This paper presents a simple method to increase the sensitivity of protein family comparisons by incorporating secondary structure (SS) information. We build upon the effective information theory approach towards profile-profile comparison described in [Yona & Levitt 2002]. Our method augments profile columns using PSIPRED secondary structure predictions and assesses statistical similarity using information theoretical principles.Our tests show that this tool detects more similarities between protein families of distant homology than the previous primary sequence-based method. A very significant improvement in performance is observed when the real secondary structure is used.Integration of primary and secondary structure information can substantially improve detection of relationships between remotely related protein families. Detecting an evolutionary relationship between proteins is the basis for functional inference. Existing methods most often rely on sequence information in an attempt to quantify the evolutionary divergence or similarity between the sequences compared. A significant similarity would suggest that the proteins are related. However, in many cases sequences have diverged to the extent that their similarity is undetectable by standard sequence comparison algorithms. Nevertheless, they may still have similar structures and functions ,2.It has long been postulated that evolutionary pressure acts upon the three-dimensional structure of proteins and intra-protein interactions rather than at the level of the primary sequence ,4. IndeeThere have been many attempts to build algorithms that predict protein structure from amino acid sequence. Unfortunately, this is a hard problem, and existing methods are only partially successful . On the Alternatively, one can use both representations to assess protein similarity. Incorporating secondary structure information into protein comparison is not a new idea. Several researchers have attempted to boost performance and sensitivity of various methods by adding this extra degree of information with some success. Yu et al. encoded functionally conserved sequence patterns into probabilistic structural models (that comprise a family of hidden Markov models) . The modThese and similar studies have indicated that the incorporation of secondary structure information, even if predicted, can increase sensitivity and specificity of a protein comparison model. Here we describe a method that integrates secondary structure information with primary sequence information in a single scoring scheme, using a single statistical representation. The model can be applied to any protein family and does not require the application of expensive threading algorithms. Our method extends our previous work on profile-profile comparison . SpecifiWe use a data set of domain families derived from the SCOP classification of protein structures , releasen-tuple of probability distributions of amino acids, derived from a group of related proteins, where n is the length of the multiple alignment of these proteins. It is represented in software as a two dimensional matrix of 20 rows and n columns, where each column (known as a profile column), is a probability distribution p over the 20 amino acids in one position in the multiple alignment. These profile columns form the basis of profile-profile comparisons.The PSI-BLAST profiles are the basis for our representation of a protein family. Each profile is a true secondary structure information is gleaned from the PDB files of the seed proteins using STRIDE  is the Kullback-Leibler (KL) divergence γ described in section 'Integrating secondary structure with primary structure'. The significance measure reflects the probability that the source distribution, r, could have been obtained by chance. The higher r is, the more distinctive the common source distribution, and the lower the probability that it could have been obtained by chance.In this study the background distribution is composed of two components: the background distribution of amino acids (estimated from a large sequence database) and the background distribution of secondary structure elements . The components are mixed using the same mixing parameter similarity score of two probability distributions p and q as a combination of the divergence score and the significance score:We define the D → 0) whose common source is far from the background distribution (S → 1), tends to one. On the other hand, the similarity score of two dissimilar distributions (D → 1) whose most likely common source distribution resembles the background distribution (S → 0) tends to zero. This scoring scheme also distinguishes two distributions that each are similar to the background distribution (D → 0 and S → 0 giving Score - 1/2) from two dissimilar distributions, but whose common source is similar to the background distribution (D → 1 and S → 0 giving Score = 0). In a recent study [With this expression, the similarity score of two similar distributions (nt study  it has bp over a set X that is obtained by "mixing" two probability distributions over two disjoint subsets: p1 over the subset X1 and p2 over the subset X2 (where X = X1 ∪ X2 and X1 ∩ X2 = θ). Let γ be the mixing parameter, i.e. the total weight of the first distribution p1 in the combined distribution p. Assume q is obtained in a similar manner from q1 and q2. Then,Note that our measures are functionals of the probability distributions, based on variations of the entropy function, and specifically the KL divergence function. One of the nice properties of this function is that it is additive in the following sense. Assume we have a probability distribution X1 (the secondary structure) and X2 (the primary structure).In other words, this measure preserves independence between the two subsets. Therefore, with our extended profile representation, the new functionals are simply a weighted sum of the individual functionals over the subsets γ and (1 - γ) respectively.Note however that this property holds for the divergence and the significance measures but not for the final similarity score that is a combination of the divergence and the significance scores. An alternative would be to compute the divergence, significance and similarity scores independently for the secondary and primary structures, and then combine the two similarity scores into one, with weights i of the seed sequence (see section 'Data sets') as the seed amino acid of the i-th profile column.It is interesting to compare the similarity scores before and after the addition of secondary structure information. To assess the impact of this information, we computed the distribution of similarity scores for five types of profile columns, depending on the type of their seed amino acid. We refer to the amino acid at position Two seed amino acids are defined as similar, neutral, or dissimilar based on their BLOSUM62 scoring matrix , with poidentical columns), (2) different columns that are associated with the same seed amino acid (strongly similar columns) (3) different columns that are associated with similar seed amino acids (similar columns), (4) different columns with mutually neutral seed amino acids , and (5) different columns with dissimilar seed amino acids (dissimilar columns). We repeated this calculation before and after the integration of true secondary structure information  and the results are plotted in Figure The five types of column pairs are: 1) a column with itself .ALLR (Average Log Likelihood Ratio) scoring function that was suggested in [p and q, their ALLR score is defined asWe also tested the ested in . This scnp (nq) is the number of total counts from which p (q) is derived, and P0 is the background distribution.where We computed the correlation scores and ALLR scores for the same sets of columns defined in the previous section and compared it to the information-theoretic scores Figure . Note thprof_ss) depends on several parameters: (1) a shift parameter is introduced to convert the similarity scores to scores that are suitable for local protein comparison . The tradeoff between the primary sequence information and secondary structure probabilities was varied from zero to one. With zero dependence on secondary structure the method is equivalent to prof_sim (profile-profile comparison based on just primary structure). The results are shown in Figure γ = 0.055 (i.e. 0.055 weight on the secondary structure information and 0.945 on the sequence information) gave the best performance. (Note that if each secondary structure was given as much weight as a single amino acid γ would be  or ~0.13).To estimate the best value for γ = 1), the performance was much worse than when only sequence information was used (γ = 0). These corner-case results and the fact that the best results were obtained with γ << 0.5 suggest that for protein family comparison, the coarse-grained secondary structure information is noisier and less reliable than sequence information. However, as the graphs indicate, using both sources of information clearly improves performance. Our tests were done using actual secondary structure information in the profile; however, similar results were obtained when the predicted information was used for one or both of the profiles  Marquardt-Levenberg algorithm.To differentiate true similarity values from those that may be observed by chance, it is essential to establish a baseline empirical distribution for the scores. Here we used the statistical framework of the extreme value distribution (EVD). Although rigorous mathematical proof has not been found for local gapped similarity scores, empirical studies have shown that the distribution of these scores can be approximated by this distribution. An empirically fit EVD also has the benefits of being a true fit to the quirks of a particular protein family. Three such distributions were established to assess the significance of the profile-profile matches. All distributions were fit with the 'fit' function in gnuplot uniform approach (uniform parameters).The first distribution is based upon comparisons between unrelated families . This distribution is useful in that it can be used to assess the significance of a score in comparing any pair of protein families, without further need for computations. Practically, this aggregates all comparisons between non-related families into a single list. This is essentially the distribution of similarity scores of random profiles, as shown in Figure The second distribution is similar to the first, except a correction was made for the length of a profile, similar to the approach employed by FASTA . By chanThe third distribution proves to be the best approach in assessing significance of matches with a particular profile. This distribution is created on a per-family basis. The scores of each family against all (unrelated) SCOP families were fit to an EVD. Since many of the family profiles are unrelated to the query family, the corresponding scores provide a relatively reliable baseline distribution. This approach is a robust method to assessing the significance of matches for a