A multiple sequence alignment (MSA) generated for a protein can be used to characterise residues by means of a statistical analysis of single columns. In addition to the examination of individual positions, the investigation of co-variation of amino acid frequencies offers insights into function and evolution of the protein and residues.conn(k), a novel parameter for the characterisation of individual residues. For each residue k, conn(k) is the number of most extreme signals of co-evolution. These signals were deduced from a normalised mutual information (MI) value U computed for all pairs of residues k, l. We demonstrate that conn(k) is a more robust indicator than an individual MI-value for the prediction of residues most plausibly important for the evolution of a protein. This proposition was inferred by means of statistical methods. It was further confirmed by the analysis of several proteins. A server, which computes conn(k)-values is available at .We introduce H2r, which analyses MSAs and computes conn(k)-values, characterises a specific class of residues. In contrast to strictly conserved ones, these residues possess some flexibility in the composition of side chains. However, their allocation is sensibly balanced with several other positions, as indicated by conn(k).The algorithms  Most easily, conserved residues can be identified, which indicate positions crucial for function or structure. Therefore, MSAs are frequently the basis for the prediction of important residues; see e.g. ,2. For qe.g. . Even moe.g.  and quite.g. . Göbel efficient . The mutfficient ,8 or chifficient . A similfficient . In ordefficient .in silico perturbation is a constraint that limits the occurrence of amino acids at a certain position. Each choice selects a specific subset of MSA sequences and may cause variations in the column-specific occurrence of amino acids. Analysing these patterns, Ranganathan and co-workers have proposed the existence of energetically coupled residues [The above methods rely on the computation of a global co-variation statistic for the identification of correlated residues. In contrast to these concepts, methods based on the idea of "perturbations" have been introduced recently ,13. An iresidues . A similresidues . In combresidues .The enormous increase of sequence information resulting from genome sequencing projects has broadened the data basis for coupling analysis. Therefore, methods can be used that examine a large number of parameters. Even more, the existence of a high quality MSA is crucial for the analysis of correlated mutations. The sequence space of a protein has to be sampled correctly; otherwise, the quality of the predictions will deteriorate. If similar sequences originating from closely related species majorise an MSA, signals caused by a shared evolution of the proteins may be stronger than correlation patterns. Such bias will influence any calculation. However, methods based on the analysis of perturbations may be susceptible to less accented distortions. In this case, smaller sets of sequences determine predictions and may constitute signals interpreted as perturbations. If these subalignments are dominated by closely related sequences, the predictions may be wrong.H2r, a novel algorithm of that kind. H2r combines classical and well-proven concepts of computer science. It was our aim to focus on reliability even at the expense of sensitivity. We will confirm H2r's robustness and show that coupled residue pairs identified by H2r constitute tightly interconnected networks. Parameters will be introduced that allow the characterisation of these networks and individual residues. It will be demonstrated that the mode of generating MSAs does not markedly influence H2r's results. We study predictions in protein 3D-space and discuss possible reasons for the evolution of correlation patterns.This is why we prefer algorithms exploiting exhaustively the information deposited in each column of an MSA. In the following, we report k have to be found, where an amino acid X occurs with a certain minimal frequency fmin, which originates from Shannon's information theory [A large number of algorithms, utilising quite different principles have been introduced to identify correlated mutations. The co-variance algorithm proposed in  uses theduced in , which un theory  and is cn theory . Formallk is its entropy H(k); see [A parameter frequently used for quantifying the composition of an individual column (k); see  and refef of two variables (columns) k and l isThe entropy MI = H(k) + H(l) - H can be computed. MI-values have been the basis for several analyses. However, it has been shown that raw MI-values are a poor indicator for the prediction of co-evolution [MI-values. For synthetic MSAs, the ratios MI/H or MI/(H(k) + H(l)) have performed best [U, which is a measure for the dependency of k on l and vice versa:Using formulas (1) and (2), the mutual information volution . More remed best . In the U ≤ 1.0: If columns k and l are completely independent, then H = H(k) + H(l) and U vanishes. If the two columns are completely dependent, then H(k) = H(l) = H and U equals 1.0. For the analysis of correlated mutations in MSAs, high values of U indicate a strict pair-wise co-occurrence of amino acids in columns k and l. In more detail, formula (3) has been discussed in [H can directly be deduced from frequencies i = 1..20, all j = 1..20 amino acids, and all combinations of positions k and l. This implies that the MSA has to be large enough to allow a reliable estimation of these frequencies. For a similar approach, a lower limit of approximately 125 sequences has been determined [U-values range as expected; see Additional File It follows that 0 ≤ ussed in , which ctermined . For synk, l. Additionally, these values allow the assessment of individual positions k. For the ATP synthase ε subunit of Escherichia coli, it has been made plausible that residues with highest Z-scores deduced from normalised MI-values are more likely to change the activity than those with low values [i.e. for lower U-values. Merely by chance and due to random fluctuations, residue pairs might be assigned a relatively large value resulting in a strikingly high Z-score.To begin with, the outcome of a mutational analysis identifies coupled residue pairs w values . Howeveri.e. by adding up several analyses. We applied this principle for the identification of conspicuous positions. In agreement with previous findings [H2r form tightly connected networks; see Additional File conn(k) as the number of high-scoring pairs a residue k is an element of. Connectivity values differ significantly: For the MSA associated with the PFAM [conn(388) was 10 and conn(111) was 1. In order to illustrate how networks of interconnected residues are located in 3D-space, Figure conn(388)-value. The MSA of PF01053 (Cys_Met_Meta) and the related protein structure (pdb-code 1QGN) have already been a test bed for in silico analysis [conn(k)-approach is the SH3 domain: For a chi-squared approach it has been shown that 5 residues participate in 53 of 92 significant co-variations [For any noisy signal, the quality of a prediction can be enhanced by sampling, findings , high scthe PFAM  entry PFanalysis . A furthconn(k) in detail, we examined the outcome of H2r on several datasets. The first two experiments were carried out to estimate the probability of conn(k)-values for MSAs bearing no coupling signals of real proteins.In order to assess the parameter H2r_train. We used H2r to determine the occurrence and frequency of conn(k)-values for these MSAs. However, for the following assessment we had randomly assigned U-values in the range of 0.0 to 1.0 to all pairs k, l. For this test, 500 individual experiments (one MSA each) were analysed. A second test was based on PF01053 that was introduced above. Here, we did 1000 independent experiments by assigning U-values randomly and analysing the distribution of conn(k)-values. Results of both experiments are summarised in Table conn(k)-values ≥ 4 are highly unlikely to occur merely by chance. The frequency for conn(k) = 4 is < 2.5·10-3, a connectivity conn(k) > 6 was not observed in any of these experiments. Please note that only the 75 largest U-values were analysed for each MSA. This has to be considered when interpreting the above frequencies.For parameter optimisation  has been determined [SH3_filt, which consisted of 471 sequences. Its U-values were relatively low, the largest one, U was 0.28. This observation indicates that these correlations are much weaker than those observed in Cys_Met_Meta, which possesses a maximal U-value of 0.72. As PF00018 contains 3506 sequences, we used a bootstrapping approach to repeat the experiment several times and to analyse the results statistically. We generated 20 datasets by randomly selecting 210 sequences in each case. The results are summarised in Figure SH3_filt results (compare Panel B) and proposed to accept conn(k)-values ≥ 5. Furthermore, this cut-off was supported by the following correspondences of function and conn(k)-values.As a further test for the robustness of our approach, we analysed PF00018. This dataset subsumes 3506 sequences of SH3 domains. The domain consists of approximately 60 residues occurring in a large number of eukaryotic proteins involved in signal transduction. The 3D-structure of the related Fyn domain -values occurring for individual datasets of the bootstrapping approach made clear that the filtering of the input sequences is a critical step. A random selection of 210 sequences resulted e.g. for residue 135 in conn(k)-values ranging from 2 to 8.However, the extreme variation of U-value determined for an MSA indicates the strength of the coupling signal. Our analysis of synthetic MSAs allows a rough estimation of the values; see Additional File conn(k)-value ≥ 4 can be considered reliable in this case. For PF00018, the maximal U-value was 0.28. For this dataset, biochemical findings allowed us to explain the role of residues