{"text": "DNA mutates, and it's a good thing it does. If it didn't, there could only be one kind of life, not the millions there are today, and species could not adapt to new challenges. This is because mutations in genes—the coding portion of DNA—are the raw material for evolution. However, genes make up a surprisingly small fraction of our DNA. If the genome were a cookbook, its 30,000-odd genetic recipes would be scattered among millions of pages of apparently meaningless nonsense.Mutations affect all DNA, not just the genes, and this provides population geneticists with a veritable toolbox of methods useful, for example, in DNA profiling. Importantly, all these methods rely on the idea of a “molecular clock,” the notion that mutations rain down on noncoding DNA like a fine drizzle, so constantly that genetic similarity is a good measure of evolutionary time. Thus, if orangutans diverged from humans twice as long ago as did chimpanzees, on any given piece of DNA we would find twice as many differences between the orangutan sequence and the human sequence as between humans and chimps. The mutations are marking time.If the molecular clock works, scientists can do wonderful things like estimating how long ago it was that the common ancestor of all humans lived, or when birds evolved from dinosaurs. The clock assumes that mutations occur independently of each other and at a constant rate. By analyzing thousands of noncoding DNA sequences scattered throughout the human genome, Edward Vowles and William Amos have found that the clock is anything but constant. Instead, a mutation in one spot in the genome affects the chance of getting another mutation nearby.Not all noncoding DNA is made up of benign tracts of random letters. Some sequences appear to be more difficult to copy than others, and these trouble spots can give rise to alphabetic stuttering. DNA is made up of four component chemical units, called nucleotides, which are often referred to by their initial letters: A, C, G, and T. Stuttering occurs when the same pairs or triplets of letters occur together, for example ACACAC. Such regions are called microsatellites, and instead of mutating by swapping one letter for another, as most nucleotides do, these sequences evolve mainly by gaining and losing triplets or pairs like “AC.”In this study, Vowles and Amos used the published sequence of the human genome to track down and compare thousands upon thousands of microsatellites. If the molecular clock ran smoothly, they would expect to find no similarity at all between the DNA sequences surrounding any pair of unrelated microsatellites. To their surprise, they found the complete reverse, with entirely unrelated microsatellites showing widespread and obvious similarities in their flanking DNA. This meant that mutations near microsatellites were not random, but favored certain letters in certain positions. Just as a new shipwreck will attract its own special community of marine life, so microsatellites appear gradually to change the surrounding DNA towards a common pattern. The result is convergent evolution, an unusual state of affairs where, as time goes by, DNA sequences become more similar, not less.As yet, the exact mechanisms remain unclear, though it probably has something to do with how comfortably different combinations of letters sit next to each other. In English, “U” always follows “Q” and “B” never follows “V.” Similar rules may apply to DNA, albeit on a much subtler level. For example, if a microsatellite contains alternating As and Cs, the flanking regions also tend to have As at alternate positions, in phase with the As in the microsatellite. It is as if the DNA prefers the pattern in the microsatellite to extend into the flanking DNA, rather than abruptly stopping at the end of the microsatellite.These findings suggest that it may be wise to take the notion of a molecular clock at face value. With a perfect clock, two or three identical mutations would be highly unlikely, but we now know that this may be possible near microsatellites. Vowles and Amos estimate that as much as 30% of the genome may show evidence of convergent evolution, simply because microsatellites are so common. These mutation biases probably exist to a lesser extent in most sequences. Once scientists understand more fully how and where these biases operate, they may be able to estimate more accurately the risk of any given mutation occurring, be it one that causes human disease or makes a virus more virulent. These findings represent yet another windfall from the Human Genome Project, and act as a powerful reminder that unexpected results always lurk around the corner as we delve deeper into the secret world of the genome."}
