotachograph and the outer end of the ETT. Spot-check measurement of tracheal pressure were taken by introducing a pressure measuring catheter into the trachea via the lumen of the ETT, as described in detail elsewhere . The pretilation ,22, in wT was calculated by numerical integration of V'. VE was calculated by multiplying respiratory rate by VT. In 10 out of 30 patients, the exhaled carbon dioxide volume per minute was derived from the additive compound of flow and carbon dioxide samples during expiration. For subsequent statistical analysis, data for corresponding PEEP levels and identical time cues were averaged.Stored data were analyzed on a breath-by-breath basis after having rejected the breaths that were affected by transitions in PEEP level. We determined inspiratory time, expiratory time and respiratory rate by means of the flow signal, in which a combined criterion of flow and expiratory carbon dioxide concentration allowed us to differentiate between inspiration and expiration accurately . VT was post hoc analysis we investigated the PEEP-related effect on breathing pattern in patients without  and with impaired lung mechanics . Allocation was roughly based on respiratory system compliance above or below 50 ml/cmH2O . Differences in all parameters of interest were assessed by an analysis of variance suitable for repeated measures with four repeated within factors (i.e. the PEEP levels) and either no between factor  or one between factor (group) in the 2O was associated with a fall in VE from on average 11.6 ± 3.0 l/min to 10.0 ± 2.0 l/min (P < 0.0001). Whereas VT was unaffected by the increase in PEEP , the fall in VE was due to a decrease in respiratory rate from 23.6 ± 9.6 breaths/min at zero PEEP to 19.9 ± 7.3 breaths/min at 10 cmH2O PEEP (P = 0.001). The decrease in respiratory rate was due to a significant increase in expiratory time , whereas inspiratory time remained unaffected . Expiratory carbon dioxide volume significantly decreased with increasing levels of PEEP (P < 0.01), whereas the end-expiratory carbon dioxide did not differ significantly.For the investigation with 30 s intervals, a total of 175 ± 88 breaths per patient were eligible for statistical analysis. In each patient the increase in PEEP from 0 to 10 cmHE and expiratory time in individual patients. An identical pattern was found when the PEEP pattern was applied for intervals of 10 s or 60 s . Effects were more pronounced in patients with impaired respiratory system compliance than in the control group (P < 0.025). In both groups, the changes became fully established at the latest within the second breath after a change in PEEP.In patients with decreased respiratory system compliance, respiratory rate and VE, and vice versa. According to our findings, the magnitude of the effect is about 0.4 breaths/min per 1 cmH2O change in PEEP in either direction, and so it is about 10 times smaller than the effect found in anaesthetized animals [The findings of this clinical study, conducted in a heterogeneous population of adult intensive care patients, indicate that the level of PEEP significantly influences resting short-term breathing patterns by selectively affecting the duration of expiration. Thus, a reduction in PEEP is paralleled by an increase in respiratory rate and subsequently in V animals . Our fin animals -14, in wE).The PEEP-related effect upon breathing pattern found in our heterogeneous study population was not only significant but also substantial. However, whether these findings are of clinical importance remains unclear. On the one hand, this is because in our study we focused on short-term rather than on long-term effects. From a theoretical point of view, the short-term responses seen here might simply be offset by a subsequent shift in blood carbon dioxide concentration beyond the time frame of 60 s. Such an hypothesis is strongly supported by the slight but significant decrease in exhaled carbon dioxide volume with increasing levels of PEEP  associated with the prolonged duration of investigation. Consequently, because our study was not designed to be a long-term investigation, we are unable to draw any conclusions on whether the observed short-term responses are preserved over time or whether they are offset by slowly adapting reflexes or behavioural responses over a longer period of observation. On the other hand, the PEEP-related short-term effect is sufficiently substantial