with conn(k)-values ≥ 5. As a (conservative) rule of thumb, we propose a cut-off of 0.5: If the maximal U-value is ≥ 0.5, H2r lists residues with conn(k)-values ≥ 4 otherwise those ≥ 5.The maximal conn(k)-values. In order to characterise the outcome of H2r for a sampling on filtered data, we created datasets by randomly choosing 75% or 60% of the remaining sequences. Resulting conn(k)-values showed that the composition of these MSAs did not markedly affect H2r's results -values remained stable.The above random sampling of sequences without any filtering induced a large variation of ε subunit of E. coli has been extensively mutated and the effects of mutations have been characterised and compiled -values and normalised MI-values were identical for all high scoring residues. This indicates that both parameters allow equally well to quantify the coupling of residues. The maximal U-value was 0.37 in this case. Therefore, H2r considered conn(k)-values ≥ 5 as reliable. This was true for positions 12, 65, 72, and 81. For positions 65 and 81 their susceptibility to mutational effects is known, none has been reported for the remaining two positions. If the four largest U-values were used for predicting conspicuous residues k, a comparison of this approach and the conn(k) method gave the following result: In both cases, 2 positions known to be susceptible to mutational effects were predicted correctly. It is unknown, how mutations affect the other two residues. Thus, if one utilises the concordance with known mutational effects as an indicator for prediction quality, both approaches have a similar performance.The ATP synthase led (see  and refeZ-scores . For eaceviously  and deteconn(k) and the maximal U-value deduced from PF00018 representing the SH3 domain. The largest U-value is 0.28. For none of the extra residues possessing maximum U-values ranked 2 or below 3, a clear function has been reported in [conn(k)-values are more robust and more reliable than individual U-scores.However, if the coupling signals were less pronounced the number of unclear predictions increased drastically for the maximum score approach. In Table orted in . In summε subunit had in both of the above experiments high MI-values; however no high Z-score has been reported [Interestingly, residues 72 and 73 of the ATP synthase reported . This diH2r to a perturbation based method, we analysed the MSA of globin sequences, as compiled in [U-value was 0.72. 9 residues gained a conn(k)-value ≥ 4. These were – projected onto 2DN1 – residues 97, 40, 57, 93, 131, 37, 85, 2, 39. Only 3 of these predictions (printed in bold) were in agreement with previous findings as reported in [In order to compare the output of piled in . For thiorted in .1QGN) [1SHF) [ε subunit  [in silico methods. Please note that we did not compile specific MSAs but used the precompiled full PFAM alignments for the following tests.For the following comparisons, we used the above introduced proteins represented by a PFAM entry and a related protein structure: Cys_Met_Meta  , SH3 dom) [1SHF) , and ATP3, 1AQT) . We seleHSRP (data not shown). CorrMut is a server identifying correlations in the evolution of amino acid sequences [HSRP in all three cases (data not shown).The server based on ,24 did nequences . After sH2r: 85 (15), 86 (-), 97 (-), 114 (22), 115 (-). Thus, H2r did not confirm any of the 14 top ranking predictions. Interestingly, our implementation of a Göbel like algorithm [MI-based methods like H2r and the above algorithms differ quite significantly in their predictions. This statement is further supported by an analysis of a larger dataset reported in the Additional File Based on a chi-squared statistical method, the 25 top co-varying SH3 residues have been computed and ranked . Our resH2r: 85 5, 86 (-)αββα tryptophan synthase complex, which catalyses the final reaction from indole-3-glycerole phosphate + L-serine to L-tryptophan + H2O. The α subunit (TrpA) cleaves indoleglycerol-3-phosphate to glyceraldehyde-3-phosphate and indole. The latter is transported through a hydrophobic tunnel to the associated β subunit (TrpB), where it is condensed with L-serine to yield L-tryptophan. A sophisticated mechanism of allostery links the α and β monomers of the synthase[H2r for TrpA and TrpB are plotted as projected onto pdb-entry 1KFJ [conn(k)-value, is an element of the TrpA/TrpB interface [As illustrative examples, we analysed three enzymes of the tryptophan synthesis pathway. TrpA and TrpB constitute the  synthase. Both pr synthase. In Figutry 1KFJ . For Trpnterface . Residuenterface . Residueconn(k)-value ≥ 4; conn(90) = 8 is the highest value. Residue 90 is near the lysine, which binds PLP and catalyses the reaction. Residue 19 is an element of the TrpA/TrpB interface [For TrpB, 5 residues possessed a nterface  and is anterface  and partnterface . The rolH2r predicted 5 residues as suspicious; see Figure Conn(284) was 11. Please note that the residues 235, 297 (distmin = 0.89 Å) and 50, 54 (distmin = 0.72 Å) are contacting residue pairs. For all these residues, the reason for high conn(k)-values is unclear.The anthranilate phosphoribosyl transferase (TrpD) catalyses the group transfer of 5'-phosphoribose from D-5-phosphoribosyl-1-pyrophosphate to the nitrogen atom of anthranilate, which is the third step in L-tryptophan biosynthesis. For TrpD, H2r as a web tool [H2r determines bootstrap supported conn(k)-values and reports the results via email. The web-interface can be utilised to change parameters like the number of HSRPs or the usage of pseudo counts. For parameter selection, please see the Additional File We have implemented a server offering web tool . After ue.g. the prediction of protein 2D-structure [e.g. relevant for the identification of binding sites [H2r supplements the repertoire of entropy-based methods of single residues by extending it to residue pairs. The information associated with high conn(k)-values is comparable to that of strictly conserved positions: Both signals, which are based on statistical analyses, identify  suspicious residues. However, in both cases the origin of these signals can only be elucidated by exploiting additional knowledge. A typical example for this enigmatic information is TrpD. 4 of the 5 residues constitute two contacting residue pairs, which supports the significance of the related signal. Nevertheless, the conn(k)-values alone do not explain the function of these residues or the origin of the signals.Incorporating MSAs turned out to improve the outcome of many applications like tructure  or fold-tructure . The reang sites -38. A prng sites ,40. Corre.g. sequence logos are frequently used to assess individual columns in MSAs [MI (as defined by formula (4)) was the basis for the work presented in [MI describes the extent of association between residues k and l.Shannon's theory of communication has turned out to be useful in many fields of application. In computational biology,  in MSAs . A mutuaented in . In biolMI-values are a poor indicator for the prediction of co-evolution [MI-values have been introduced [However, it turned out that unfiltered volution . Therefotroduced .U as it takes into account the entropy values H(k) and H(l), which express the degree of conservation at positions k and l. U-values are normalised and the results deduced from synthetic MSA_1  has two major advantages: 1) It is less susceptible to signals of a common evolution that might dominate those sequences constituting a perturbation. Generally, these signals are quite strong [U-values plotted in Additional Figure 2 clearly illustrates the inferiority of the perturbation approach. Comparing e.g. the columns representing frac values 0.4 and 0.8 illustrates the loss of information. If (say) a perturbation is due to an amino acid occurring in 40% of the sequences, the information content of the remaining 60% of the sequences is ignored. If a second amino acid induces a similarly strong perturbation, the U-value increases significantly; compare Additional Figure 2. The same is true for other combinations. A perturbation-based approach does not distinguish between these cases. This example makes clear that the analysis of all frequencies We prefer e strong ,42. In aA well-known problem in the analysis of MSA is the interpretation of columns containing gaps. For the identification of correlation patterns, a gap cannot be treated as 21st amino acid when calculating frequencies. In this case, columns consisting mostly of gaps would be identified as strictly coupled. Figure H2r, frequencies U-values. These frequencies are deduced from those sequences possessing a gap neither at position k nor at l. Thus, all dependencies, where gaps are not involved, are determined in a correct manner. Therefore, the U-values will at all positions solely depend on the signals induced by the amino acid propensities. Thus, ignoring gaps is equivalent to an analysis with 20 instead of 21 symbols. This limitation has to be considered when interpreting conn(k)-values.On the other hand, ignoring gaps is not appropriate, too. It could be that a substitution of a small side-chain with a large one induces the loss of a residue position. Columns 5 and 6 of Figure HSRPs, simple algorithms are sufficient for cluster and network generation; see Additional File γ synthase (1QGN) consists of two domains [1QGNA01 and 1QGNA02. 1QGNA01 binds PLP and consists of residues 48 – 307. 