{"text": "Sequences flanking microsatellites of similar length show remarkable levels of convergent evolution, indicating shared mutational biases. These biases extend 25–50 bases either side of the microsatellite and may therefore affect more than 30% of the entire genome. To explore the extent and absolute strength of these effects, we quantified the observed convergence. We also compared homologous human and chimpanzee loci to look for evidence of changes in mutation rate around microsatellites. Most models of DNA sequence evolution assume that mutations are independent and occur randomly. Allowances may be made for sites mutating at different rates and for general mutation biases such as the faster rate of transitions over transversions. Our analysis suggests that these models may be inadequate, in that proximity to even very short microsatellites may alter the rate and distribution of mutations that occur. The elevated local mutation rate combined with sequence convergence, both of which we find evidence for, also provide a possible resolution for the apparently contradictory inferences of mutation rates in microsatellite flanking sequences.Microsatellites are a major component of the human genome, and their evolution has been much studied. However, the evolution of microsatellite flanking sequences has received less attention, with reports of both high and low mutation rates and of a tendency for microsatellites to cluster. From the human genome we generated a database of many thousands of (AC) An analysis of sequences flanking microsatellites in the human and chimpanzee genomes suggests that mutations do not occur independently and randomly, as is commonly assumed in models of DNA sequence evolution DNA base substitutions do not occur randomly . InsteadMicrosatellites are sequences of repeated 1–6-bp motifs that mutate primarily through the gain and loss of repeat units, in a process thought to depend on DNA replication slippage . PreviouTo our knowledge, no one has yet conducted a systematic study of mutational biases operating around microsatellites. The direct study of naturally occurring mutations in flanking sequences is virtually prohibited by their slow rate of accumulation, and inferences based on comparisons between homologous microsatellite loci rely on small numbers of sequences. However, an indirect approach is possible, based on comparisons among very large numbers of microsatellite flanking sequences from the finished human genome. If microsatellites have little or variable influence on their flanking regions, among-locus similarities will be minimal or absent. Conversely, if microsatellites generate similar local mutation biases, nonhomologous loci should betray evidence of convergent evolution. With the publication of large blocks of sequence from the chimpanzee genome, one can extend this approach to ask questions about rate of divergence between homologous flanking sequences.Here we use a combination of these indirect approaches to show that microsatellites appear to create regions around them in which both the rate and spectrum of mutations are modified.n, and for simplicity considered only ‘isolated’ repeats, defined as those at least 100 bp from the nearest AC repeat as small as two units in length. 47% of AC repeats on human Chromosome 1 match these criteria. From the human genomic sequence, maximum sample size was set at 5,000 randomly selected loci for length classes (AC)2 to (AC)5. For longer microsatellites, of which fewer than 5,000 could be found, all sequences encountered were included. 2+ repeats, was generated from Chromosome 1.We studied the most abundant class of human dinucleotide repeats, (AC)n and (CA)n repeats, (AC)n microsatellites were divided into subclasses, termed ‘cassettes’, according to their immediate 5′ and 3′ flanking bases such that 5′-X(AC)nY-3′ is referred to as cassette X/Y. Hence, a (CA)3 repeat would be classed as cassette C/A around (AC)2. Such a distinction may appear pedantic, but since both DNA replication fidelity and repair efficiency are known to be influenced by base order and local sequence context (n and (CA)n are equivalent. This nomenclature also helps to resolve the problem of defining microsatellite length because (CA)3 equals (AC)2. nY is viewed as comprising two dinucleotides, XA and CY. The probability of observing XA and CY jointly is then calculated from the individual frequencies of XA and CY estimated using 1 Mb of randomly sampled sequence from Chromosome 1 containing no (AC)2+ repeats.To distinguish between (AC) context , it seem2 microsatellites, the cassette frequencies are broadly similar to those expected from the frequencies of the component dinucleotides in unique sequence DNA, though eight cassettes show significant differences (Around (AC)ferences A. Howeve5, since this exhibits the strong patterns while at the same time retaining sufficient sample sizes for analyses to be conducted on the rarer cassette types. Several features are apparent. First, levels of patterning can be remarkably strong, with the probability of observing a given base at a given site varying from less than half of that in unique sequence to more than double, often at adjacent sites. Second, many of the patterns show strong dinucleotide periodicities, presumably reflecting the dinucleotide structure of the microsatellite. Third, there are several examples of clear 5′ to 3′ asymmetry, indicating that mutational patterns on one side of a microsatellite may not be the same as those on the other.We consider flanking sequences to extend 50 bases either side of a microsatellite. To compare flanking sequences, we first divided microsatellites