that it could be detected by attentive clinicians, and so it could be used to adapt ventilatory settings for weaning from mechanical ventilation. Furthermore, short-term effects are of the utmost importance with respect to the automated weaning procedures and closed-loop ventilation strategies that now are increasingly being applied worldwide .The question then arises as to the mechanism by which changes in PEEP can affect the breathing pattern, as found in this study. Based on findings in animals and humans, it is likely that the PEEP-related effect is best explained by the Hering–Breuer inflation/deflation reflex ,10,26-28E) does not support this hypothesis. In addition, if predominantly chemical feedback responses were at work, then different responses would be expected for the different periods of time for which the PEEP levels were applied (i.e. slight responses with durations of 10 s but stronger responses with durations of 60 s). Finally, the occurrence of the effect within the first breath after PEEP adjustment could hardly be accounted for by chemical feedback mechanisms.Second, the PEEP-related effect could also be due to responses of the chemical feedback system. Based on both alveolar gas exchange  and the circulatory time , any change in alveolar gas composition will influence the response of peripheral (mainly oxygen) and central (mainly carbon dioxide) chemoreceptors ,8,24. InA third alternative explanation for the observed PEEP-related effect might relate to persistent inspiratory muscle activity during exhalation ,30. The In conclusion, the PEEP-related short-term effect on breathing pattern found in the present is best explained by neuronal reflex mechanisms (i.e. the Hering–Breuer inflation/deflation reflex).post hoc analysis we found a similar response to changes of PEEP in patients with normal and those with decreased respiratory system compliance. For the latter, however, the effects were stronger. Consistent with our results, Tryfon and coworkers [In the oworkers  found a oworkers ,31,32. EE was at a similar level as during the observational period of the study. In addition, careful examination of the expiratory flow pattern during the investigation did not suggest expiratory flow limitation as an indirect sign of the occurrence of intrinsic PEEP.An alternative hypothesis for our unexpected results centres on the potential occurrence of intrinsic PEEP during the investigation. If this is the case, then intrinsic PEEP would have occurred predominantly in patients with normal rather than in those with decreased respiratory system compliance. Consequently, the increase in external PEEP would have reduced the work of breathing, and thus would have attenuated changes in the PEEP-related effect on breathing pattern predominantly in patients with normal respiratory system compliance (as found in our and Tryfon's study). However, there was no evidence for the occurrence of intrinsic PEEP, at least during the preceding period of controlled mechanical ventilation, in which VIn tracheally intubated, spontaneously breathing adult patients, the level of PEEP significantly influences the resting short-term breathing pattern by selectively affecting expiratory time. The mechanism is probably based on the Hering–Breuer inflation/deflation reflex. Further studies are needed to address counteracting behavioural and/or slow responses of chemical respiratory control, and therefore to elucidate the clinical importance of our findings.• 	In spontaneous breathing patients upwards steps in PEEP significantly decrease respiratory rate and minute ventilation whereas downward steps have just opposite effects• 	The PEEP-related effects are exclusively due to alteration of the expiratory time• 	Effects become fully established within the first two breaths after PEEP adjustment and went on for minimally one minute• 	Findings of this study and theoretical considerations strongly suggest a reflex related response by the Hering-Breuer inflation/deflation reflex2 = fractional inspired oxygen; PEEP = positive end-expiratory pressure; PSV = pressure support ventilation; VE = minute ventilation; VT = tidal volume.ETT = endotracheal tube; FiOThe author(s) declare that they have no competing interests.CH designed the study, carried out the measurements, performed the statistical analysis, and drafted the manuscript. JG conceived the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.