1QGNA02 binds the substrate cysteine [conn(k)-values ≥ 4 are located at the interface of these two domains; compare Figure conn(k)-value of 10, is an element of 1QGNA02, which is not the PLP binding domain. However, this residue is located directly opposite of PLP. For this example, the findings support the notion that conn(k)-values identify residues that signal the concerted co-evolution of domains to form a novel protein function. The functional role of residues possessing high conn(k)-values in the SH3 domain indicates their importance, too. The same is true for most of the conspicuous TrpA/TrpB residues.Correlation signals can be used to compile networks of residues . In the  domains , which h domains  – been dcysteine  and consH2r were not markedly affected by the mode of MSA generation. This indicates that state of the art programmes and datasets like PFAM offer MSAs of similar quality, which proved to be adequate for coupling analysis. Nevertheless, the composition of the samples fed into an algorithm has to be controlled. Assessing the local quality of MSAs as introduced with T-Coffee [For a reliable prediction of residues that play a major role in protein function or evolution, robustness has to be implemented on all levels of algorithmic design. For success, the generation of high quality MSAs is a critical step. Both the advent of novel algorithms ,49 and mT-Coffee  and the T-Coffee  could bee.g. by applying a BLOSUM-like scoring function. This is why we are planning to model the biological context more specifically.In addition, it should be possible to improve the above core algorithm. Shannon's theory does only consider the frequency of symbols and does not regard the features of the represented objects. In the case of MSAs, it would be reasonable to analyse the composition of columns and to assess the properties of occurring amino acids conn(k) is a novel parameter for the characterisation of a specific class of residues. In a robust way, it indicates the strength of co-variation detectable among residues. In contrast to strictly conserved residues, amino acid composition is allowed to vary for these residues. However, the instrumentation of these positions is sensitively balanced with several other ones. Just as strictly conserved residues, these ones offer an enigmatic signal of protein evolution or function. For a complete decoding, knowledge about the protein, its function, and evolution has to be considered.k, whose values are linked to a discrete set of frequencies f(ai) of amino acids, the entropy H can be computed according to formula (1). The entropy H of two variables (columns) k and l is defined by formula (2). In order to measure the dependency of k and l, the coefficient U can be computed according to formula (3). All frequencies have to be deduced from an MSA. An implementation for computing U is described in [For a random variable (column) ribed in .f is the total occurrence of all amino acids at position k, S are Blosum50 [λ is a weight factor, with 0 ≤ λ ≤ 1.0. For H2r, we used λ = 1.0.Blosum50  scores aThe frequencies ai at position k and aj at position l. n(k) is the sum of all ncorr-values were normalised so that the sum of the ncorr-values was equal to the sum of the (uncorrected) ai was determined. If fmax(ai) ≥ 0.95, the column and the related residue were regarded as strictly conserved.For each column of an MSA, the largest frequency of any amino acid S1 ... Sn be the n sequences constituting the input (MSA) sequ_in. For the computation of sequence identity values ident, the number of identical residues (ignoring gaps) was determined. The two parameters identmin and identmax defined the minimal and the maximal sequence identity values used for comparison. In pseudo-code the algorithm works as follows:Let Input: sequ_in = {S1,..., Sn}Output: The set filteredS1 to filteredAdd i = 2 n doFor {Si to all sequences of filtered and determine ident   Compare identmin ≤ ident ≥ identmax for all comparisons   If Si to sequ_in      Add }identmin was 20% and for identmax it was 90%. Columns possessing more than 25% gaps were masked and not processed further. Please note that the first sequence of the input is always an element of the set filtered.Due to the results of parameter optimisation  of two residues k, l as the minimal space between van der Waals radii of any pair of atoms belonging to k or l, respectively. Thus, a distance of 0 Å indicates that at least two atoms of k and l are in direct contact in 3D-space.For measuring distances of residues, we used routines compiled by M. Gerstein . We defiMZ prepared datasets and multiple sequence alignments and assisted in manuscript writing. RM designed and implemented the algorithm and wrote the manuscript. Both authors read and approved the final version.H2r. Computations used for parameter optimisation and additional performance tests.Parameter optimisation and performance tests for Click here for file


Retinal perfusion variability impacts ocular disease and physiology.To evaluate the response of central retinal artery (CRA) blood flow to temperature alterations in 20 healthy volunteers.Non-interventional experimental human study.Baseline data recorded: Ocular surface temperature (OST) in °C (thermo-anemometer), CRA peak systolic velocity (PSV) and end diastolic velocity (EDV) in cm/s using Color Doppler. Ocular laterality and temperature alteration (warming by electric lamp/cooling by ice-gel pack) were randomly assigned. Primary outcomes recorded were: OST and intraocular pressure (IOP) immediately after warming or cooling and ten minutes later; CRA-PSV and EDV at three, six and nine minutes warming or cooling.Repeated measures ANOVA.: Pre-warming values were; OST: 34.5 ± 1.02°C, CRA-PSV: 9.3 ± 2.33 cm/s, CRA-EDV: 4.6 ± 1.27 cm/s. OST significantly increased by 1.96°C (95% CI: 1.54 to 2.37) after warming, but returned to baseline ten minutes later. Only at three minutes, the PSV significantly rose by 1.21 cm/s (95% CI: 0.51to1.91). Pre-cooling values were: OST: 34.5 ± 0.96°C, CRA-PSV: 9.7 ± 2.45 cm/s, CRA-EDV: 4.7 ± 1.12 cm/s. OST significantly decreased by 2.81°C (95% CI: −2.30 to −3.37) after cooling, and returned to baseline at ten minutes. There was a significant drop in CRA-PSV by 1.10cm/s (95% CI: −2.05 to −0.15) and CRA-EDV by 0.81 (95% CI: −1.47 to −0.14) at three minutes. At six minutes both PSV (95% CI: −1.38 to −0.03) and EDV (95% CI: −1.26 to −0.02) were significantly lower. All values at ten minutes were comparable to baseline. The IOP showed insignificant alteration on warming (95% CI of difference: −0.17 to 1.57mmHg), but was significantly lower after cooling (95% CI: −2.95 to −4.30mmHg). After ten minutes, IOP had returned to baseline.This study confirms that CRA flow significantly increases on warming and decreases on cooling, the latter despite a significant lowering of IOP. The ocular circulation is geared to meet the nutritional needs and respond to physiological changes to allow the specialized tissues to function optimally. UnderstaOcular warming in healthy individuals increases the retinal blood flow (RBF), while decreasing the choroidal blood flow (CBF). The decrChilling the eye is often resorted to, as in cyclocryotherapy and retinal cryopexy. People at high altitude, polar explorers or those exposed to snowstorms, avalanches or freezing waters risk cryo-injury. No studies were found evaluating the effect of cooling on ocular blood flow (MEDLINE). Katsimpris demonstrated in rabbits that trans-palpebral ocular cooling significantly lowered aqueous and vitreous temperatures, comparable to the effect of direct corneal chilling.Utilizing CDI, we studied the changes in central retinal artery (CRA) blood flow on warming and cooling normal eyes.The study was approved by the institutional review board of the Institute of Ophthalmology, Aligarh Muslim University and informed consent was taken. Twenty healthy young volunteers were recruited from amongst the junior residents of the ophthalmology department. The inclusion criteria were best corrected visual acuity (BCVA) of 20/20 Snellen, with normal anterior segment on slit-lamp biomicroscopy and a normal fundus on indirect ophthlamoscopy. Any volunteer with fever, history suggestive of a rheological disorder, such as diabetes or hypertension, glaucoma, maculopathy, pathological myopia or tear film abnormalities (on Schirmer testing), subjects who had undergone any ocular surgery or were contact lens wearers were excluded. A carefully supervised pilot study was done on five volunteers, where warmth and cooling was done for gradually increasing periods (up to ten minutes) and VA and biomicrosopy carried out at each interval to assess any adverse effect of the intervention.Subjects abstained from drinking tea, coffee and smoking two hours prior to the test and rested for ten minutes before the tests. The study was performed in an air-conditioned room with a controlled temperature range of 20°C-24°C, humidity range of 20-25%, and constant brightness. Baseline measures recorded were: Ocular surface temperature (OST) in °C, using a thermo-anemometer  with resolution of 0.1°C, range of −20 to 200°C and an accuracy of ±0.8°C. The probe was placed in contact with the lower bulbar conjunctiva  and the CRA velocity (cm/sec) was measured by Color Doppler images Figs.  and 3 obThe laterality of the eyes (right or left) and the temperature alteration (warming or cooling) were randomly assigned by the toss of a coin. Between temperature alterations each subject was allowed a ten-minute break. For ocular warming an electric lamp having one 60 watt tungsten bulb was placed ten cm from the closed lids for ten minutes. The cooling was achieved by using an ice-gel pack  with surface temperature ≤5°C placed in contact (avoiding pressure) with the eye through closed lids for ten minutes.The following primary outcome parameters were recorded sequentially:Immediately after warming (or cooling) at time = 0 min: OST.The intraocular pressure (IOP) was recorded as a secondary outcome parameter with the Pulsair Easy Eye air puff tonometer , as the median of three readings, taken within a minute of warming or cooling.After 3, 6 and 9 min-Peak systolic and end diastolic CRA velocity.After 10 min-OST and IOP were re-measured.P < 0.05 (GraphPad Prism version 5.02); 95% confidence intervals are quoted.Data was analyzed using repeated measures ANOVA (RANOVA) and Tukey test for multiple comparisons with significance set at We chose ten male and ten female healthy postgraduate-volunteers from the department. Their mean age was 26.4 years (SD 1.6). Baseline