according to (AC) repeat number and cassette type, and then calculated the frequency of each of the four nucleotides at each of the 100 possible positions. Any mutation biases present should be revealed by locally changed base composition, and this appears to be the case. For many microsatellite length–cassette combinations, the flanking sequences exhibit strong deviations from random. The observed patterns can be placed in six broad classes according to the strength of a two-base periodicity and the degree of 5′ to 3′ asymmetry. These patterns are illustrated in 9 but then declines in longer microsatellites. As well as showing variation between cassettes, flanking sequence patterning also changes with microsatellite length. For those cassettes where sample size is sufficient to show a trend, that is, T/A and to some extent C/A and A/A, both amplitude of deviation from random and the breadth of patterning tend to increase with increasing AC repeat number. We do not see dramatic changes such as a reversal in 5′ to 3′ asymmetry or the emergence of new patterns. For cassette T/A, pattern strength increases up to (AC)In view of the strong two-base periodicities seen for some base–cassette combinations, we next examined the distribution of frequencies of all 16 possible dinucleotide motifs. As expected from the single nucleotide patterning, dinucleotides also tend to show periodic patterning  and S2, From 3, then increases towards a peak in strength at (AC)9 before diminishing as AC repeat number increases further. Cassettes A/A and C/A show similar trends and together suggest that, where patterning occurs, it is apparent by (AC)6. When data were plotted without first categorizing them by cassette type, although a number of the dinucleotide patterns in As with the mononucleotide patterning, the periodicity in dinucleotide frequencies changes in amplitude and width 5′ to 3′) as repeat number increases. For most cassettes, the paucity of long microsatellites precludes study of how the patterning changes with repeat number. However, cassette T/A is sufficiently abundant for the progression to be described, and cassettes A/A and C/A, although less common, nonetheless yield meaningful results. ′ to 3′ a5. Classifying loci by cassette type, length, and whether they occurred in LINE/L1, SINE/Alu, or unique sequence DNA yielded in most classes sample sizes too low to be of use. However, where sample sizes were adequate, that is, cassette T/A, dinucleotide AT, patterning in unique sequence loci was indistinguishable from that in LINE and SINE microsatellites 5 to provide strong patterning and large sample sizes. The results of deleting all flanking sequences containing AT microsatellites with n or more repeats, where n = 2, 3, 4, and 5, are given in n allowed, suggesting that the dominant AT patterning results from (AT)n microsatellites developing immediately adjacent to the AC tract.The strongest two-base periodicities we find tend to involve motif AT. Such periodicity might arise in two main ways: either because AT motifs in phase with the microsatellite tend to expand through slippage to form (AT)n repeats. This is an important test given that our strict definition of a microsatellite restricts our analysis to pure AC repeats and allows the possibility that compound repeats, for example, (AT)n(AC)n(AT)n are included. To do this, we plotted the distribution of single AT motifs around (AC)3+ microsatellites in the subset of flanking sequences with no (AT)n microsatellites, where n > 1. Summed over all AC microsatellite lengths, single AT dinucleotides are overrepresented in 5′ sequences at odd numbered positions and at even numbered positions in 3′ sequences (We next examined whether the phase of AT dinucleotides was determined solely by their tendency to form clusters next to (AC)equences . Excesse2 or longer were assigned back to their own class 57% of the time, showing that these sequences consistently differ from those near to AC repeat tracts. Remarkably, this figure falls to almost half (32%) for sequences flanking (AC)2, indicating that, even with just two repeats, similarities to other microsatellite flanking sequences already exist. The same pattern extends to other length microsatellites, with flanking sequences tending to be preferentially assigned back to their own or to an adjacent length class. When a flanking sequence is not assigned back to its own class, it is usually assigned to one of three other classes: the ‘1’ class of random sequences, the ‘9’ class where patterning is strongest, or to the longest class, class 21 (unpublished data).To assess the level of any convergent evolution, we used a simple assignment test to determine how often individual sequences resemble others flanking unrelated microsatellites of similar length see . Figure These analyses reveal a tendency for microsatellite flanking sequences to be similar to each other, but fail to quantify the level of sequence change involved. To do this, we sought to estimate similarity among three classes of sequence: (1) blocks of 50 bp lying immediately adjacent to a microsatellite; (2) blocks of 50 bp chosen randomly to lie between 500 and 600 bases downstream from a