particular profile since it allows for any unusual properties of the query profile  and the parameters are adjusted accordingly  that can be detected with our method. Specifically, each family in our test set is compared with all other protein families and the results are sorted based on the p-value. Given the sorted list we count the number of true family-family relationships that are detected before the orted in ).true relationship if both families belong to the same superfamily, a possible relationship if both families belong to the same SCOP fold, a weak relationship if they belong to the same class, suspicious if they belong to different classes  and an error if one family is all-alpha and the second is all-beta. We repeat the procedure described above, each time using a different definition of a false positive. The results are summarized in Table Usually a false positive is defined as a relation between families that do not belong to the same superfamily. This popular criterion, however, is somewhat strict as relations between families that belong to the same fold can also be considered as positives. We use the following terminology to distinguish between the different types of "false positives". We define a relationship between two protein families to be a prof_ss improves over prof_sim  although the improvement is smaller compared to the one reported in Table γ was set to 0.055), the new algorithm almost doubles the number of pairwise relationships that are detected.The second measure we use is the receiver operator characteristic (ROC) measure, a common measure in assessing sensitivity and selectivity. Given a sorted list of results, the ROC index measures the area under the curve that plots the positives versus the negatives. Maximal performance translates to a perfect separation and a maximal normalized ROC score of 1. The ROC-N measure is a variation over the ROC measure, where the plot is truncated at N negatives. In other words, the ROC-N measure is the number of true positives detected up to N false positives. Here we used the popular ROC-50 measure. To obtain the ROC-50 scores for each method we pool together all pairwise comparisons for all protein families, and sort them by their normalized e-value. The number of true positives is aggregated until 50 false positives occur. As before, we repeated this procedure with different definitions of false positives, and the results are summarized in Table In this section we give several interesting examples of alignments between remote protein families that exemplify the differences between sequence-based profile-profile alignments and the new generalized profile alignments.prof_sim is able to roughly match up the helices but not the beta strands with a rms of 11.96. The predicted secondary structure does not improve the alignment in this case, however, when the true secondary structure is used, prof_ss is able to completely align the helices as well as most of the strands with a much better rms of 4.45 , and the replication terminator protein . Although designated as all-alpha, proteins in this superfamily contain a small beta-sheet at the core. The similar substructures have three alpha helices and a couple beta strands, 5 Figure .This superfamily belongs to the concanavalin A-like lectins/glucanases fold, characterized by a sandwich structure with 12–14 strands in 2 sheets. We compared two families in this superfamily: the beta-Glucanase-like family  and the vibrio cholerae sialidase, N-terminal and insertion domains .prof_ss both when using the predicted information and the true secondary structure information. On the other hand, prof_sim is unable to align the sheets at all  and the bromoperoxidase A2 family . These are large and complex proteins with many helices and strands. This paper presents a simple method to improve remote homology detection between protein families. We use statistical models of protein families in the form of profiles, and by incorporating secondary structure information within that model, we can reuse existing comparison methods for comparing profiles. It is shown that this method improves over the previous method that is based only on primary sequence information.As opposed to other methods that compare single proteins, our method compares models of protein families. Instead of summing over different models, our model combines structural and primary sequence information within the profile itself. Our method allows us to explore a wide range of scenarios, between purely sequence-based representation and a purely secondary-structure based representation. The optimization of the single mixing parameter shows that the slight incorporation of predicted secondary structural information is invaluable. Since predicted structure information in PSIPRED comes from neighboring profile columns, this proves that each profile column confers extra information that is relevant to its neighbors and is useful to inferring protein relationships.Furthermore, it is shown that if true secondary structure information is used, performance improvements are very significant and the number of relationships that can be detected is almost doubled. We conclude that despite the high overall accuracy of the secondary structure prediction method, its imperfect nature can greatly affect the performance. However, our method can be generalized to any secondary structure prediction method that produces estimated probabilities for secondary structure, so should a new prediction method be found that performs better than the current methods, the model presented here is expected to reflect the improved performance and consequently improve homology detection.prof_sim program and integrated secondary structure information, optimized the model, ran experiments, and analyzed the result sets. GY conceived of the study, designed the model and analyzed the results.RC extended the


However, knockout in the mouse of cftr-/- mouse. Lungs showed decreased compliance and increased airway resistance in young animals as compared to cftr+/+ littermates. These changes were noted in animals less than 60 days old, prior to any long term inflammatory effects that might occur, and are consistent with structural differences in the cftr-/- lungs. Surprisingly, the cftr+/- animals exhibited a lung phenotype distinct from either the homozygous normal or knockout genotypes. The heterozygous mice showed increased lung compliance and decreased airway resistance when compared to either homozygous phenotype, suggesting a heterozygous advantage that might explain the high frequency of this mutation in certain populations.Using measurements of pulmonary mechanics, a definitive lung phenotype was demonstrated in the cftr results in distinct differences in pulmonary mechanics of the adult. Distinct phenotypes were demonstrated in each genotype, cftr-/-, cftr +/-, and cftr+/+. These results are consistent with a developmental role for CFTR in the lung.In the mouse the gene dosage of  CF is one of the most common autosomal recessive diseases in Caucasians with a carrier rate of 3–4% [1), functional residual capacity (FRC), and other parameters of lung function prior to the onset of recurrent infection [Cystic fibrosis (CF) is a progressive disease primarily affecting the intestines, lungs, and pancreas. The gene responsible for CF was identified in 1989 [ of 3–4% , and is nfection .cftr knockout mouse does not develop overt lung disease, which has severely limited its usefulness. However, the availability of new methods for pulmonary testing in rodents [cftr knockout mouse for functional lung changes. In the present study, therefore, we examined pulmonary function in young adult cftr -/-, cftr +/-, and cftr+/+ S489x mice in an effort to establish a lung phenotype.Soon after the CF gene was discovered, a knockout mouse was developed. This mouse demonstrates subtle changes in epithelial cell phenotype, including alterations in secretory glycoconjugates and changes in secretory vesicles . Monocyt rodents ,9 now prPV) curve which is dependent upon both lung structure and interfering pathology. PV curves were measured in triplicate, starting from positive end-expiratory pressure (PEEP) values of 0, 3, and 6 cmH2O in S489X mice at 30–60 days of age following genotyping for the normal and mutant cftr alleles. The 3 cmH2O PEEP curves obtained for each genotype are presented in Figure PV curves all begin at V = 0 ml, which is the FRC defined by the 3 cmH2O PEEP. The PV curves obtained at PEEP levels of 0 and 6 cmH2O were similar.Routine evaluation of dynamic lung function employs the stepwise variation in air volume on both the inflation and deflation phases of a single breath. Measurement of airway pressures at each step results in the classic pressure-volume (Cst) of the lungs, which reflects elastic recoil at a given pressure, was calculated from the slopes of the PV curves. As shown in Figure Cst was significantly reduced in both cftr+/+ (p < 0.001) and cftr-/- (p < 0.001) as compared to age-matched cftr+/- mice. Hysteresis was altered among the 3 genotypes  and cftr+/+ mice (p < 0.05) compared to cftr-/- mice. These data suggest the presence of a gene dosage effect in which an altered lung structure in the heterozygous animals leads to an elevated compliance relative to the two homozygous animals. Lung weights were measured and no statistically significant differences were observed among the three genotypes  and determined values for airway resistance (Raw), tissue damping (G), and tissue elastance (H). Figure Raw, G, and H were significantly reduced in the cftr+/- mice as compared to both cftr+/+ and cftr-/- animals  at all PEEP levels. Raw also decreased with PEEP in a similar fashion in all three genotypes. In contrast, Raw, H and G were significantly increased in the cftr-/- mice compared with cftr+/+ and cftr+/-, and showed a greater dependence on PEEP . The ratio G/H, termed hysteresivity (both the low and high frequency), was not significantly affected by either genotype or PEEP (Panel E). Thus, the absence of either 1 or 2 copies of the cftr gene had significantly different effects on the phenotype of the lung. Paradoxically, the absence of only one cftr copy resulted in a greater lung compliance (lower elastance) than if neither or both copies were present.We applied the forced oscillation method to the mice and determined respiratory mechanical input impedance ,11. We fcftr knockout mouse as a model of cystic fibrosis has been severely limited by its failure to demonstrate readily measurable lung disease, the primary cause of morbidity and mortality in humans [cftr in the lung of young adult animals resulted in decreased Cst and η and increased Raw, G and H as compared to normal littermate controls.The usefulness of the n humans . HoweverCst and hysteresis in cftr+/- mice was significantly higher than in cftr+/+ animals while G and H were decreased. As this was not associated with any pathology such as emphysema, we conclude that it represents a functionally different lung from that of the cftr+/+. Our data thus reveal a remarkable inverse correlation between the effect