measures of pulse (μ ± SD: 81.6 ± 6.3 beats/min); systolic BP (μ ± SD: 119.1 ± 8.7 mmHg) and diastolic BP (μ ± SD: 77.1 ± 6.7 mmHg) were within normal range. The baseline values of OST, CRA PSV, EDV and RI and IOP are shown in The OST increased to a mean of 36.4 ± 0.90°C immediately after warming. This reflects a significant mean rise of 1.96°C (95% CI 1.54 to 2.37). Ten minutes after cessation of warming, the OST had returned to near baseline values (μ: 34.4 ± 0.92°C). Compared to baseline, the changes in CRA parameters on warming are shown in The OST decreased to a mean of 31.7 ± 1.38°C immediately after cooling. This reflects a significant mean decrease of 2.81°C (95% CI −2.24 to −3.37). Ten minutes after cessation of cooling, the OST had returned to near baseline values (μ: 34.2 ± 0.93°C). Compared to baseline, the changes in CRA parameters are shown in P = 0.15; 95% CI −0.17to1.57). On cooling there was a significant drop in IOP by 3.32 mmHg  The IOP had returned to pre-cooling levels ten minutes after cessation of cooling.At baseline, in 20 eyes, the IOP ranged from 10-19 mmHg  and showWe studied the effect of warming (20 eyes) and (for the first time) cooling (20 eyes) on the CRA parameters of 20 healthy young volunteers.Our baseline OST 34.5 ± 0.98°C) recorded with a thermo- anemometer were similar to those of Nagaoka  measured with non-contact infrared radiation thermography. Mori obt8°C recorOur baseline CRA-PSV of 9.5 ± 2.4 cm/s was similar to that reported by numerous authors.5–15 The mThe baseline CRA-EDV of 4.7 ± 1.2 cm/s in our series appears higher than that of Guthoff . In Nagaoka's study the OST rose by 3.3°C from a baseline of 34.5 ± 0.2°C to 37.8 ± 0.3°C on warming for ten minutes and like us, returned to baseline within ten minutes after cessation of warming.[In our study, after ten minutes of warming, the OST rose significantly  on cooling but returned to near baseline values after ten minutes (95% CI: −0.93 to 0.21). Ortiz demonstrated a much greater lowering by 21.5°C using extremely cold air stream of −19°C for 40 min. In anothBoth CRA-PSV and EDV significantly decreased on cooling at three minutes, but had returned to baseline values by six . No signP = 0.7). The IOP rose to match the baseline values at ten minutes after cessation of cooling. Perhaps of greater importance is that despite a decrease in IOP the CRA, PSV and EDV were significantly lower on chilling. This suggests that cold exposure of eyes causes a significant reduction in ocular blood flow to the posterior segment and cryo-trauma to the eye would likely be an ischemic insult. Although Goldmann applanation tonometer is considered the gold standard, we preferred the air puff tonometer on account of it being quicker: And time was important for us, since the very nature of the study demanded numerous measurements in a limited time. Equally importantly, we were not studying IOP per se, but the change induced by our experimental intervention. For ‘change’ we felt the air puff tonometer would suffice.It is important to consider changes in IOP on warming or cooling the eyes, since alterations of IOP may modify ocular perfusion. IOP has been reported to both decrease and showOur study lacks in not having been able to co-evaluate the choroidal circulation and flow, largely on account of a lack of equipment. Subjective estimates of the choroidal blood flow although done, were considered to lack the objectivity to be seriously analyzed. This may be of greater import since choroidal perfusion abnormalities are reported in pathologies like age-related macular degeneration.25Our study confirms that CRA blood flow significantly rises in response to ocular warming. In addition this is the first study to demonstrate a significant lowering of the CRA blood flow on cooling the eye. Cryo-injury to the eye has great importance for people who live in high altitudes or in extreme climatic zones like Ladakh (Tibet) and the Antarctic. The effect of cooling the eye on retinal blood flow sheds some light on this little researched aspect of ophthalmology. Additional research will help us understand the effects of heating and cooling modalities used in ocular therapy. Moreover, we can better predict their adverse effects.


Here we studied the activity of α-bisabolol in acute leukemia cells.We previously demonstrated that the plant-derived agent α-bisabolol enters cells ex vivo blasts from 42 acute leukemias  for their sensitivity to α-bisabolol in 24-hour dose-response assays. Concentrations and time were chosen based on CD34+, CD33+my and normal peripheral blood cell sensitivity to increasing α-bisabolol concentrations for up to 120 hours.We tested 50) included mainly Ph-B-ALL cells. AML cells were split into cluster 2 and 3 (45 ± 7 and 65 ± 5 μM IC50). Ph+B-ALL cells were scattered, but mainly grouped into cluster 2. All leukemias, including 3 imatinib-resistant cases, were eventually responsive, but a subset of B-ALL cells was fairly sensitive to low α-bisabolol concentrations. α-bisabolol acted as a pro-apoptotic agent via a direct damage to mitochondrial integrity, which was responsible for the decrease in NADH-supported state 3 respiration and the disruption of the mitochondrial membrane potential.A clustering analysis of the sensitivity over 24 hours identified three clusters. Cluster 1 (14 ± 5 μM α-bisabolol ICOur study provides the first evidence that α-bisabolol is a pro-apoptotic agent for primary human acute leukemia cells. Aliquots of the homogenates  were loaded on SDS-polyacrylamide gels at the appropriate concentrations. Electrophoresis was performed at 100 V with a running buffer containing 0.25 M Tris-HCl (pH 8.3), 1.92 M glycine, and 1% SDS. The resolved proteins were electroblotted onto a nitrocellulose membrane using the iBlot™ system . Membranes were then incubated with a mouse monoclonal IgG antibody to poly(ADP-ribose) polymerase (PARP) , with a rabbit polyclonal IgG antibody to BID  or with a rabbit polyclonal IgG antibody to α-tubulin . The membranes were then washed and incubated with an anti-mouse or anti-rabbit IgG peroxidase-conjugated antibody . The blots were washed again and then incubated with enhanced chemiluminescent detection reagents  according to the manufacturer's instructions. Proteins were detected using the ChemiDoc XRS Imaging System (Bio-Rad).Cells were homogenized at 4°C in 50 mM Tris-HCl (pH 8) containing 0.1% Nonidet-P40 (NP-40), 200 mM KCl, 2 mM MgCl2, 10 mM Tris-HCl, pH 7.5, 1 mM sodium orthovanadate, and complete EDTA-free protease inhibitor cocktail . Cells were then chilled on ice for 10 minutes and gently lysed by adding 0.3% (v/v) NP-40. In order to restore an isotonic environment, a solution containing 525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl, pH 7.5, 2.5 mM EDTA, and protease inhibitor cocktail was added. Lysates were first centrifuged at 600 × g at 4°C in order to remove nuclei and then the supernatants were centrifugated at 17,000 × g for 30 minutes at 4°C. The obtained supernatants were collected and used as the cytosolic fraction. The pellets, that contained mitochondria, were washed once with the same buffer and then were resuspended in sample buffer. The cytosolic and the mitochondrial fractions were separated on a 15% SDS-PAGE and probed using a rabbit polyclonal IgG antibody to BID . Then, the membrane with the cytosolic and mitochondrial fractions were probed with a rabbit polyclonal IgG antibody to α-tubulin  and with a mouse monoclonal IgG antibody to Hsp60 , respectively.Cell pellets were suspended in 100 μL of solution containing 10 mM NaCl, 1.5 mM MgClg) and washed with ice cold buffer A . The pellet was resuspended in 2 mL of buffer A containing 80 μg of digitonin. After a 1-minute incubation on ice, 8 mL of buffer A were added and cells were centrifuged . The pellet was resuspended in 100 μL buffer A containing 1 mM ADP, 2 mM KH2PO3 (respiration buffer) and immediately used for the polarographic assay. Cell number and permeabilization was measured by the trypan blue exclusion method.Leukemic cells and normal lymphocytes were centrifuged  was started by adding 5 mM glutamate plus malate (G/M) and 5 mM succinate plus glycerol-3-phosphate (S/G3P), which are complex I and complex III/glycerol-3-phosphate dehydrogenase substrates, respectively. The maximal respiration rate (uncoupled respiration) was empirically determined by the addition of 200 nM carbonylcyanide-4- (trifluoromethoxy)-phenylhydrazone (FCCP). Oxygen consumption was completely inhibited by adding 4 μM antimycin A at the end of the experiments .6/mL were treated with 40 μM α-bisabolol for 3 or 5 hours at 37°C. They were then washed with pre-warmed CM, 4 μM of the potential sensitive dye JC-1  was added, and they were then placed back into the incubator. After 30 minutes they were washed twice with pre-warmed PBS. An aliquot of each sample was spotted onto a slide, mounted with a coverslip and immediately recorded by an Axio Observer inverted microscope . Visualization of JC-1 monomers (green fluorescence) and JC-1 aggregates (red fluorescence) was done using filter sets for fluorescein and rhodamine dyes (emission 488 and 550 nm respectively). Image captures of random fields using fixed imaging parameters were performed, and previously unviewed areas of cells were captured to avoid photobleaching [Cells resuspended in CM at 1 × 10leaching . Image aleaching .6 cells were resuspended in 0.3 mL of culture medium containing 10% FBS and incubated for 90 minutes at 65°C and then overnight at 37°C in the presence of 0.4 M NaCl, 5 mM Tris-HCl (pH 8), 2 mM EDTA, 4% SDS and 2 mg/mL proteinase K. The lysates were brought to a final concentration of 1.58 M NaCl and centrifuged twice for 10 minutes at 6,000 × g to separate the DNA fragments from intact DNA. The supernatants were recovered, and DNA was precipitated by the addition of three volumes of absolute ethanol at -80°C for 1 hour. The DNA pellets were recovered by microcentrifugation  and resuspended in a minimal volume