microsatellite (the random selection aims to remove possible complications of exact position and phase with the microsatellite); and (3) blocks of 50 bp randomly selected from around the genome. Comparisons within class 3 define the average level chance similarity in the genome, here estimated at 12.77 ± 3.28 (sd) bases out of 50. Comparisons within class 1 estimate how much convergent evolution is apparent at any given repeat number, and reveal a profile that rises to a maximum of 14.31 at a length of seven repeats, followed by a gentle decline with increasing length thereafter A. SimilaThus, in all cases, sequences immediately flanking a microsatellite show greater similarity to each other, to sequences nearby, and to sequences elsewhere in the genome than randomly selected sequences do to each other, a trend that is maximal for microsatellites around 7–10 repeats in length. We believe these similarities are generated primarily by the enhanced simplicity of microsatellite flanking regions and their tendency to gain AT motifs. Such characteristics allow unusually high matches when compared with random blocks of 50 bases that have high simplicity or contain polyA tracts. Over and above this background level of elevated similarity, proximity to any microsatellite appears to increase similarity, implying that microsatellites tend to lie more generally in regions of similar base composition. This might reflect either a tendency for microsatellites to arise preferentially in certain broad sequence contexts, or modification of the local base composition by the microsatellite itself. Moreover, similarity is further enhanced when a flanking sequence is compared with a neighbouring block. This suggests a local context effect such as might arise through the isochore structure of the genome, with neighbouring blocks being located in the same isochore and hence ‘coloured’ by the same nucleotide biases.2 flanking sequences to their own class is significant up to ten bases away from the microsatellite , and only when the window reaches 24 bases from the microsatellite does the assignment level fall to the value of 4.8% expected of random sequences.To define more precisely the regions where convergent evolution is occurring, we repeated the assignment test but instead of using the full 50 bases either side we now used a symmetric pair of moving 25-base windows placed either side of the AC microsatellite . Close tftp://ftp.ncbi.nih.gov/blast/executables) to compare microsatellites identified in completed sections of the chimpanzee genome against homologous loci identified in humans. If homologous loci are identified through comparisons among their immediate flanking sequences, an element of circularity will be introduced, since loci with lower rates of evolution will be identified preferentially. To circumvent this problem, we used a region 300 bases in length and 220 bases downstream of the microsatellite to conduct each Megablast search.So far we have considered only the nature of the mutations that affect flanking sequences, and not their rate. To examine whether mutation rates are affected by the presence of a microsatellite, we used Megablast ; A total of 8218 chimpanzee loci were identified, and they yielded 5537 unique human homologues. Since microsatellite flanking sequences may contain insertions or deletions, we adopted the following approach so as to minimise problems of alignment. The chimpanzee flanking sequence was divided into 20 contiguous blocks of 20 bases, ten on each side of the microsatellite. Each block was then compared against 1,000 bases of human sequence, downstream from the region identified by Megablast, to find the best possible match. To filter sequences with major rearrangement we required both that all 20 blocks match with at least 15/20 bases, and that matched blocks all lie in the same order as their homologues. Differences between pairs of homologous flanking sequences were then quantified, at each of the 20-block positions, as the proportion of perfectly matching blocks and the average number of matches within each block .2–3 there is little apparent variation in either measure of similarity with block position apart from a small tendency for 5′ blocks to show an increasing average percentage match closer to the microsatellite. However, although the trends are by no means strong, around longer microsatellites similarity of blocks near to the microsatellite is reduced, particularly when measured using average percentage matches , and among these, approximately 77% of the blocks were identical. Around (AC) matches .Our analysis of dinucleotide frequencies around microsatellites shows patterns of similarity on a smaller scale than might be revealed with 20-base blocks of sequence. To investigate the number of changes in the immediate flanking bases, we also calculated the proportion of mismatches occurring at a given base for each 5′ and 3′ sequence in the immediate flanking blocks 9, but appears present even around sequences as short as (AC)2 and may extend as many as 50 bases either side. Some patterning is more or less symmetrical, but we also found several examples showing strong 5′ to 3′ asymmetry, implying