of one and two non-functional copies of the cftr gene.A particularly intriguing further observation was that η[cftr is a chloride channel and is thought to be involved in water balance, a change in surface tension in the lung, and consequently in η, might be expected. However, as shown in Figure G and H both increase, they do so in the same proportion so there is no significant change in η between the three cftr genotypes. On the other hand, lung weights were not different among the different groups of mice, so the decreased compliance and increased resistance of the cftr-/- animals was not simply due to their having smaller lungs than control animals. This suggests that the parenchymal structure in the lungs of the homozygous and heterozygous animals were organized differently, in a manner that affected G and H similarly.What do these data mean in terms of lung structure? The knockout animal has cystic fibrosis by definition, and our data now show it to also have lung disease manifest as a reduced compliance and increased resistance. These changes could reflect changes in the intrinsic mechanical properties of the parenchyma, or simply a reduction in lung volume. The former effect could include alterations in the biophysical properties of the air-liquid interface in the lungs, and would be expected to result in a change in η. Indeed,cftr levels are highest in the developing lung and decrease 75-fold at birth. In utero over-expression of cftr has also been shown to affect lung growth and development[cftr may affect the early development of the lung in a manner that is affected by the interaction of other genes.As documented in numerous publications, the mouse strain used in the present study does not develop chronic inflammatory disease up to the age (30–60 days) used in this study (for review see ). On thevelopment, and thevelopment. These dcftr animals? Interestingly, there is no decrement in lung function in human heterozygotes [cftr, something we term a "Goldilocks Effect". That is, while two defective copies of the gene are detrimental and two normal copies are satisfactory, one normal and one defective gene may results in an optimal dosage for lung development.Are there any functional consequences for increased lung compliance in the heterozygous ozygotes -21. Alsoozygotes ,23. Indeozygotes . When onozygotes  is perhaozygotes . The rescftr knockout mice should reveal additional genetic loci that modulate the influence of cftr on lung growth and development. Corresponding studies in humans should be useful in evaluating the effect of therapies on reversing altered pulmonary function in the CF patient.Further studies of pulmonary mechanics in cftr knockout mouse. In addition, the cftr+/- mouse had a distinguishable pulmonary function phenotype from that observed in either the homozygous normal or mutant genotype mice. These data are consistent with CFTR-dependent, physiologic changes in the structure and function of the lung.Using sophisticated techniques to a evaluate rodent pulmonary function; a distinct, readily quantifiable lung phenotype was identified in the th generation backcross to C57Bl/6 has been maintained by random mating for the past 8 years. This colony has a 100% mortality rate among cftr knockouts by 45 days of age unless the animals are placed on an elemental liquid diet and corncob bedding upon weaning [cftr alleles. Age and litter matched cftr+/+ and cftr+/- were used for each cftr-/- mouse examined. Six animals were included in each group. All experiments were approved by the animal care and use committee.The S489X mouse 5 weaning . Mice 30flexiVent, SCIREQ Inc. Montreal, PQ, Canada) and ventilated with a tidal volume of 10 ml/kg; inspiratory:expiratory ratio of 66.67%, respiratory rate of 150 breaths/minute, and maximum pressure of 30 cmH20. PEEP was controlled by submerging the expiratory limb from the ventilator into a water trap. Each animal was paralyzed with pancuronium bromide (0.5 mg/kg) and allowed to equilibrate on the ventilator until spontaneous breathing ceased (5 minutes).The mice were anesthetized with intra-peritoneal pentobarbital (90 mg/kg) and the trachea was dissected free of surrounding tissue and cannulated with a 20-gauge cannula. The animals were then connected to a small animal ventilator , i is the imaginary unit, α links G and H, and f is frequency. We also calculated a quantity known as hysteresivity (η = G/H), which is believed to increase when regional heterogeneities develop in the lung [Raw, G and H were all normalized by multiplication by lung weight.where g airways, Iaw is the lung . Raw, G FRC defined by the PEEP, the flexiVent was programmed to deliver seven inspiratory volume steps for a total volume of 0.8 ml followed by seven expiratory steps, pausing at each step for 1 s. Plateau pressure (P) at each step was recorded and related to the total volume (V) delivered to produce a quasi-static PV curve. Cst was calculated from the slope of each curve [Starting at the ch curve , and wasZrs measurements at each PEEP level and PV curves were obtained in triplicate. Data were statistically evaluated using paired t-test with p < 0.05 being taken as significant


Caenorhabditis elegans retroviruses, are class II viral fusion proteins. Comparisons of divergent viral fusion proteins can reveal features essential for virion:cell fusion, and suggest drug and vaccine strategies.The Bunyaviridae family of enveloped RNA viruses includes five genuses, orthobunyaviruses, hantaviruses, phleboviruses, nairoviruses and tospoviruses. It has not been determined which Bunyavirus protein mediates virion:cell membrane fusion. Class II viral fusion proteins (beta-penetrenes), encoded by members of the Alphaviridae and Flaviviridae, are comprised of three antiparallel beta sheet domains with an internal fusion peptide located at the end of domain II. Proteomics computational analyses indicate that the carboxyl terminal glycoprotein (Gc) encoded by Sandfly fever virus (SAN), a phlebovirus, has a significant amino acid sequence similarity with envelope protein 1 (E1), the class II fusion protein of Sindbis virus (SIN), an Alphavirus. Similar sequences and common structural/functional motifs, including domains with a high propensity to interface with bilayer membranes, are located collinearly in SAN Gc and SIN E1. Gc encoded by members of each Bunyavirus genus share several sequence and structural motifs. These results suggest that Gc of Bunyaviridae, and similar proteins of Tenuiviruses and a group of  Two classes of viral envelope proteins that mediate virion:cell fusion have been described. Class I and II fusion proteins (aka α-and β-penetrenes) are distinguished, in part, by the location of the "fusion peptide," a cluster of hydrophobic and aromatic amino acids that appears critical for fusing viral and cell membranes. The fusion peptides of class I fusion proteins are located at or near the amino terminus, whereas fusion peptides of class II fusion proteins are internal. The overall structures of these two classes of viral fusions proteins are also distinct. Class I fusion proteins have a pair of extended α helices that are separated by sequences variable in length, but usually containing one or more dicysteine linkages. Several otherwise disparate viruses, including orthomyxoviruses, paramyxoviruses, retroviruses, arenaviruses, filoviruses and coronaviruses encode class I fusion proteins -4. ClassCaenorhabditis elegans retroviruses, are class II viral fusion proteins (β-penetrenes).The Bunyaviridae family of enveloped RNA viruses includes five disparate genuses. Orthobunyaviruses, phleboviruses, nairoviruses and tospoviruses are spread by insect vectors, whereas hantaviruses are spread by rodent vectors . MembersCaenorhabditis elegans, including Cer13 . We also compared Bunyavirus M sequences to structural proteins of Flaviviruses, including members of the flavivirus genus tick-borne encephalitis virus, strain Neudoerfl ; Japanese encephalitis virus, strain JaOARS982 , yellow fever virus, strain 17D-204 , dengue virus type 2, strain PR-159/S1 , and West Nile virus, strain NY 2000-crow3356 . The prototype hepaciviruses, strain H (subtype 1a) of hepatitis C virus , and several pestiviruses, including the Alfort 187 strain of classical swine fever virus, aka hog cholera virus , bovine viral diarrhea virus genotype 1 aka pestivirus type 1, stain NADL  and border disease virus, strain BD31 , were used in other comparisons.For sequence and structural comparisons of Bunyavirus M encoded proteins representatives of the five genuses were used, including pleboviruses Sandfly fever virus, Sicilian strain  and Rift Valley fever virus , orthobunyavirus Bunyamwera virus , hantavirus Hantaan virus, strain 76–118 , nairovirus Crimean-Congo hemorrhagic fever virus, strain IbAr , and tospovirus tomato spotted wilt virus, ordinary strain . Additional phlebovirus M sequences compared included those of Uukuniemi virus  and Punta Toro virus . Bunyavirus M sequences were compared to sequences encoded in Alphavirus subgenomic RNA, including structural proteins of Sindbis virus , Semliki Forest virus , Venezuelian equine encephalitis virus, strain TC-83 , Western equine encephalitis virus, strain McMillan , O'nyong-nyong virus, strain GULU , Mayaro virus, strain TRVL4675 , Barmah Forest virus strain BH2193  and Ross River virus, strain NB5092 . Comparisons were also made with proteins encoded by RNA2 of the HZ isolate of Rice stripe virus, a Tenuivirus , and with certain retroviruses of ) was the preferred method of secondary structure prediction [). TMpred is based on a statistical analysis of TMbase, a database of naturally occurring transmembrane glycoproteins [Xplorer version 2.2a from the Stephen White laboratory using default settings [ was used to predict mucin type GalNAc O-glycosylation sites. RasMac , developed by Roger Sayle, was used to render a 3D model of SFV E1, which was extrapolated to SIN E1 and SAN GC.Methods to derive general models of surface glycoproteins have been described previously . PRSS3, ediction . PHDsec proteins . Sequencsettings . The NetSimilar sequences or common structural/functional motifs are located collinearly in the carboxyl terminal glycoprotein of Sandfly fever virus and Sindbis virus envelope glycoprotein E1.Previously, Gallaher and coworkers modeled the structure of the retroviral transmembrane glycoprotein (TM)  onto thePrior X-ray crystallographic studies have demonstrated that SFV E1 is a class II viral fusion protein (β-penetrene) . BecauseTo provide support for the proposed alignment of SAN Gc and SIN E1, another proteomics computational tool was used to compare potential membrane interactive domains in the glycoproteins. Besides fusion peptides, a motif that can be important in virus:cell fusion and is present in many class I and class II viral fusion proteins is an aromatic aa rich domain proximal to the transmembrane anchor . The preRecently, Gibbons and coworkers determined the structure of a fragment of the SFV E1 ectodomain  after exposure to low pH and liposomes . Under tThere are many possible alternatives to the cysteine