of 40 μl of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 1 mg/mL DNase-free ribonuclease A. Aliquots of 5 μg of DNA were then loaded onto a 1% agarose gel containing 0.25 μg/mL ethidium bromide. After electrophoresis, the DNA was visualized by UV light using the ChemiDoc XRS Imaging System (Bio-Rad).For internucleosomal DNA laddering analysis, 5 × 10p values < 0.05. The 24-hour IC50 was approximated by using mean cytotoxicity data in the different groups .Student's t-test for means, chi-squared tests, Mann-Whitney U test and Kruskall-Wallis analysis of variance by ranks were considered significant for Due to the lipophilic properties of α-bisabolol, a preliminary evaluation was performed of the dose-dependent solubilization in the culture medium over 24 hours by a RP-HPLC method. The addition of α-bisabolol at time 0 was followed by a rapid increase of the measured concentrations during the first 3 hours. After 24 hours, concentrations may be considered roughly constant, though with a slightly downward trend Figure . A doubl50 of 59 ± 7 μM and were only marginally sensitive to 40 μM α-bisabolol over 120 hours.The viability of normal blood cells was evaluated after different times and doses of exposure to α-bisabolol. The cytotoxicity increased in a dose- and time-dependent manner. Figure +my and CD34+/33+ or CD34+/19+ cells from 5 normal bone marrow samples. These subpopulations were assumed to represent the normal counterpart of acute leukemia blasts and the hematopoietic compartment that is responsible for bone marrow renewal and, eventually, drug toxicity. The 24-hour α-bisabolol IC50 was 95 ± 7 and 62 ± 9 μM in CD33+my and CD34+ cells, respectively (p < 0.05). By contrast, no difference was observed between CD34+/33+ and CD34+/19+ cells .Figure ex vivo dose-response  cytotoxicity assays at 24 hours in 42 different samples of leukemic cells  obtained from patients before any treatment. Table -B-ALL, Ph+B-ALL, and AML cells. The 24-hour dose-response assays showed that α-bisabolol was cytotoxic to primary Ph-B-ALL cells (33 ± 15 μM IC50). Though less sensitive, Ph+B-ALL, including Ph+-cells resistant to imatinib mesylate, and AML cells were also killed . Thus, α-bisabolol is a pro-apoptotic agent for acute leukemia cells ex vivo, particularly for Ph-B-ALL.Based on these data from normal cells, we performed p < 0.05) by comparing differences among experimental samples with regard to responsiveness to apoptotic signals induced by α-bisabolol. The group with the highest sensitivity to α-bisabolol (cluster 1: 14 ± 5 μM IC50) included 2 Ph+ and 6 Ph- B-ALL cases. Thus, a proportion of the Ph-B-ALL cases shared a high sensitivity to α-bisabolol, although some other Ph-B-ALL were scattered over different sensitivity groups. The AML cases were split into two groups with intermediate  and lower  sensitivity. Unlike Ph-B-ALL, AML cases as a whole were less sensitive to α-bisabolol. The Ph+B-ALL cases were scattered all over the three groups but were mainly clustered with intermediate sensitivity AML. Interestingly, introducing both CD34+ and CD33+my cell sensitivity to α-bisabolol  in clustering analysis made it evident that ALL cells as a whole were more sensitive to α-bisabolol than their normal counterpart (grouped into cluster 3 among less sensitive cells). This analysis demonstrated that some Ph-B-ALL cases may be highly sensitive to the apoptotic mechanisms activated by α-bisabolol and indicated that the Ph+B-ALL cases and especially the AML cases  may well be characterized by variable degrees of resistance to these mechanisms. Still, all leukemia cases were eventually responsive to 65 μM α-bisabolol for 24 hours  alone or α-bisabolol associated with imatinib mesylate (3 μM for 24 hours representative of in vivo effective concentration). In contrast, cells sensitive to imatinib mesylate shared a significant increase in cytotoxicity to α-bisabolol. For instance, cells from patient Ph+B-ALL #05 , and the combination was clearly synergistic, denoted by CI values <1 for any given Fa [α-bisabolol was active against PhWe have previously demonstrated that α-bisabolol binds to the BCL-2 family member BID . To eval-B-ALL, 1 Ph+B-ALL, 2AML) and healthy lymphocytes from 6 donors to determine whether α-bisabolol treatment affects mitochondrial state 3 and uncoupled respiration. Figure vs. 280.7 ± 11.9 pmol O2/minute/106 cells; p < 0.05). In contrast, the oxygen consumption sustained by S/G3P oxidation was not affected by α-bisabolol treatment, and the mitochondrial respiration was not stimulated by the addition of FCCP. These data are in line with a loss of mitochondrial integrity in treated leukemic samples, which is responsible for the matrix NADH decrease. This behavior is confirmed by the observation that the respiration in the presence of S/G3P was unaffected. Healthy lymphocyte respiration was not statistically modified by α-bisabolol treatment in state 3 using G/M and S/G3P as substrates and FCCP as a mitochondrial uncoupler. This is in agreement with the resistance to α-bisabolol observed in lymphocytes . In fit cells, JC-1 is more concentrated in the mitochondria (driven there by the ΔΨm), where it forms red-emitting aggregates, than in the cytosol, where it exists as a green-fluorescent monomer. Accordingly, the ratio red/green JC-1 fluorescence can be used as a sensitive measure of ΔΨm [c translocation and the start of the apoptotic process) is indicated by a loss of red fluorescence and an increase in green fluorescence. Figure -B-ALL #01 out of the 6 tested. Microscopy revealed that in untreated leukemic cells well-polarized mitochondria were marked by punctate red fluorescent staining . Blasts exposed to 40 μM α-bisabolol underwent a progressive loss of red fluorescence, indicated by a shift right and downward over 3  and 5 hours . In contrast, normal lymphocytes used as a negative control did not suffer any changes in their microscopy or cytofluorimetric pattern when exposed to a similar α-bisabolol concentration, indicating that there was no mitochondrial damage . The third subgroup included mainly, but not exclusively, AML samples with an IC50 value of 65 ± 5 μM. Thus, Ph-B-ALL cases were definitely more sensitive than AML cases, whose IC50 was near to that observed in vitro also in normal leukocytes, except lymphocytes, and in hematopoietic precursors. Nevertheless, previous studies in animal models suggested that similar α-bisabolol concentrations may be safely administered through daily oral supplementation even on a long-term basis [in vitro are also lower than, or similar to, the concentrations that we measured in the blood and in the brains of healthy mice sacrificed after treatment with 1.4 g/Kg α-bisabolol. In these mice the blood parameters of liver and kidney fucntionality were preserved and, remarkably, the concentration in the brain exceeded 50 μM without toxicity. Therefore, an active concentration of α-bisabolol safely accumulated in a body environment where lymphoid blasts have a tendency to localize and survive protected from a number of curative drugs [By cluster analysis, we separated out three subgroups of leukemias with different sensitivities over 24 hours. α-bisabolol was effective with an ICrm basis . The α-bve drugs . A dose +B-ALL cells were also sensitive to α-bisabolol. In three cases (Ph+B-ALL #01, #04, #06 in Table + human cell line CML-T1. It is not clear, however, whether the synergism depends on internalization mechanics or on intracellular modulation of the damaging actions of each or both drugs. A compound like α-bisabolol - and others [Phd others  - could d others ,28,29.m, which induces outer membrane permeabilization and leads to the apoptotic death of blasts. Our data not only implicate α-bisabolol for the first time in mitochondrial impairment in human leukemic cells but also suggest that this goes through a peculiar model of cell death, i.e., the formation of a cellular population with intermediate DΨ m which is a feature of apoptosis seen only in a few cell types and never described to date in leukemic blasts [Our biochemical data suggest a direct effect on mitochondrial integrity as a possible mechanism of α-bisabolol damage to leukemic cells. This behavior is supported by the observed oxygen consumption decrease in the presence of glutamate/malate and by the unaffected respiration rates in the presence of succinate/glycerol-3-phosphate. Microscopy and flow cytometry data show that α-bisabolol disrupts ΔΨc blasts .In all leukemia samples treated with α-bisabolol, BID was found to be expressed in a full-lenght form that was suitable for binding to α-bisabolol. We failed to demonstrate full-length BID translocation to the mitochondria in leukemic cells as a pro-apoptotic mechanism . Neverthvia lipid rafts and directly involves mitochondrial permeability transition pore opening [Thus, according to our previous and present work, α-bisabolol enters cells  opening , which i opening  remains +B-ALL. It is also active against primary AML cells at slightly higher concentrations. Our findings support α-bisabolol as a possible candidate for the treatment of acute leukemias and establish a basis for studies in animal models.We provide here the first evidence that α-bisabolol is an effective pro-apoptotic agent in primary ALL cells at concentrations and durations that spare normal blood and bone marrow cells. It retains cytotoxic potential in both imatinib mesylate-resistant and -sensitive PhThe authors declare that they have no competing interests.EC, AR, ACdP performed the research, analyzed data, and performed statistical analysis; MB, EG, CB, RF contributed analytical tools, performed selected experiments and analyzed data; GP contributed criticism; HS suggested the research, contributed ideas and critical scientific knowledge, analyzed and interpreted data; FV chose the clinical setting, designed and performed the research, analyzed and interpreted data, and wrote the paper; all authors checked the final version of the manuscript.