The statistical analysis of protein structures requires datasets in which structural features can be considered independently distributed, i.e. not related through common ancestry, and that fulfil minimal requirements regarding the experimental quality of the structures it contains. However, non-redundant datasets based on sequence similarity invariably contain distantly related homologues. Here we provide a reference dataset of non-homologous protein domains, assuming that structural dissimilarity at the topology level is incompatible with recognizable common ancestry. The dataset is based on domains at the Topology level of the CATH database which hierarchically classifies all protein structures. It contains the best refined representatives of each Topology level, validates structural dissimilarity and removes internally duplicated fragments. The compilation of Nh3D is fully scripted.The current Nh3D list contains 570 domains with a total of 90780 residues. It covers more than 70% of folds at the Topology level of the CATH database and represents more than 90% of the structures in the PDB that have been classified by CATH. We observe that even though all protein pairs are structurally dissimilar, some pairwise sequence identities after global alignment are greater than 30%..Nh3D is freely available as a reference dataset for the statistical analysis of sequence and structure features of proteins in the PDB. Regularly updated versions of Nh3D and the corresponding PDB-formatted coordinate sets are accessible from our Web site  The number of structures in the Protein Data Bank (PDB)  has growA number of PDB subsets have been proposed to address this need. The most often used subset of structures in the PDB is the PDBSELECT ,3. This In order to define a reference dataset of non-homologous protein structures, we have constructed a tool to extract coordinate subsets from the PDB, driven by CATH  domain tThe CATH database is a hierarchic classification of protein domains. At the Topology level of the hierarchy (similar to the Fold-level of SCOP ), the doTo create Nh3D Figure , represe1) X-ray or neutron-diffraction structure, resolved at 2.2 Å or better ;2) Domain has a minimum length of 50 residues ;3) Engineered structures are excluded, unless no alternatives are available .When two or more structures meet the selection criteria, the one with the better resolution is chosen as the representative. For structures that have the same resolution, the one with the better Ramachandran Z score  secondary structure assignment using DSSP  and STRIb) relative side-chain solvent accessible surface areas using the Shrake and Rupley  approach2are identified and flagged)c) regions with high B-Factor (where the average B-factor of the backbone atoms of three consecutive residues is found to be greater than 60 Å d) beta and gamma turn assignments using PROMOTIF Residues in the PDB coordinate files that have missing backbone residues are identified and removed from the dataset. Modified residues that appear as 'HETATM' records in the PDB files are treated as chain breaks; a distance check between bonded backbone atoms is implemented to check for additional discontinuities in a chain.Nh3D is freely available in an easily parseable, fully specified flatfile format and as PDB-formatted coordinates. Differently formatted output for specific needs can be made available upon request to the authors.Our dataset uses coordinates from 477 proteins consisting of 570 of 820 CATH Topologies and contains 90780 residues. The chosen toplogies represent over 90% of the structures in the PDB classified by CATH version 2.5.1.Interestingly, we observe numerous domains with striking sequence similarity , with completely dissimilar structure. A single likely homologue  has been identified in CATH Version 2.5.1. However, the domain 1nos03 was already excluded from the database in a previous step, due to the high residue B-factors over the length of its domain.Significant internal repeats were removed from 38 domains. For example, repeats were found in representatives of Porin, Tachylectin-2, and MutS topologies. Other repeats, most notably those of TIM barrel proteins, did not meet our cutoff criteria for recognizable homology and hence were retained.A comparison of our results with a sequence based PDB subset (PDBSELECT of October 2004) shows the large number of redundant, similar structures that cannot be eliminated based on sequence dissimilarity alone. The most redundant is the Rossman fold , the second most redundant are immunoglobulin-like domains . While it has been a matter of discussion whether such "superfolds" might be the result of convergent evolution [see e.g. ], we takNh3D is freely accessible as a reference dataset for the unbiased statistical analysis of sequence and structure features of proteins in the PDB. While our own motivation was to construct a source dataset for an analysis of short peptide conformations, we anticipate many uses of Nh3D, including the calibration of algorithms for the detection of distant gene relationships, for the recognition of false positives in sequence or structure alignments, or for the prediction of protein structure.. Nh3D Version 2.0 based on CATH 2.6.0 has since been made available.The dataset, a list of best representatives for CATH Topologies that did not meet our criteria, documentation, format specifications and supplementary information can be downloaded from CATH-A hierarchical classification of protein domain structuresDSSP-Definition of Secondary Structure of ProteinsEMBOSS – European Molecular Biology Open Software SuiteNMR-Nuclear Magnetic ResonanceSCOP-Structural Classification of ProteinsSTRIDE – STRuctural IDEntification methodPDB-Protein Data BankRADAR – Rapid Automatic Detection and Alignment of RepeatsRMSD – Root Mean Square DeviationWWW-World Wide WebBTG wrote the second version of the code to generate Nh3D and helped to draft the manuscript.GQ wrote the first version of the code and helped to prepare the manuscript.SAS participated in the design and helped to draft the manuscript.BS conceived of the study, and participated in its design and coordination and drafted the manuscript.