linkages and secondary structures of SAN Gc drawn in Figure -18). As noted previously [-8). As with phlebovirus Gc, the prototype of the hantavirus genus, Hantaan virus (HAN), showed a modest sequence alignment (p < 0.05) with SIN E1, further supporting the proposed similarities between Bunyavirus Gc and Alphavirus E1. Significant alignments were not detected between Bunyavirus Gc or Alphavirus E1 and TBEV E or other flavivirus class II viral fusion proteins. Limited local similarities were observed between some Bunyavirus Gc and pestivirus E2. It is noteworthy that the significance of the overall sequence similarities between certain phlebovirus Gc and Alphavirus E1 is higher than some similarities among Gc of some prototypic members of the Bunyaviridae . These results further validate the use of the PRSS3 algorithm to identify limited similarities amongst viral proteins. Alignment of the carboxyl terminal portion of the pvc2 protein of rice stripe virus (RiSV), a Tenuivirus, and the envelope protein encoded by Cer13 retrovirus with two phlebovirus Gc reveals a collinear arrangement of fusion peptide consensus sequences . HAN Gn also showed a significant alignment with Gn of RVF, a phlebovirus. Both HAN Gn and Gn of TSWV, a tospovirus, also showed significant alignments with envelope protein 2 (E2) of SIN. SIN E2 has been implicated as the virion protein responsible for binding to the cell surface receptor [The longest open reading frames of M segments of all members of the Bunyaviridae are antisense to the virion RNA. mRNAs transcribed from Bunyavirus M segments are translated into large polyproteins that are subsequently cleaved by into functional proteins ,43. Gc oreceptor . These rThe simplest M polyprotein, encoding only Gn and Gc, is that of hantaviruses Fig. . In addiC. elegans retroviruses previously shown to have remarkable sequence similarities to phlebovirus Gc. These results also indicate that Gallaher's "Rosetta Stone" strategy can be used to identify potential class II viral fusion proteins, as demonstrated previously for class I fusion proteins [Proteomics computational analyses suggest that Bunyavirus Gc proteins are class II viral fusion proteins (β-penetrenes), with a structure similar to the fusion proteins of Alphaviruses and Flaviviruses. Similar sequences or common structural/functional motifs are collinearly located in Bunyavirus Gc and Alphavirus E1. Features common to other class II fusion proteins, including an internal fusion peptide, a carboxyl terminal transmembrane domain and regions with a high propensity to interface with bilayer membranes, are conserved and in similar locations in Gc of viruses in each genus of the Bunyaviridae. These features are also present in glycoproteins encoded by nonenveloped Tenuiviruses of plants, and a group of proteins ,49. The Many viral fusion proteins fit neither class I or II and it is likely that other classes of viral fusion protein also exist. However, among major classes of enveloped RNA viruses, there are at least six, myxoviruses, retroviruses, paramyxoviruses, filoviruses, arenaviruses and coronaviruses, that encode class I viral fusion proteins -4. AlphaAlphaviruses appear to use separate envelope proteins for fusion (E1) and attachment (E2) . BecauseThe remarkable similarities in both the pre- and post-fusion forms of the fusion proteins of SFV E1, an Alphavirus, and DEN and TBEV, members of the flavivirus genus of the Flaviviruses, in the absence of detectable sequence similarities, suggest that Alphavirus and Flavivirus class II fusion proteins may have diverged from a common progenitor. Alternatively, there may have been convergent evolution towards the common structure. Likewise, the sequence similarities detected between phlebovirus Gc and SIN E1 are consistent with divergent evolution from a common progenitor, but are insufficient to directly establish a phylogenic relationship. The results presented here suggest that Gc of members of the Bunyaviridae may have a common ancestor. Gn and Gc are in analogous locations in the polyproteins encoded by the five genuses of the Bunyaviridae. The simplest Bunyavirus M polyprotein, that of hantavirus members, encodes only Gn and Gc, whereas M of members of other Bunyavirus genuses encode several additional proteins. Therefore, divergence of Bunyavirus M segments may have occurred either through acquisition of sequences and/or lose of sequences in a cassette manner constrained in part by the locations of the major glycoproteins.Comparisons of divergent viral fusion proteins with internal fusion peptides can reveal features essential for virion:cell fusion. Regions of high membrane interfacial propensity including the fusion peptide and the transmembrane anchor, appear in similar locations in Bunyaviruses, Alphaviruses and Flaviviruses. The presence of several additional sequences with the propensity to interact with bilayer membranes in class II viral fusion proteins has not been considered in previous virion:cell fusion models ,11,54. CCurrent fusion models do not consider that the transmembrane domain and fusion peptide, while anchored into the viral and cellular membranes, would still be free to move laterally without distorting the membranes. More importantly, the virion is quite small compared to the cell, and would be freely mobile. Rearrangement of the fusion proteins may simply draw the virus closer to the cell without distorting either the viral or cellular membranes. An alternative to the models involving apposing membrane nipple formation is suggested by the observation that sequences of class II viral fusion proteins, including the fusion peptide, the transmembrane anchor and other sequences with high WWIHS scores, potentially form a nearly continuous track of membrane interactive regions that could channel the movement of lipids during virion:cell fusion Fig. , black. In the absence of structural determinations by X-ray crystallography, models such as proposed here can provide useful hypotheses to guide experimental strategies for development of vaccines or drugs to prevent or treat infection by viruses with class II fusion proteins. Prior to the availability of X-ray structural data, several potent HIV-1 TM inhibitors were developed ,57 basedThe authors declare that they have no competing interests.CEG performed the sequence alignments and assisted in the preparation of figures. RFG supervised the work and wrote the manuscript.


Grafting is a powerful but complex means to study the spread of RNA silencing Dactylosphera vitifoliae devastated European grapewine varieties over the course of the late 1800s and early 1900s, the varieties were saved by grafting them onto resistant rootstocks from the New World. Since then, these rootstocks have been used to maintain the susceptible Old World cultivars. But grafting is also an excellent tool for scientists studying systemic signals traveling between the rootstock and distal parts of the plants, and vice versa. For example, two important studies  refers to the phenomenon whereby specific gene transcript levels are reduced in the presence of a related RNA. From studies of RNA silencing in several systems, much is now known about the mechanisms involved , but theAs expected, the discovery of this process triggered a quest for the “systemic inducer” of the process: a signal that travels through the plant and is able to initiate silencing in a remote location within the plant. Grafting was an obvious tool to use in the quest for this signal, as it allowed silencing source and sink tissues to be of different origin.Nia. Some of the transgenic lines generated always showed higher levels of Nia transcripts than the wild type—as expected from the presence of an additional gene—and were termed class I lines. However, other transgenic lines had undergone silencing for both the endogenous and exogenous Nia genes and those were termed class II lines. Nia transgene, which has an endogenous counterpart in the wildtype plant, green fluorescent protein has no homolog in nontransgenic lines. Therefore, silencing spreading in the wild-type “spacer” in the sandwich grafts could not be assisted by an endogenous sequence. Rather, the systemic signal must have traveled all the way to the scion and induced gene silencing there. The establishment of systemic silencing took 4 wk in the “direct” grafts and 6 wk in the sandwich grafts. However, silencing spread to the scion only in some of the grafts: in ten out of 16 direct grafts and five out of 11 sandwich grafts.Related experiments by I found these papers were very important not only for what they proved—the existence of a systemic signal of silencing—but also because they gave an unequivocal answer to the scientific questions they posed, using relatively simple methodology. Although excited by these successful examples of the transmission of silencing, I kept coming back to two questions: (1) what prevents transmission in some of grafts, and (2) why does it take longer to transmit silencing to the scion than it takes systemic silencing to reach the most remote parts of an intact plant?When we started working on the silencing signal ourselves, we repeated some of the above experiments but found somehow lower efficiencies in the initiation of silencing in the scions. We soon realized that our results were influenced by the developmental stage of our scions. A paper from Jeff Meins's laboratory in Switzerland shed light on some aspects of the grafting puzzle. Researchers there introduced additional chitinase genes using bolistics, in sense or antisense orientation under the control of a strong promoter (35S) into chitinase transformant lines of tobacco that never exhibited spontaneous gene silencing . In lineSurprisingly, only top grafting resulted in scions that were systemically silenced by a rootstock signal. Furthermore, even transmission after top grafting was less effective than expected; in one stock/scion combination only 27 out of 71 grafts exhibited transmission of the silencing signal. The authors also found that antisense-induced silencing was never transmitted to the scion. These findings do not answer the many questions about the mechanisms underlying systemic silencing, but they point us in certain directions.The individual parts of a whole plant are, in terms of import and export, in an equilibrium that changes with development. When grafting takes place, how this equilibrium is altered depends on the individual “parts” that contribute to the “new” whole plant. In addition, there is now indirect evidence  that whaFrom the grafting experiments to date, it is now evident that the transporting capacity of the vascular tissue bypass that is formed at the graft junction does not fully reach the level of the original vascular tissue. The basis for these restrictions is not known. In a way, the graft interface functions as an unintentional filter. If the specificities of this “filter” were known, it would help us comprehend some transmission inconsistencies. Keeping in mind these limitations, grafting remains an invaluable tool in the search for the systemic silencing signal.