Road traffic accidents are the second largest cause of burden of disease in Thailand, largely attributable to behavioural risk factors including drinking and driving, speeding, substance abuse and failure to use seatbelts. The aim of this study was to assess the prevalence and associated factors of non-seatbelt use among drivers during Songkran festival in Thailand.A cross-sectional survey has been performed to determine the prevalence of seatbelt use among Thai drivers (N=13722) during four days of the Songkran festival. For this sample the population of drivers was consecutively selected from 12 petrol stations in four provinces from each of the four main geographical regions of Thailand. The study was conducted at petrol stations at roads in town, outside town and highway at different time intervals when trained field staff administered a structured questionnaire and performed an observation checklist on seat belt use.An overall prevalence of 28.4% of non-seatbelt use among drivers was found. In multivariable analysis demographics , environmental factors , seatbelt use experiences and attitudes  and lower exposure to road safety awareness (RSA) campaign  were associated with non-seatbelt use.Rates of non-seatbelt use by Thai drivers during Songkran festival was 28.4%. Lower exposure to the RSA campaign was found to be associated with non-seatbelt use among drivers during the Songkran festival. The Road Traffic Injury (RTI) fatality rate in Thailand was 40 per 100,000 populations, i.e., double the world average for low and middle income countries . A numbeSongkran is the New Year celebration in Thailand, set by the solar calendar since ancient times. It takes place between 13 and 15 April. At Songkran festival are major holidays that encourage a million of travellers who travel to/from their hometown and doing the activities during these holiday periods . UnfortuA cross-sectional survey has been performed to determine the prevalence of helmet use among drivers. The recruitment period of this project was during four days of the Songkran festival from 13–16 April 2007. For this sample the population of drivers from 12 petrol stations were selected from four provinces from each of the four main geographical regions of Thailand excluding Bangkok. Provinces were Chiang Mai, Lampang, Nakhon Sawan and Phichit in the northern region, Nakhon Ratchasima, Khon Kaen, Udon Thani, and Loei in the Northeastern region, Songkhla, Phuket, Surat Thani, and Trang in the southern region, and Phra Nakhon Si Ayutthaya, Chonburi, Chachoengsao, and Phetchaburi in the central region. In total 48 petrol stations (three petrol stations per province) was selected using quota sampling. In town, the petrol station on the road with the largest shopping mall was selected; out of town the petrol station on the road leading to the largest district was selected; in terms of petrol station on the highway, each province only has one highway. If there was more than one petrol station on the selected road or highway, the largest petrol station was selected. The study team spent four days at each petrol station road venue  from 7:00–9:00, 13:00–15:00, 17:00–19:00, 22:00–24:00. All consecutive motor vehicle occupants who entered the petrol station were asked to participate by trained personnel (who were students from Chiang Mai University that were trained by the research team) while they were having their gas tank filled. The number of vehicles and time interval for vehicle selection were determined by the availability of field staff to conduct a motor cycle rider observation, interview and alcohol test. The target sample size was 100 drivers from each of the petrol stations per time period, except during 22:00–24:00 for which 50 drivers were targeted. Trained field staff administered a structured questionnaire and performed an observation checklist. The project was approved by the Ethics Committee for research in human subjects of the public health programme, Chiang Mai University.The primary outcome of the study was seatbelt use. Seatbelt use was assessed by observation. The questionnaire covered demographic data, vehicle characteristics, history of road traffic accidents, known risk factors such as, age, sex, environmental factors, seatbelt use experiences and attitudes, and exposure to the road safety awareness (RSA) campaign.2 are presented to describe the amount of variance explained by the multivariable model. Probability below 0.05 was regarded as statistically significant.Data were analyzed using Statistical Package for the Social Sciences (SPSS) for Windows software application programme version 19.0. Frequencies, means, standard deviations, were calculated to describe the sample. Data were checked for normality distribution and outliers. For non-normal distribution non-parametric tests were used. Associations of non-seatbelt use were identified using logistic regression analyses. Following each univariate regression, multivariable regression models were constructed. Independent variables from the univariate analyses were entered into the multivariable model if significant at P<0.05 level. For each model, the RThe total sample included 13722 drivers ; 77.4% of the drivers were male and 22.6% female. The majority of the drivers (79.9%) were between 26 to 59 years old and about half (50.7%) were driving a pickup. Driver participation in the study was equally distributed across four of Thailand’s four regions, four data collection times during the day, four dates of data collection and three locations of data collection. The overall prevalence of non-seatbelt use was 28.4% , followed by passenger (22.5%) and pedestrian (2.0%). A large group of participants (46.6%) indicated that they had not usually been using a seatbelt before and 41.5% had not intended to use a seatbelt. The majority (73.7%) perceived a danger of not wearing a seatbelt and 53.0% were highly aware of the danger of not wearing a seatbelt. A significant number of 26.4% indicated that they had been caught by the police because of not wearing a seatbelt and 67.3% perceived a moderate to high risk about being caught by the police because of not wearing a seatbelt. Almost all (90.4%) had heard about the RSA campaign and more than one-thirds (36.3%) had frequently heard or seen the RSA campaign on the radio or on TV. More than half (57.0%) of the participants had been talking to others about the RSA campaign. One-thirds (33.3%) liked the RSA campaign very much, 31.4% frequently followed the TV news reports on road traffic injury (RTI) statistics and more than half (54.7%) believed perceived that the RSA campaign had a high effect , environmental factors , seatbelt use experiences and attitudes  and lower exposure to RSA campaign  were associated with non-seatbelt use  were in this study associated with non-seatbelt use. Phillips et al.  found frCaution should be taken when interpreting the results of this study because of certain limitations. As this was a cross-sectional study, causality between the compared variables cannot be concluded. A further limitation was that some variables were assessed by self-report and desirable responses may have been given. Other examples of limitations include that other substance use (illicit drugs) were not assessed, as found to be prevalent in other studies in Thailand . Future Rates of non-seatbelt use by Thai drivers and passengers during Songkran festival was 28.4%. Lower exposure to the RSA campaign was found to be associated with non-seatbelt use among drivers during the Songkran festival.The authors declare that they have no competing interests.PS, KP and SP were the main contributors to the conceptualization of the study. KP, PS and SP contributed significantly to the first draft of the paper and all authors contributed to the subsequent drafts and finalization. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/608/prepub


The aim of this study was to assess helmet use and associated factors among motorcycle riders during Songkran festival in Thailand. A cross-sectional survey was conducted to determine the prevalence of helmet use among Thai motorcycle riders  during four days of the Songkran festival. For this sample, the population of motorcycle riders was consecutively selected using quota sampling from 12 petrol stations in four provinces from each of the four main geographical regions of Thailand. The study was conducted at petrol stations at roads in town, outside town and highway at different time intervals when trained field staff administered a structured questionnaire and performed an observation checklist. Results indicate that 44.2% of the motorcycle riders and 72.5% of the motorcycle passengers had not been using a helmet. In multivariable analysis demographics, environmental factors, helmet use experiences and attitudes and recalling a lower exposure to road safety awareness (RSA) campaign were associated with non-helmet use among motorcyclists. It appears that the RSA campaign may have some positive effect on reducing non-helmet use among motorcycle riders during the Songkran festival. A nat belts ,7. Youthat belts . Survey at belts . Other sat belts , India 6at belts , Iran 89at belts , Nigeriaat belts , Vietnamat belts  and geneat belts ). Based at belts ,16, gendat belts ,15), beiat belts , locatioat belts ), time oat belts ,18, weekat belts  and haviat belts .et al. [According to the Road Traffic Act 1979, section 122, motorcyclist and passenger are obliged to wear a helmet to protect themselves from harm during driving . In a stet al.  found thet al. .From 1997 an active public education programme was undertaken on a national scale to raise awareness about road safety and to support law enforcement. This included dissemination of knowledge through multiple channels, e.g., roadside posters, stickers on the back of vehicles, sporadic radio and TV programmes or spots, public announcements and press reports . After 2Songkran is the New Year celebration in Thailand, set by the solar calendar since ancient times. It takes place between 13 and 15 April. Songkran festivals are major holidays that encourage a million travellers who travel to/from their hometown and doing the activities during these holiday periods . UnfortuA cross-sectional survey was conducted to determine the prevalence of helmet use among Thai motorcycle riders. The recruitment period of this project was during four days of the Songkran festival for 13–16 April, 2007. For this sample the population of motorcycle riders from 12 petrol stations was selected using quota sampling from four provinces from each of the four main geographical regions of Thailand excluding Bangkok. Provinces were Chiang Mai, Lampang, Nakhon Sawan and Phichit in the northern region, Nakhon Ratchasima, Khon Kaen, Udon Thani, and Loei in the northeastern region, Songkhla, Phuket, Surat Thani, and