that the two ends of a microsatellite are by no means equivalent. The net result is that sequences flanking microsatellites of a given length tend to be more similar to each other than to random sequences or to sequences flanking microsatellites of different lengths. Thus, there appears to be convergent evolution. Finally, we compared large numbers of homologous flanking sequences between humans and chimpanzees, and found evidence that mutation rates near microsatellites tend to be somewhat elevated.We have studied very large numbers of (AC)n tracts exhibit remarkable levels of patterning, with any given dinucleotide motif tending to be much more likely to occur at even numbered positions rather than odd, or vice versa. For several reasons, we believe that the patterning arises due to the structural properties of the microsatellite (see below), becoming more pronounced as repeat number increases. These reasons include the following: the consistently central placement of microsatellites within the patterning, the dependence of the strength of patterning on AC repeat number, the similarity between microsatellites in LINE and SINE elements and those elsewhere, the weakness of the patterning around (AC)2, and the strong influence of cassette type on the form of patterning. Unfortunately, it is surprisingly difficult to eliminate the alternative hypothesis, namely that the patterning arises due to some other force and that AC repeats then either form or expand more rapidly when placed centrally within the pattern. This ambiguity is particularly relevant to the question of cassette distribution, where it seems reasonable both that (AC)n tracts might cause biased interconversion between cassettes and that certain cassettes may allow slippage more than others. For example, while the structural properties of AC repeats are known to generate mutational biases in adjacent bases (Sequences surrounding (AC)nt bases  capable nt bases .2 already showing a small but significant bias in the distribution of cassette types and greater similarity to other sequences flanking AC microsatellites than to random sequences. In addition, the moving window assignment test indicates that significant convergence exists even when the ten bases closest to the microsatellite are excluded. Such a wide influence around such a common, short motif is remarkable and suggests that a high proportion of the genome may be affected by these and similar forces. To illustrate, (AC)2 is expected to occur every 250 bases, as is (GT)2. Taking the sphere of influence on each side as ten bases plus half the 25-bp window yields a value of 45. This predicts that over 30% (approximately 45 bases of every 125) of the genome will be affected by (AC)2 on one strand or the other, a figure that will only increase with inclusion of longer arrays and other microsatellite motifs.The relationship between an AC microsatellite and its flanking sequences begins surprisingly early, with (AC)5 and peaking in strength at (AC)9. Although patterning is seen in several different dinucleotide motifs, even in the human genome there are insufficient data to study any but the commonest cassette–motif combinations over a wide range of microsatellite lengths. Focusing on the motif AT, we found evidence that the strongest patterning was due to the development of AT microsatellites abutting AC tracts. However, this is not the only effect. After removal of all (AT)2 or longer microsatellites, there remains a significant tendency for single AT motifs to appear in phase with AC tracts, suggesting that mutation bias as well as slippage is involved.As AC repeat number increases, so does the strength of patterning, becoming pronounced by (AC)2 and (AC)9, it might seem logical that the pattern would become stronger and stronger as repeat number increases further. Instead, (AC)9 appears to be the peak strength, with longer microsatellites showing lower amplitude but a broader spread of patterning. It is interesting that this peak coincides with the length at which microsatellites begin to become polymorphic: a common rule of thumb for marker development in mammals is that primers are designed for loci carrying ten or more repeats (Given the increase in strength of patterning between (AC) repeats . This ma repeats . Again,  repeats .The rich patterning we find presumably arises through local mutation biases. Previous work on mutation biases has tended to reveal either generic effects such as isochors , where sn and (A)n induce local mutation biases  or A to Y (pyrimidine) transversion mutations, and also the increase in pattern strength around longer microsatellites, with interruptions in longer arrays more likely to be internal to the repeat tract and hence be excluded from the analysis. However, we suggest that this model is unlikely for two reasons. First, such a model fails to accommodate the strong asymmetry in patterning that is observed for some dinucleotides and specific cassette bases around the (AC)n repeat tract. Polarity has been noted for minisatellite mutations, with mutational processes differing between the two ends of the repeat tract at tract , but a mat tract .The patterning we describe appears to