Tamoxifen is being used successfully to treat breast cancer. However, tamoxifen also increases the risk of developing endometrial cancer in postmenopausal women. Raloxifene also decreases breast cancer in women at high risk and may have a lower risk at developing cancer of the uterus. Tamoxifen has been shown to stimulate arachidonic acid release from rat liver cells. I have postulated that arachidonic acid release from cells may be associated with cancer chemoprevention.3H] arachidonic acid. The release of the radiolabel from these cells during incubation with tamoxifen and the raloxifene analog LY117018 was measured. The prostaglandin I2 produced during incubation of the rat liver cells with μM concentrations of tamoxifen and the raloxifene analog was quantitatively estimated.Rat liver, rat glial, human colon carcinoma and human breast carcinoma cells were labelled with [2 production and that induced by lactacystin and 12-O-tetradecanoyl-phorbol-13-acetate. LY117018, however, blocks the tamoxifen stimulated prostaglandin production. The stimulated prostaglandin I2 production is rapid and not affected either by preincubation of the cells with actinomycin or by incubation with the estrogen antagonist ICI-182,780.Tamoxifen is about 5 times more effective than LY117018 at releasing arachidonic acid from all the cells tested. In rat liver cells only tamoxifen stimulates basal prostaglandin ITamoxifen and the raloxifene analog, LY117018, may prevent estrogen-independent as well as estrogen-dependent breast cancer by stimulating phospholipase activity and initiating arachidonic acid release. The release of arachidonic acid and/or molecular reactions that accompany that release may initiate pathways that prevent tumor growth. Oxygenation of the intracellularly released arachidonic acid and its metabolic products may mediate some of the pharmacological actions of tamoxifen and raloxifene. The successful treatment and prevention of estrogen-dependent breast cancer in women by tamoxifen is attributed to its estrogen receptor (ER) occupancy . In tTamoxifen and raloxifene have several properties in common; e.g. prevention of tumors in the DMBA induced rat mammary model, maintenance of bone density in the ovariectomized rat and reduction of low density lipoprotein cholesterol. The partial estrogen agonist activity of tamoxifen on uterine tissue, however, increases the risk of developing endometrial cancer. This does not appear to occur with raloxifene.2 production by the rat liver cells. Although both compounds release AA from these cells, LY117018 is less effective. Only tamoxifen stimulates both basal and PGI2 production induced by incubation of rat liver cells with lactacystin in the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). LY117018, however, inhibits the PGI2 production stimulated by tamoxifen.Tamoxifen stimulates arachidonic acid release from rat liver cells . In thisThe intracellular release of AA and/or the cellular reactions that accompany that release may initiate pathways that prevent tumor growth. The tissue specific effects of tamoxifen and LY117018 may be associated with the AA or with cyclooxygenase (COX) activity and/or one of the many bioactivities resulting from oxygenation and metabolism of the released AA.3H]AA (91.8 Ci/mmol) was purchased from NEN Life Science Products, Inc. ; ICI-182,780 from Tocris Cookson, Inc. ; tamoxifen and 4-OH-tamoxifen were from Sigma Chemical Co. . LY117018 was obtained from Dr. David A. Cox, Eli Lilly and Co. . Raloxifene was extracted from EVISTA® tablets with dimethylsulfoxide.The C-9 rat liver and BT-20 human breast carcinoma cells were purchased from the American Type Culture Collection  and maintained in MEM supplemented with 10% fetal bovine serum. The C-6 rat glial cell line was obtained from Dr. Elaine Y. Lai in the Department of Biology, Brandeis University and maintained in medium 199. The human colon carcinoma cells (HT-29) were obtained from Dr. Basil Rigas, American Health Foundation, Valhalla, NY and maintained in McCoy's medium.  AA (0.2 μCi/ml) was added and the cells incubated for 24-h. The cells were washed 4 times with MEM and incubated for various periods of time with 1.0 ml of MEM, medium 199 or McCoy's containing 1.0 mg bovine serum albumin (BSA)/ml and different concentrations of each test compound. The culture fluids were then decanted, centrifuged at 2000 × g for 10 min, and 200 μl of the supernate counted for radioactivity. Radioactivity recovered in the washes before the 6-h incubation was compared to input radioactivity to calculate the % radioactivity incorporated into the cells  released from radiolabelled methylcholanthrene transformed fibroblasts (about 20% after a 3-h stimulation by serum), 92% was AA, 4% was PGE2, 0.6% and 1% were PGF2α and phospholipids respectively [3H] AA had been incorporated into phosphatidylcholine (50%), phosphatidylethanolamine (36%), phodphatidylserine (9%) and triglycerides (10%) [3H] AA label released from human colorectal cancer cell lines (HCT116 and SW180) was AA as measured by TLC  AA, was added after the first 24-h incubation. The cells were incubated for another 24-h, washed three times with MEM, then incubated with lactacystin and TPA in the presence and absence of the test compounds in MEM/BSA for various periods of time. The culture fluids were decanted and analyzed for 6-keto-PGF1α, the stable hydrolytic product of PGI2, by radioimmunoassay  AA is presented as a percentage of the radioactivity incorporated by the cells. Except for the time-course experiments, which used duplicate dishes  would be 0.25 nM (the specific activity of the [3H] AA is 92 Ci/mmole). Since TLC or HPLC analyses were not done, the concentration of the total released AA could not be quantitatively estimated. However, even treatment of a colorectal cancer cell line with 200 μM AA for 48-h leads to apoptosis . Similar to AA release [2 production by tamoxifen is not affected by preincubation of the cells for 2-h with 1 μM actinomycin D. The induction obtained by incubation with lactacystin in the presence of TPA is inhibited  of basal activity is shown in Fig.  μM of ba release , stimulated Fig. . Stimula780 Fig. . This es, GW7845 . The rap2 production stimulated by tamoxifen  or Aramidex ® (anastrozole) do not release AA from cells in culture (unpublished data). These two drugs inhibit estrogen synthesis by blocking aromatase enzymes and also prevent estrogen-dependent breast cancer [These studies demonstrate that, at μM concentrations, tamoxifen and the raloxifene analog, LY117,018 stimulate the release of AA from cells in culture. At nM levels, the release is not observed. The effectiveness of these two compounds at prevention of estrogen-dependent breast cancer reflects competition for the ER . In addit cancer . In view2+ ionophore A-23187 reflects phospholipase activities. It is regulated by phosphorylation of the enzyme [A mechanism that most simply explains the release of AA by tamoxifen and LY117018 is the ability of such compounds to intercalate into cell membranes and affect phospholipase activities. The release of AA from endothelial cells by the Cae enzyme . The enze enzyme  and woulThe AA release by tamoxifen and other reagents studied in my laboratory occurs with μM concentrations ,19,23,24When measured after a 6-h incubation, 10 μM tamoxifen stimulates deesterification of membrane phospholipids as measured by extracellular AA release, Fig. . The 6-h2 produced by the rat liver cells can be quantitatively determined. The effects of tamoxifen and LY117018 on the COX activity of the rat liver cells may be similar to their effects on other cells that express COX. Tamoxifen and LY117018 affect COX activity differently; only tamoxifen stimulates PGI2 production. AA, which regulates the production of lipoxygenase, COX and cytochrome P-450 epoxygenase products could impact many of the pharmacological actions of these two selective estrogen receptor modulators. AA can be oxygenated by COX isoforms, lipoxygenases, and cytochrome P-450 epoxygenases and their products converted to the prostaglandins, leukotrienes, epoxyeicostetranoic acids [2 to the major physiologically active products, PGD2, PGE2, PGF2α, PGI2 and thromboxane A2 [Some tissue specific effects of tamoxifen and raloxifene, e.g. on endometrial cells of the uterus, may be related to COX activity. At the low cell densities used in the present studies, the PGIic acids  and AA tic acids . In the oxane A2 . They, ioxane A2 ,31.None declared.L.L. is the sole author and all of the experiments, with the exception of the western blots referred to as Levine L and Tashjian A (unpublished data), were conducted by L.L.The pre-publication history for this paper can be accessed here:


Cervical cerclage is a surgical procedure involving suturing the cervix with a purse type stitch to keep it closed during pregnancy. This procedure has been used widely in the management of pregnancies considered at high risk of preterm delivery. Several observational studies into the efficacy of cervical cerclage have claimed high rates of successful pregnancy outcome in women with a poor obstetric history attributed to cervical incompetence. However, a recent aggregate data Cochrane review found no such conclusive evidence from seven included randomised studies. Current data suggests that cervical cerclage is likely to benefit women considered to be 'at very high risk' of a second trimester miscarriage due to a cervical factor, however identifying such women remains elusive and many women may be treated unnecessarily. Undertaking an individual patient data (IPD) meta-analysis of the studies will allow us to investigate whether treatment is more effective in particular subgroups. Such an analysis will also provide a more powerful analysis of the predictors of preterm delivery and pregnancy loss, including ultrasound measurement of cervical length, and will allow a more complete analysis of 'time to event' outcomes.The analysis will include data from randomised trials comparing the intervention of elective cerclage versus no cerclage or bedrest to prevent miscarriage or pre-term labour. A specific list of data will be requested for each trial, including demographic and obstetric history data. The primary outcomes of interest will be neonatal mortality/morbidity. Attention will also be given to secondary outcomes such as time from randomisation to delivery, preterm delivery before 32 weeks and maternal morbidity. An intention to treat analysis will be performed, with attention paid to assessing clinical and statistical heterogeneity. Multilevel models with patients and trials as the two levels will be explored to investigate treatment effect on various outcomes. Patient-level covariates will be incorporated into the models in an attempt to account for statistical heterogeneity as well as to investigate interactions with treatment effect.Predictive models generated from our analysis should lead to more effective counselling of women at risk and a more cost effective use of cerclage. Cervical cerclage is a surgical procedure carried out during pregnancy. The operation involves suturing the neck of the womb (cervix) with a purse type stitch to keep the cervix closed. This surgical procedure has been used widely in the management of pregnancies considered to be at high risk of preterm delivery.Several observational studies in the last 50 years have claimed high rates of successful pregnancy outcome in women that had a poor obstetric history attributed to cervical incompetence. However, a recent Cochrane review found no conclusive evidence from seven included randomised studies that inserting a cervical stitch in women perceived to be at risk of preterm birth or second trimester pregnancy loss attributed to cervical factors, reduces the risk of pregnancy loss, preterm delivery or morbidity associated with preterm delivery (Drakeley 2003).In the Cochrane review, the data for important clinical outcomes including preterm delivery and maternal infection showed significant heterogeneity due to inconsistency in clinical definitions used, including