Trang in the southern region, and Phra Nakhon Si Ayutthaya, Chonburi, Chachoengsao, and Phetchaburi in the central region. In total 48 petrol stations (three petrol stations per province) were selected. In town, the petrol station on the road with the largest shopping mall was selected; out of town the petrol station on the road leading to the largest district was selected; in terms of petrol station on the highway, each province only has one highway. If there was more than one petrol station on the selected road or highway, the largest petrol station was selected. The study team spent four days at each petrol station road venue  for 7:00–9:00, 13:00–15:00, 17:00–19:00, and 22:00–24:00. All consecutive motorcycle riders who entered the petrol station were asked to participate by trained personnel (who were students from Chiang Mai University that were trained by the research team) while they were having their gas tank filled. The number of vehicles and time interval for vehicle selection were determined by the availability of field staff to conduct a motorcycle rider observation, interview and alcohol test. The target sample size was 100 motorcycle riders from each of the petrol stations per time period, except during 22:00–24:00 for which 50 motorcycle riders were targeted. Trained field staff administered a structured questionnaire and performed an observation checklist. The project was approved by the Ethics Committee for research in human subjects of the public health programme, Chiang Mai University.The primary outcome of the study was helmet use. Helmet use was assessed by observation. The questionnaire covered demographic data, motorcycle characteristics, history of road traffic accidents, known risk factors such as age, sex, environmental factors, helmet use experiences and attitudes, and exposure to road safety awareness (RSA) campaign.P < 0.05 level. For the model, the R2 is presented to describe the amount of variance explained by the multivariable model. Probability below 0.05 was regarded as statistically significant.Data were analyzed using Statistical Package for the Social Sciences (SPSS) for Windows software application programme version 19.0. Frequencies, means, and standard deviations were calculated to describe the sample. Data were checked for normality distribution and outliers. Interaction between predictor variables was also examined and it was found that none of the variables had a Variance Inflation Factor (VIF) value above 2.5. Associations of non-helmet use were identified using logistic regression analyses. A multivariable regression model was constructed. Independent variables  from the univariate analyses were entered into the multivariable model if significant at The sample included 18,998 motorcycle riders  of whom 320 refused to participate, giving a response rate of 98.3%. Overall, 44.2% of the motorcycle riders had not been using a helmet. Almost half of the motorcycle riders (49.6%) had a passenger  of which 72.5% had not been wearing a helmet. The largest group of the motorcycle riders were between 18 to 25 years old (43.9%), followed by 26 to 59 years olds (41.8%), 2.2% were below the legal motorcycle riding age (<15 years), and among those who were 15 to 17 years old 46.1% were illegally riding a size of motorcycle with more 110 cc. The data collection was equally distributed across four regions, four days of data collection; three locations of data collection and data collection across three different times during the day, only data collection in the evening yielded a smaller sample see . About one third of the motorcycle riders (33.4%) indicated that they had been in an accident before, mostly as a rider (75.5%), followed by passenger (22.8%) and pedestrian (1.5%). Almost half had had the intention to use a helmet (44.8%). A large group agreed with the danger of non-helmet use (75.0%) and almost two-thirds were highly aware of the danger of not using a helmet (55.8%). Almost half (47.2%) had been caught with the non-use of a helmet before. The majority (83.9%) had heard about an RSA campaign, 29.2% had frequently heard or seen the RSA campaign on the radio and/or TV, and 29.3% frequently followed TV news reports on road traffic injury (RTI) statistics. Two in five (40.1%) had also been talking to others about the RSA campaign and most (89.0%) liked the RSA campaign see .In multivariable analysis, it was found that the highest proportion of non-helmet use among motorcyclists was in the age group 15 to 17 years old and in riders from the northern region in Thailand. Motorcyclists who were having a passenger were significantly more often not using helmet than those who had no passenger see .Non-helmet use was found to be more frequent earlier during the Songkran festival, later during the day, on roads out of town and on highways. Respondents who had been in an accident before, had low awareness of the danger of non-helmet use and having been caught for non-helmet use were more likely to not wear a helmet. Motorcyclists who recalled a lower exposure to road safety awareness campaign  were more likely not to wear a helmet compared to those who recalled a higher exposure to road safety awareness campaign. et al. [This study among a large sample of motorcyclists in Thailand found that 44.2% of the motorcycle riders and 72.5% of motorcycle passengers had not been wearing a helmet, which is similar to a survey of 2007 in Thailand, with 46% and 69.1% non-helmet use among motorcyclists and passengers, respectively ,10,11, aet al.  who founet al. [In contrast to other studies, this study did not find any association between gender ,11,15 anet al.  found frCaution should be taken when interpreting the results of this study because of certain limitations. Since the sampling procedure was not truly random, this may be a limitation of the study. As this is a cross-sectional study, causality between the compared variables cannot be concluded. A further limitation is that some variables were assessed by self-report, and desirable responses may have been given. Other examples of limitations include that substance use  ,37,38, uRates of non-helmet use by Thai motorcycle riders and passengers during Songkran festival seemed to be high. It appears that the road safety awareness campaign may have a slight positive effect on reducing non-helmet use among motorcycle riders during the Songkran festival. The presented information concerning different peaks of unhelmeted motorcyclists will be helpful in devising specific countermeasures against such risky behaviour.


Supplemental Digital Content is available in the text Anorectal melanoma (AM) is a rare type of melanoma that accounts for 0.4% to 1.6% of total malignant melanomas. The incidence of AM increases over time, and it remains highly lethal, with a 5-year survival rate of 6% to 22%. Considering the rare nature of this disease, most studies on AM comprise isolated case reports and single-center trials, which could not provide comprehensive assessment of the disease. Therefore, we conducted a population-based study by using the Surveillance, Epidemiology, and End Results (SEER) program to provide the latest and best available evidence of AM.We extracted all cases of AM registered in the SEER database from 1973 to 2011 (April 2014 release) and calculated age-adjusted incidence. Only cases with active follow-up were included to predict factors associated with prognosis. Survival outcomes were also compared among different types of surgery.We identified 640 AM cases, which consisted of 265 rectal melanoma and 375 anal melanoma. The estimated annual incidence rates of AM per 1 million population were 0.259 in males and 0.407 in females, and it increased with advanced age and over time. Tumor stage and surgical treatment were independent predictors of survival. Results implied that surgery improved the prognosis of patients with local- and regional-stage AM but could not prolong the survival of patients with distant-stage AM. Moreover, the outcome of less extensive excision was not statistically different from that of more extensive excision.This study provides an up-to-date estimation of the incidence and prognosis of AM by using SEER data. The incidence of AM continuously increases over time, despite its rarity. This disease also exhibits poor prognosis. Thus, AM must be further investigated in future studies. We also recommend surgery as the optimal treatment for local- and regional-stage AM patients but not for those with distant metastasis. Primary mucosal melanomas behave more aggressively and have poorer prognosis than cutaneous melanomas and are most common in the head and neck, anorectum, and female, with distribution of approximately 55%, 24%, and 18%, respectively.4 As the most frequent location of primary gastrointestinal tract melanoma, anorectal melanoma (AM) accounts for 0.4% to 1.6% of all malignant melanomas;6 the incidence rate of AM is about 2.7 cases per 10 million population per year in the United States.7 AM is likely to be unnoticed and diagnosed at an advanced stage because of its unspecific symptoms, such as bleeding, presence of a mass, and sensation of tenesmus, which are clinically consistent with benign hemorrhoid diseases.10 Only 20% to 30% of AM is located in the rectum, and the other melanomas are found within the anal canal or anal verge.12 Therapy for AM has not been standardized because of the low incidence of this disease and the lack of clinical experience. Generally, surgical excision is the primary treatment option for AM, but selection of either abdominoperineal resection or wide local excision remains controversial.13 Currently, AM remains a highly lethal disease, with a 5-year survival rate of 6% to 22%.13Melanoma is an aggressive, therapy-resistant malignancy of melanocytes. Melanoma is a major public health concern, and its incidence has continuously increased over the past 4 decades.14 The use of this large population-based database can avoid the limitations of small size, as well as selection or treatment bias. Moreover, the results can be readily generalized and are considered more valid than institutional data because patients were treated in all types of clinical settings.17 This study aims to provide the best available evidence to help clinicians have a better understanding of AM.Information on epidemiology and prognosis of AM, particularly rectal melanoma, is limited because of the rarity of this disease. Most studies in the literature include isolated case reports and single-center trials, which cannot accurately reflect the situation of AM. In this study, we provide insights into the epidemiology and survival outcomes of AM by using the Surveillance, Epidemiology, and End Results (SEER) Program. We also investigated surgical treatment for AM, particularly in terms of survival differences among different