the cut off gestational age defining preterm delivery, and different patient populations studied.Practically, methods of undertaking a meta-analysis of several studies may involve collecting either aggregate data, or data on each patient individually. The advantages of the latter approach, described as the 'yardstick'  include One of the main concerns regarding current evidence related to cervical cerclage and other interventions for preventions of preterm delivery is a possibility that the 'primary outcomes' may have been selected to give results in greatest accord with the a priori beliefs of the authors. The evidence to support this phenomenon of within-study selective reporting comes from empirical research, which demonstrates discrepancies between research protocols and subsequent publications . A regreThe aim of this project is to undertake an IPD meta-analysis of randomised trials of cervical cerclage. Specific objectives are as follows.1. To estimate the effect of cervical cerclage on gestational age at delivery.2. To investigate whether cervical cerclage is more likely to prevent extreme prematurity (<28 weeks) or delivery at later gestations.3. To investigate risk factors for preterm delivery.4. To investigate interactions between risk factors and cervical cerclage.5. To model the effect of cervical cerclage and other risk factors on neonatal and maternal morbidity.The types of studies considered for inclusion in the analysis will be all randomised trials comparing cervical cerclage with expectant management or no cerclage during pregnancy. The previous Cochrane review (Drakeley 2003)  identifiThe data collected in the studies will relate to women with confirmed, or suspected of having, cervical incompetence who desire future pregnancies and women who present as an emergency and are thought to have a diagnosis of cervical incompetence. The intervention investigated in the studies will be elective cerclage by whichever method , versus no cerclage or bed rest as interventions to prevent miscarriage or pre-term labour as defined in the original Cochrane review (Drakeley 2003).The methods of trial identification described in the original Cochrane review (Drakeley 2003)  were excluded in the original review.The following data for each woman/infant pair will be requested from all trials: date of randomisation and gestational age, maternal demographics and obstetric characteristics at randomisation including cervical length on ultrasound, fibronectin and bacterial vaginosis data if available, treatment allocated, complications during pregnancy including ruptured membranes, maternal pyrexia or chorioamnionitis, date of delivery, gestational age at delivery and all neonatal data including birthweight, length of stay at NICU and morbidity related to prematurity.The following methodological data will also be requested for all trials: method of generation of randomisation list, method of concealment of randomisation, stratification factors and blinding methods.Data will be accepted either in electronic (floppy disk/CD/internet) or paper form. A desired format and coding will be specified but trialists may supply data in the most convenient way open to them, providing details of coding are sent with the data.A copy of the original data sent (before checking) will be held in a separate file. The following procedures will then be performed and documented for all trial data supplied. Trial details will be crosschecked against any published report of the trial. Range and consistency checks will be applied – missing data, errors and inconsistencies will be followed up with a nominated individual. The chronological randomisation sequence will be reviewed. The balance of prognostic factors will be checked, taking into account of factors stratified for in the randomisation procedure.The primary outcome of interest will be neonatal mortality/morbidity. Choice of primary outcome is about what should determine clinical decision-making. However it is recognised that trials to date may have insufficient power and there is a need to consider secondary outcomes of time from randomisation to delivery, preterm delivery before 32 completed weeks (<32+0 weeks) and maternal morbidity as defined in the Cochrane Review (Drakeley 2003). We willReporting of these outcomes in the original trial report is not an eligibility requirement for this review.Data on all randomised patients will be requested to perform an intention-to-treat analysis as far as possible. Clinical heterogeneity will be assessed by reviewing the differences across trials in characteristics of randomised patients.Initially, an aggregate data analysis will be undertaken although treatment effect estimates will be obtained from the individual patient data. Binary outcomes will be summarised in terms of odds ratios or relative risks, depending on the degree of heterogeneity observed. Time-to-event outcomes will be summarised in terms of the log (hazard ratio). The I square statistic and chi-square test for statistical heterogeneity will be applied to these summary data.Regression models, stratified by trial, will be used to explore the effects of treatment, risk factors and treatment-covariate interactions on the various outcomes of interest. These will include Cox and accelerated life models for time-to-event outcomes  treatment effect across trials. Patient-level covariates (as listed above) will then be incorporated into the model in an attempt to account for some of the remaining statistical heterogeneity. An attempt will be made to incorporate these covariates first of all by assuming their effect to be constant across all trials and subsequently by assuming some heterogeneity in the covariate effect across trials by modelling them either as fixed or random effects. Finally, treatment-covariate interactions will be investigated by including additional variables and adopting a similar approach.If IPD are not available for some trials, the potential for bias will be investigated as follows. The reasons for not being able to obtain the data will be assessed for the potential for bias. Results using aggregate data from these trials will be compared with results using aggregate data from trials where IPD have been supplied, and any difference investigated. The analysis plan will be reviewed in light of the availability of IPD but prior to any comparative analyses.Predictive models generated by our analysis should allow more effective counselling of women at risk of preterm delivery and thus more cost effective use of cerclage.ZA and PRW were authors of a paper that will be included in the IPD meta-analysis . ZA was an author of the non-IPD systematic review on this topic . The autPRW conceived the idea, CTS drafted the initial protocol, and all authors commented on and approved this final version.The pre-publication history for this paper can be accessed here:


A 38-year-old woman presented with acute hematological toxicity from her anticonvulsants, even though she had been taking them for many years Six months prior to admission, her weight was 175 lbs, and her body mass index was 36. On admission, her complete blood count was normal, but over a 2-wk period she developed acute pancytopenia . During 6/l; and WBC, 10.7 × 106/l. After starting carbamazepine, her WBC dropped to 4.5–5.5 × 106/l, and her absolute neutrophil count dropped from 9 × 106/l to about 2.5 × 106/l. When the sodium valproate was added, her MCV increased to 112 fl, her platelet count fell to 100–150 × 106/l, and her Hb dropped to 110–120 g/l.She had been taking carbamazepine for 17 y and sodium valproate for 13 y for a mixed seizure disorder. At age 22 y, before starting any anticonvulsants, her baseline hematological parameters were as follows: Hb,123 g/l; MCV, 106 fl; platelets, 296 × 10The patient's other medications on admission were carnitine and low-dose L-thyroxine for hypothyroidism; she had been taking both for several years. She had not been recently exposed to any new medications, environmental toxins, or over-the-counter dietary supplements. There was no family history of aplastic anemia. The patient lived at home with her mother, who cares for her and has legal guardianship.A bone marrow biopsy  showed a6/l and her platelet count to 248 × 106/l. Serial complete blood counts showed worsening anemia over the next 15 wk, requiring two packed red blood cell transfusions at week 6 and week 14, when her Hb was 31 and 53 g/l, respectively. Throughout this period, her reticulocyte count was low, at about 0.18% , while her WBC and platelet count were normal. We made a diagnosis of pure red cell aplasia (PRCA). A second bone marrow biopsy under minimally conscious sedation was unsuccessful, and the patient declined further attempts. After the first transfusion she was started on prednisone and erythropoietin for her PRCA. Meanwhile, because of her worsening seizures, we increased her sodium valproate dose, increasing her serum valproate level from 70 mcg/ml to 110 mcg/ml. After the second transfusion, we discontinued her sodium valproate and started her on clonazepam and oxcarbazepine as alternative anticonvulsants.The patient's carbamazepine was discontinued. Within 10 d, her WBC had risen to 6.7 × 10The patient had a brisk reticulocyte response. Her reticulocyte count rose from 0.36% to 0.78% in the second week and to 6.61% in the fourth week after stopping the valproate. Six weeks after stopping the drug, her Hb was 125 g/l and her MCV was 106 fl, suggesting replacement of transfused red blood cells with newly formed red blood cells (which have a higher MCV than older transfused cells). Over the next 30 mo of follow-up, she had no relapse of her aplastic anemia.We were unable to identify a specific cause for the patient's anorexia and weight loss. We found no evidence of malignancy on admission or subsequent follow-up. Within 1 wk of stopping her sodium valproate, her appetite improved and she put on 15 lbs over the next 6 wk. By the fourth month after stopping the drug, she had gained 45 lbs.6/l; Hb, 147 g/l; MCV, 104.9 fl; and platelets, 262 × 106/l.We last saw the patient in August 2004. Her seizure control had worsened, and she had developed signs of early dementia. Her current anticonvulsants are clonazepam, oxcarbazepine, and zonisamide, and she continues taking synthroid. Her last complete blood count was stable: WBC, 6.4 × 10Sodium valproate and carbamazepine are associated with rare but potentially lethal hematological complications. There are five case reports of PRCA shortly after initiation of sodium valproate, with the longest interval between the initiation of therapy and the onset of aplasia being 2 y ,2,3,4,5.Our investigations ruled out most of the known causes of acute bone marrow suppression, making the anticonvulsants the most likely cause. Malnutrition was an unlikely cause: her WBC and platelet counts had recovered before any increase in appetite or weight gain; her body mass index was normal despite her weight loss; and her bone marrow iron stores were adequate and her serum folate and vitamin B12 levels were high, suggesting adequate nutrient supply.We considered and rejected the possibility of Down syndrome–associated aplastic anemia. There have been six case reports of this condition, and in all cases it occurred in young children, suggesting a genetic predisposition . Half ofThe brisk return of her WBC and platelet counts upon discontinuing carbamazepine, and her brisk reticulocytosis upon discontinuing sodium valproate, were both consistent with previous reports of hematological toxicity due to these drugs ,4,5,6,7.We found no cause for the patient's anorexia and weight loss, but her appetite returned and she gained weight after stopping the sodium valproate. There is a known association between this drug and anorexia .Acute hematological complications of anticonvulsant therapy can still occur after many years of therapy, so continued vigilance is warranted.Although sodium valproate is generally associated with increased appetite and weight gain, it can also be associated with anorexia, nausea, vomiting, and weight loss.


Asparagus acutifolius L. is a dioecious and native plant species, widely distributed in the Mediterranean Basin. It is known for its fine flavour and could represent an important resource for cultivation programs in desert areas. Few molecular studies have been performed on this species. In the present paper, the ISSR technique was employed to study genetic diversity in Italian A. acutifolius.ST (0.4561) and Theta B (0.4776) values indicate a wide genetic variation among the samples examined. The distance UPGMA tree grouped together the genotypes strictly according to their geographical origin, showing that each sample is genetically structured and can be considered a distinct population. AMOVA analysis further confirmed genetic structuring of the populations. Population-specific fragments were also detected.Twenty-three primers produced a total of 228 polymorphic fragments used to evaluate genetic variation. FA. acutifolius according to geographical origin, and confirm the importance of genetic studies for designing germplasm conservation strategies.The results suggest that ISSR markers are useful in distinguishing the populations of  The availability of a variety of DNA markers, such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and intersimple sequence repeat (ISSR) has enabled researchers to investigate genetic diversity among various plant species across natural populations -5. Amongin situ and ex situ of genetic diversity in natural populations is imperative to guarantee sustainable development [Local populations of traditional cultivars provide a valuable resource for plant breeding as well as for the preservation of genetic diversity . The expelopment .Asparagus acutifolius L. (Liliaceae) is a native, perennial plant species widely distributed throughout the Mediterranean areas, whose flowers are classified as dioecious and are mainly bee-pollinated; it generally does not reproduce by self-pollination. It grows in bushy and semi-dry places, sunny or semi-shade, mainly on limestone.A. officinalis and does not require rich soils for cultivation; for these reasons, it could be an economically important resource for the recovery of arid rural areas where controlled introduced programs could be achieved.This species is known for its strong taste compared to the cultivated A. officinalis, for which many molecular markers have been characterized  [A. acutifolius are drawn from RAPD analyses [Viola pubescens [Oryza [To date, there is little information available on the genetic variability of this species. At present, the most widely studied species is P, AFLP) . The fewanalyses  and the analyses . The ISSanalyses . They haanalyses , Viola pubescens , potato ubescens , and Orys [Oryza .A. acutifolius collecting samples in eight different scattered rural areas: six continental and one each from the Italian islands of Sardinia and Sicily.In this study we used ISSR markers to analyse the genetic diversity of Italian A. acutifolius was collected and their characteristics.Figure A. acutifolius is summarized in Table Among the 42 primers tested, 23 proved useful to characterize the samples  was exhibited in the Sassari and the lowest (35.09%) in the Recco samples.Genetic structuring was evident due to the detection of specific bands in each sample examined. Spoleto and Caserta samples showed one and three fixed specific fragments, respectively, found to be statistically significant (P < 0.0001). For the other samples, 27 ISSR specific polymorphic fragments were detected, with a varying degree of statistical significance ranging from P < 0.0400 to P < 0.0001.Genetic distances  were exaSamples collected at different geographic site grouped together, as shown in the UPGMA tree Fig. , and theThe values of gene diversity are summarized in Table T) was 0.2618 ± 0.0240 and the average variation within samples (average HS) was 0.1424 ± 0.0084. The mean diversity among the samples (DST) was 0.1194. The fixation index FST = (HT-HS)/HT was 0.4561, indicating a reduction of genetic diversity of about 45%. The Theta-B value obtained by Hickory analysis is an estimate of FST under a random-effects model of population sampling. Its mean value is 0.4766 ± 0.0173; the HT and HS values are, respectively, 0.2859 ± 0.0115 and 0.1619 ± 0.0026 showing that there is a general agreement between the results obtained using the two different approaches.As summarized in Table A. acutifolius in an Italian population. ISSRs also revealed diversity within each sub-population. The results obtained are in accordance with the principle that the number of individuals used to estimate average heterozygosity can be very small if a large number of loci is studied [ISSR markers can be used in population genetic studies of plant species as they effectively detect very low levels of genetic variation . They al studied .The gene diversity values Table  ranged fST and the Theta-B value (0.4766) demonstrated a very great genetic differentiation among samples, possibly caused by random genetic drift. Further statistical support to the genetic structuring of the samples examined comes from the AMOVA analysis. Despite the continuous distribution of A. acutifolius, the eight samples represent genetically differentiated populations.The fixation index is 0.4561, which indicates a substantial reduction of genetic diversity (about 45%), probably due to the high genetic isolation of samples analysed. The FA. acutifolius populations.In each population it was possible to identify ISSR specific fragments. As shown in Table A. acutifolius throughout the Italian peninsula, there is poor gene flow through the isolates. The high genetic differentiation of the A. acutifolius populations examined might be attributed to the kind of pollinators (mainly bees) that can act at short distances, preventing the gene flow, and to the effects of anthropogenic habitat fragmentation.The high degree of genetic differentiation is confirmed by the UPGMA tree topology, in which all accessions from the same population grouped together Fig. . The popA. acutifolius populations [The results obtained using ISSR markers are in agreement with the RAPD analysis that also identified population-specific fragments in different Italian ulations .A. acutifolius has well-differentiated populations, despite their morphological low variability. These results show that to maintain genetic diversity within A. acutifolius it is necessary to conserve many populations.Information about the spatial organization of genetic variability is essential for the conservation of genetic resources . Our resA. acutifolius collected in the eight different locations in Italy  and 1X Taq DNA polymerase buffer, in a total volume of 25 μL. The amplification programme was 1.5 min at 94°C; 35 × 40 s at 94°C, 45 s at the primer annealing optimal temperature  and negative and positive controls were loaded and run at constant voltage (150 V) for 2 hours. After running, the gels were UV visualised and recorded using a Kodak Digital Science dS1D DC40/DC120 Camera. To verify the repeatability of the results, each DNA extraction, PCR amplification, and gel running was repeated twice.Unequivocally scorable and consistently reproducible amplified DNA fragments were transformed into binary character matrix .T), heterozygosity within population (HS), diversity among populations (DST), fixation index (FST), and genetic distance [Genetic variation within and among sub-populations was analysed on the basis of the banding profile using various parameters such as percentage polymorphism (P), total heterozygosity (Hdistance ,22-24, udistance . Since Idistance  based onAMOVA analysis, implemented in Arlequin , was conThe Mantel test of genetic and geographic distances was carried out to evaluate the correlation between the two data matrices.The UPGMA tree was generated using the PAUP*4.0 software , and BooMS carried out part of the sample collection, designed ISSR primers and carried out ISSR work; GG did part of DNA extraction; SM carried out part of the sample collection and DNA extraction; LG participated in the manuscript preparation and revision; SA conceived the study, carried out data analysis, co-ordination and interpretation of the results.


In a previous study, when adult subjects were exposed to a level of 400 lux light for more than 30 min or a level of 300 lux light for more than 2 hours, salivary melatonin concentration during the night dropped lower than when the subjects were exposed to dim illumination. It was suggested that such light exposure in adolescents or children during the first half of subjective night in normal life might decrease the melatonin level and prevent the falling into sleep. However, there has been no actual study on the effects of light exposure in adolescents.Effects of exposure to the bright light (2000 lux) from fluorescent light bulbs during a period of three hours from 19:30 to 22:30 in one evening were examined on evening salivary melatonin concentrations from 19:45 to 23:40. The control group was exposed to dim light (60 lux) during these three hours. Both the dim light control group [DLCG] and the bright light experimental group [BLEG] consisted of two female and three male adolescent participants aged 14–15 y.The salivary melatonin level increased rapidly from 3.00 pg/ml at 21:45 to 9.18 pg/ml at 23:40 in DLCG, whereas it remained at less than 1.3 pg/ml for the three hours in BLEG. Melatonin concentration by BLEG at 22:30 of the experimental day was lower than that at the same time on the day before the experimental day, whereas it was significantly higher in the experimental day than on the day before the experimental day in DLCG.Bright lights of 2000 lux and even moderate lights of 200–300 lux from fluorescent light bulbs can inhibit nocturnal melatonin concentration in adolescents. Ancient Japanese lighting from a traditional Japanese hearth, oil lamp or candle (20–30 lux) could be healthier for children and adolescents because rapid and clear increase in melatonin concentration in blood seems to occur at night under such dim light, thus facilitating a smooth falling into night sleep. Night sleep duration of Japanese children aged 10–18 y has become shorter by one hour during the last 30 years in Japan . Student"Convenience stores" are open for 24 hours and provide several kinds of food and other goods for general civilian life. Convenience stores are now common all over Japan even in suburban areas. Illumination inside the convenience stores is very bright (2000 lux or more at the level of the eyes). Bright lighting in retail stores seems to be a merchandising technique which has been in use worldwide for at least 60 years. Unconscious use of bright light in the evening or at night inside the convenience store may promote a circadian phase delay in students exposed to the bright light during the first half of subjective night. This hypothesis is based on a "light-pulse" experiment in the laboratory  as folloMelatonin, which is synthesized in the pineal organ and secreted to the blood, is well known as a key substance which may be effective in promoting the falling into night sleep by humans . Blood mCurrently, more than 80% of junior high school students of the third grade aged 14–15 y in Kochi go to private school in the evening. If they take a short stop at the convenience store to get some fast food and enjoy talking with their colleagues in front of the store before or after going to the private school in the evening, they suffer the double exposure to bright lights at the school and at the convenience store. Such bright lights are from fluorescent light bulbs and include blue or blue-green lights with 470–500 nm wave lengths which were reported to be powerful to suppress melatonin concentration . Based oIn this study, two light conditions were investigated. One was bright and high color-temperature light of more than 2000 lux, which is used in the evening and at night inside the convenience stores, at preliminary and private school for entrance examination, and at rental video shops in Japan. The other condition was a dark and low color-temperature light with less than 60 lux which is usual in the evening for traditional Japanese settings .Experimental participants were ten Japanese junior high school adolescent students  aged 14–15 y who were attending Motoyama junior high school located in the mountain area of Reihoku district (33.5°N) in Kochi Prefecture. They had enjoyed New Year holidays for 7 days before the experiment. They were