surgery types. SEER is an authoritative source of information on cancer incidence and survival in the United States; this program contains data collected from 18 cancer registries, which cover 28% of the US population.We got internet access to SEER database with the reference number 13504-Nov2013. And our study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine. This observational study did not publish any information on an individual patient. Therefore, informed patient consent was not required.18 The included patients satisfied the following criteria: anatomic sites of rectum or anus ; histologically diagosed as melanoma (Histologic type ICD-O-3 codes: 8720–8772); malignant behavior (Behavior code ICD-O-3 code: 3); and microscopic confirmation of diagnosis . In the second part of the study, cases without active follow-up  were excluded to predict factors associated with overall survival (OS) and cause-specific survival (CSS). Patients whose data were sourced solely from a death certificate (Type of Reporting Source code: 6) or those who had 2 or more primaries in their lifetime (Sequence number code: 1–99) were also excluded. The screening procedure is shown in Figure The SEER program is the largest publicly available cancer dataset and is updated annually. The program contains data on patient demographics, tumor characteristics, first course of treatment, and follow-up information. In this research, the SEER dataset from 1973 to 2011 (April 2014 release) was used for case extraction.In this study, surgery types were categorized into less extensive excision (LEE) and more extensive excision (MEE) to investigate differences in survival outcomes. LEE refers to tumor resection without dissection of lymph nodes, and MEE indicates tumor resection with lymph node removal. Specifically, in rectal melanoma, cases with RX Summ Surg Prim Site (1998+) codes of 10–28 or Site specific surgery (1983–1997) codes of 10–20 are identified as LEE; by contrast, cases with Summ–Surg Prim Site (1998+) codes of 30–70 or Site specific surgery (1983–1997) codes of 30–60 are categorized as MEE. Moreover, LEE (Summ–Surg Prim Site codes: 10–27 or Site specific surgery codes: 10–40) and MEE  in anal melanoma were extracted similarly.χ2 test or independent sample T test was employed to investigate significant differences between 2 groups. The incidence of AM in the United States was calculated as the number of new patients per 1 million people per year, with adjustment to the US 2000 population, and presented in terms of sex and tumor sites. Briefly, we extracted population data and calculated incidence rates by using the SEER∗Stat Version 8.2.0 software . We also analyzed the incidence trends using 9 age groups  and 4 observation periods .Pearson P value of <0.05 was considered statistically significant. All tests were 2 sided, and confidence intervals (CIs) were set as 95%.Univariate and multivariate models were constructed to evaluate factors correlated with survival. Survival was defined as the number of months between the date of diagnosis and the date of death of any causes (OS) or of their cancer (CSS). In the univariate model, Kaplan–Meier curves were plotted to display survival rates over time; the curves were then compared using log-rank statistics. Age at diagnosis, sex, race, stage, surgery, tumor location, and year of diagnosis were included as covariates. The multivariate Cox proportional hazards model was then fitted to estimate hazard ratios (HRs) between survival and covariates, which included variates with statistical significance in the univariate model. Cox regression method was further applied to control the influence of covariates and compare survival rates between the 2 groups. All statistical analyses were conducted using SPSS version 19.0 software . A P = 0.662) and sex (P = 0.476) were not statistically different between the 2 groups. About 83.9% of the included cases were White, and more Asian patients were observed in the anal group (P = 0.027).A total of 640 patients were assessed as eligible for inclusion in the study by using the patient selection algorithm described in the Methods section Figure ; these pP < 0.001) and less distant ones (P = 0.018). In addition, surgery was administered in 84.1% of the total patients. The number of patients with AM almost doubled from 1973–2000 (N = 221) to 2001–2011 (N = 419). Radiation and lymph node were excluded from the analysis because of the absence of corresponding data in 80.8% and 70.6% of the cases, respectively.In terms of stage, 232 (36.3%) patients were diagnosed at the localized stage, followed by the regional  and distant  stages. Compared with rectal melanoma, patients in the anal group were diagnosed at earlier stage, with more cases at the regional stage . The incidence rate increased with advanced age in both sexes and at all tumor sites. Specifically, no patient with AM younger than 20 was reported, and the overall incidence rate increased from 0.013 (20–29 years) to 2.818 (≥85 years), followed by 0.000 to 2.397 and 0.026 to 3.000 for male and female, respectively . Therefore, these 485 patients were used in log-rank and Cox proportional hazard analyses to determine potential factors that affect the prognosis of AM.About 75.8% of the cases were eligible for survival analysis. Patient demographics and therapies were not different between the total patients (N = 640) and included cases , and patients undergoing surgical resection exhibited improved survival (P < 0.001). Moreover, tumor location and year of diagnosis were related to survival.As shown in Table P = 0.023) and 0.69 (P = 0.048) in OS and CSS models, respectively . In the CSS model, regional stage could be a risk factor for survival .Multivariable analysis was then performed, and the results implied that patients with AM who underwent surgery showed improved prognosis, with HR = 0.66 P = 0.02 and 0.69P < 0.001 and P = 0.03, respectively). Conversely, survival benefits were not statistically different among patients with distant metastasis  of rectal melanoma than previous studies. Moreover, the incidence of rectal melanoma was higher than that of anal melanoma between 2006 and 2011.19 A total of 27 states and 1 metropolitan area participated in this study, and data covered more population than those of the SEER program. However, this program had no updated evaluation of the incidence of AM in the past decade. In Sweden, a population-based study covered about 95% cancer patients and covered 253 cases during a 40-year period (1960–1999).20 The reported incidence rates were 1.0 and 0.7 per million for female and male, respectively, and were significantly higher than those reported in the United States. Investigators in Australia also reported that the incidence of AM is 0.28 per million in 1985 to 1995, similar to the present results in the same period.21The estimated annual incidence of AM was 0.343 per 1 million, as well as 0.259 and 0.407 for male and female, respectively. The incidence increased with age and over time. Specifically, the incidence peaked in patients over 85-year old and in the period between 2006 and 2011, with incidence rates of 2.818 and 0.460, respectively. This finding is in agreement with the incidence data obtained from the North American Association of Central Cancer Registries; in this report, the incidence rate is 0.4 per million between 1996 and 2000 (based on 299 cases) and was age adjusted to the 2000 U.S. population standard.22 However, a simplified clinical staging system was used to categorize vaginal and anorectal melanomas because of their rarity. Specifically, AM was classified based on disease distribution as stage I , stage II , or stage III (distant metastasis).23 This finding is consistent with the SEER summary staging system adopted in the present study.We conducted univariate and multivariate analyses to predict the prognosis of AM. As predicted, stage was an independent factor of OS and CSS. Patients with local-stage AM showed improved survival than those with regional- and distant-stage AM. For the staging system of mucosal melanoma, the American Joint Committee on Cancer Tumor, Node, and Metastasis classification is used to stage mucosal melanoma of the head, neck, and vulva.26 In 2010, Iddings et al9 analyzed 145 patients from the SEER database between 1973 and 2003 and concluded that the type of surgery did not affect OS or CSS. These findings are consistent with our results based on latest information and larger population. Therefore, if technically possible, we advise patients with AM to receive LEE to avoid unnecessary injuries and improve their quality of life.Surgical resection is the standard of care for AM, and patients undergoing surgery showed improved prognosis. In this research, patients with distant metastasis could not obtain significant survival benefits from the surgery. Hence, surgical resection may not be the optimal choice for distant-stage patients. With regard to the type of surgery, MEE with dissection of lymph nodes can control lymphatic spread and result in less local relapse; however, this technique confers long hospital stay, slow recovery, and a need for permanent stoma. In addition, a few studies reported that patients failed to achieve any survival benefits by using such an aggressive surgical approach.28 Second, as patients were enrolled in SEER program from 1973 to 2011, the coding and staging system evolve and differ significantly over past 4 decades, which requires a thorough understanding of variables. Apparently, during such a long period, improvements in imaging and pathology may contribute to increased incidence rates, and improved chemotherapy and surgical techniques affect prognosis as well.30 Third, sample sizes were small in subgroup analyses, which may contribute to false positives or negatives. This limitation is inevitable for studies on such a rare tumor. Therefore, if possible, a prospective study with a large sample size must be performed in the future to validate the present results.However, this article still presents several limitations. First, as an SEER-based observational study, we were unable to get data regarding adjuvant therapy and detailed course of treatment, which are strongly associated with prognosis. Moreover, lack of information on comorbidity, recurrence, and treatment-related complications; migration of patients in and out of the SEER registry; and selection bias are factors that should be considered.This population-based study provides an up-to-date estimation of the incidence and prognosis of AM by using the SEER data. The incidence of AM increased with age and over time. Tumor stage and surgery may be independent risk predictors, and patients with distant-stage AM could not obtain survival benefits from surgical treatment. Moreover, prognosis was not statistically different between LEE and MEE.