instructed to keep usual diurnal rhythm (for example bed time and wake-up time) during the holidays. Before the experiment, participants were divided into the two groups of "bright light experimental group (BLEG)" and "dim light control group (DLCG)". Participants in BLEG were selected to show similar circadian typology to those in DLCG based on the scores in the morningness-eveningness (M-E) questionnaire of Torsvall and Åkerstedt   under fluorescent light bulbs based on our unpublished questionnaire study on 950 families having small children aged 0–6 yrs in Kochi. More than 85% of the 950 families enjoyed evening life under fluorescent light bulbs. We measured the illumination at the level of 1 m above floor just under a usual type of round-shaped fluorescent light bulb in a typical one-room apartment for students and it was 340 lux.th January 2003, all the ten participants got together in front of Motoyama junior high school at 8:00 in the morning. A wagon car took them to the experimental place which was a Japanese style hotel located at a mountain area, Yusuhara town in Kochi Prefecture, 126 km west from Motoyama town. During the driving, illumination inside the car was 350–500 lux. The car arrived at the hotel around noon. It was snowing through the day. Behavior of all the participants was controlled during the stay in the hotel till the next morning of the experimental day. All the participants played outside exposed to the sun light with 6000–7500 lux at the eye level during 12:30–13:30 and 14:00–14:50. They were allowed to have a rest in a living room in which the floor was filled with 12 tatami mats and the illumination at the eye level was 250 lux from fluorescent light bulbs during the rest of the time till 16:30. Participants took bath one by one between 16:30–18:00 and had supper all together between 18:15 and 19:20 in the living room. At 19:25, the participants of BLEG moved to a Japanese style room with 8 tatami mats where they were exposed to the light with 2000 lux at the eye level from fluorescent light bulbs, whereas DLCG group members moved to another Japanese style room with 8 tatami mats where they were exposed to the light of 60 lux and relatively low color-temperature from a electronic light bulb. All the participants included in both groups were home-working or making a small wooden folk craft object that is typical in the Yusuhara district, under each light condition till 22:30. Room temperature was controlled at 15 ± 2°C with an oil heater in both groups. Then they came back to the former living room (12 tatami mats) and stayed there under the light of 250 lux till 23:40. Then female and male participants moved to separate rooms and went to bed just before 24:00. Salivary samples were collected in collection tubes at 21:45, 22:30, and 23:40, and these salivary samplings were preserved in a refrigerator at less than -20°C. Melatonin concentration in the samples was analyzed by a professional analyzing company (MSL Co. Ltd.) which was a specialist for several chemical and microbiological analyses. All the participants from both groups were called out to get up at 7:00 in the next morning. All the participants got up between 7:00–7:15 responding to the calling out. After taking breakfast, they left the experimental place at 9:00 back for Motoyama junior high school. Throughout the study, light exposure was measured on the eye level with a digital illumination meter.On the experimental day of the 5Detailed explanation of the objectives and methods of the experiment was provided before the experimental performance to the participants and their parents. The research project received full and complete agreement from all of them.The results are shown in Fig. In Japan, bright and high color-temperature light of more than 2000 lux is available in the evening or night inside convenience stores which are open for 24 hours and private schools for the preparation to go through the entrance examination to upper schools. Also in usual life, such exposures to bright lights in the evening private school and convenience store can suppress the night increase in blood melatonin level as a direct effect and possibly delay the circadian system that drives the melatonin secretion rhythm and sleep-wake cycle.The results of this study suggest that ancient Japanese lighting in the evening and at night, which could be supplied by a traditional Japanese hearth fire or a oil lamp or candle (20–30 lux), might be healthy for adolescents and children, because the ancient lights could allow rapid and clear increase in melatonin level leading to a smooth falling into night sleep .Bright lights of 2000 lux and even moderate lights of 200–300 lux can inhibit, as a direct effect, nocturnal melatonin concentration in children. Ancient Japanese light conditions which could be supplied by a traditional Japanese hearth fire or a small oil lamp or candle might be healthy for children, because the ancient lights could allow rapid and clear increase in melatonin level in the evening, leading to a smooth falling into night sleep.None declared.


There is a possibility of clock genes having pleiotropic effects on clock period and pre-adult development time. In order to avoid such pleiotropic effects we have used wild type flies of same genotype under environments of different periodicities, which phenotypically either speeded up or slowed down the eclosion clock of D. melanogaster.In insects, circadian clocks have been implicated in affecting life history traits such as pre-adult development time and adult lifespan. Studies on the D. melanogaster, under five different light regimes, continuous light (LL), continuous darkness (DD), and light-dark (LD) cycles of 10:10 h (T20), 12:12 h (T24), and 14:14 h (T28). Although the development time was significantly different in most light regimes, except for females under T24 &T28, pre-adult survivorship remained largely unaffected. The development time was shortest under LL, followed by T20, DD, T24 and T28 regimes, in that order. Interestingly the development time showed a positive correlation with the period of eclosion rhythm, i.e., faster oscillations were associated with faster development, and slower oscillations with slower development.We assayed pre-adult development time and pre-adult survivorship of four laboratory populations of D. melanogaster in a manner that does not involve direct pleiotropic effects of clock genes on both clock period and development time.Based on these results we conclude that periodicity of imposed LD cycles, and/or of eclosion rhythm plays a key role in regulating the duration of pre-adult development in  The Kruskal-Wallis test thus confirmed the results obtained in ANOVA. Although eclosion waveforms under no two light regimes were similar, the dissimilarity was even more striking under T24 and T28. The test revealed that mean development time of males was shortest under LL, followed by T20, DD, T24, and T28, in that order. While the mean development time of females followed a similar trend, it did not differ significantly between T24 and T28.ANOVA on pre-adult development time data revealed a significant main effect of light regime  showed a significant positive correlation with the mean period of eclosion rhythm under the corresponding environments , the ancestral populations of the flies used in the present study, we had reported shortest development time under LL regime, followed by LD 12:12 h, and DD [T20, DD, and T24 and T28, in that order. As opposed to LL, eclosion under DD is gated in a circadian manner, and as a result flies took longer to develop compared to that in LL. We believe that the slight discrepancy in the results of our two studies could be due to the fact that different set of flies (about 100 generations apart) with different clock periods were used in the two experiments. The periodicity of JB populations under DD was greater than 24 h, whereas those of LL populations was lesser than 24 h. Thus, consistent with our proposal, development time of JB populations was greater under DD compared to development time in LD 12:12 h regime, whereas those of LL populations was shorter in DD compared to those under LD 12:12 h.In several insect species adult eclosion is gated in a manner that it occurs only during a narrow window of time, generally around dawn when environmental humidity is the highest . In D. m neurons , and it  neurons . Develop, and DD . In the T20, DD, T24, and T28) showed a significant positive correlation with the mean period of eclosion rhythm under the corresponding light regimes; i.e. shorter period of eclosion rhythm under T20 was associated with faster pre-adult development, followed closely by DD, whereas flies took longest to develop under the light regimes wherein their eclosion periodicities were 24 h and 28 h, suggesting that development in D. melanogaster is regulated by eclosion rhythm. The peak of eclosion under three periodic light regimes closely matched phase relationships of eclosion rhythm relative to LD cycles, suggesting that phases of eclosion rhythm also play a key role in the regulation of development time [D. melanogaster is not entirely regulated by the periodicity of the environment and/or the periodicity of eclosion rhythm. These results are in agreement with the findings of previous studies, where the duration of pre-adult development was positively correlated with clock period [The mean development time under four different light regimes , at constant temperature of 25°C (± 1°C), and constant humidity of 70 ± 5%, on a 21 day discrete generation cycle . These populations were maintained at moderate larval densities of ~ 60–80 larvae per 8 dram vial (9.0 mm height × 2.4 mm diameter) containing banana-jaggery food medium (henceforth banana food). The ancestry and maintenance of these populations has been described in detail in an earlier paper [3). From these petri plates, 60–80 eggs are collected into each of 40 vials in which larvae then develop into adults. Adults eclosing from these vials are transferred to plexiglass cages on 12th day after egg lay. On the 18th day after egg lay, adult flies are supplied with banana food supplemented with live yeast paste for two days, after which eggs are collected to initiate the next generation and the adults discarded. The breeding population typically consists of about 1500 flies.The four populations of er paper . In brieT20), 12:12 h (T24), and 14:14 h (T28). Thus a total of 200 vials were set up for the assay . These vials were introduced into five different light regimes at 20:00 h, when lights went off simultaneously in all LD regimes. Fluorescent white light of intensity ~ 100 lux was used during light phase, whereas in dark phase red light of λ >650 nm was used. Temperature and relative humidity in the five light environments monitored continuously using a Quartz Precision Thermo-Hygrograph, Isuzu Seisakusho Co, LTD, were found to be comparable. The vials were monitored for eclosion of adult flies after the pupae became dark. Eclosing adults were collected every 2 h, sexed, and counted until all the flies eclosed.From the running culture of each population (LL1..4), eggs laid on banana medium over a 2 h window were collected for the assay. Exactly 30 eggs were dispensed into 8 dram vials containing ~ 6 ml banana food and 50 such vials were set up from each population. Ten vials from each population were introduced into continuous light (LL), continuous dark (DD), light-dark (LD) cycles of 10:10 h , in which replicate populations were treated as random blocks, and light regime and sex were treated as fixed factors.In order to detect differences in the eclosion profiles of the flies under different light regime, development time data of individual flies from all four replicate populations were pooled and compared using Kruskal-Wallis test.Pre-adult survivorship was calculated as the fraction of eggs that successfully developed into adults in each vial. The mean pre-adult survivorship values of each population in each light regime were used as data in a mixed model ANOVA, with replicate populations as random blocks, and light regime as a fixed factor.DAP and AD were involved in collecting eggs, collecting and counting flies for pre-adult development time, estimating pre-adult survivorship, data entry and analyses. VKS conceived of the study and participated in its design and coordination. MKC and AJ gave valuable comments and suggestions throughout the study. All authors read and approved the manuscript.