represent an important component of the forces that shape genome evolution, both in terms of its ubiquity and the absolute strength of its effect. It follows that there are many possible practical and theoretical implications. For example, even very short microsatellites appear able to cause some level of convergent sequence evolution, and hence to confound phylogenetic analyses. Similarly, microsatellites near genes may increase local mutation rates and influence the spectrum of new mutations that arise. To explore the size of these effects we designed experiments both to measure absolute convergence and to ask about evidence for changes in mutation rate.7–10 both for assignment to their own class and for similarity to each other relative to random blocks provide a clear indication that convergent sequence evolution is occurring. Interestingly, any given flanking sequence tends to be more similar to a block 500 bases away than to a similarly placed block near a different microsatellite, suggesting longer range patterning such as might arise through placement within the same isochore isochore . FurtherIn conclusion, previous studies of microsatellite flanking sequences have identified several features, including a tendency to harbour other microsatellites, a locally increased mutation rate, and, conversely, conservation over unexpectedly large tracts of evolutionary time. Our analyses support all these trends and provide a possible resolution for the apparent contradiction between faster evolution but at the same time greater sequence conservation. Although there is evidence that mutation rates near microsatellites are elevated, we also find evidence of convergent evolution. Consequently, the increased rate of change may be to some extent neutralised and perhaps even reversed by the tendency for similar changes to occur in related lineages. Furthermore, the greatest changes appear to occur in flanking sequences around microsatellites that are below the length used as markers, at least in humans. Overall, therefore, we have been able to formalise previous anecdotal evidence and hence to document a remarkably widespread source of directional change and nonrandom evolution that undoubtedly plays an important role in shaping the make-up of our genomes.n dinucleotide repeats was extracted from the human genome  using a custom macro written in Visual Basic. Only microsatellites separated by at least 100 bp from the nearest (AC)2 or longer were included in the dataset. Thus (AC)2AT(AC)10 would not be included in the dataset, whereas ACAT(AC)10 would be included as (AC)10. Flanking sequences are here defined as the 50 bases lying either side of a microsatellite. No attempt was made to translate TG repeats with complementary AC repeats on the opposite strand. Consequently, all our microsatellites are 5′-(AC)n-3′.Our dataset of (AC)n < 200 observations) or a normal approximation (n ≥ 200 observations). Bases used to define cassette type were excluded from all calculations, and expected frequencies were taken as the average frequency across all positions.For each frequency estimate, 95% confidence intervals were derived based on the binomial distribution (5 and declined to 175 for (AC)20 C)20 see .As an index of similarity, we calculated the log likelihood of observing a given sequence based on its position-specific dinucleotide motif composition:ijkf is the frequency of dinucleotide i at position j  in flanking sequences of class k. To avoid bias, when a sequence was compared with its own class, its contribution to the dinucleotide frequencies was first removed. For each sequence in turn, A was calculated for every class and the sequence was then assigned to the class that yielded the highest index value. Under convergent evolution, we expect sequences to tend to be assigned to their own or similar length classes.where Sequences were again divided into length classes 2 to 21, and each sequence contributed four 50-bp blocks of sequence, one from each side immediately adjacent to the microsatellite but excluding the cassette bases (class 1), and one from each side displaced by a randomly selected number 500–600 bases distal (class 2). In addition, we also generated a database of 5,000 non-microsatellite-associated sequences. When making comparisons within a class, nonindependence was avoided by randomising the sequence order and then comparing sequence 1 with sequence 2, 2 with 3, …, (n − 1) with n. Our index of similarity was simply a count of the number of matching bases. A few pairs of sequences (less than 0.1%) gave high similarity scores of over 30/50 matching bases, presumably because these loci have been duplicated or lie in repetitive elements. Such sequences were discarded. As with all other analyses, sequences containing base ambiguities (marked base N) were also discarded.n repeat microsatellites were extracted from the available chimpanzee finished-quality high-throughput genomic sequence (NCBI) as outlined above for humans. A 300-base region 220 bases upstream from each chimpanzee microsatellite was used by Megablast  to identify homologous human loci in the finished genome sequence. Sequences with multiple high-scoring hits were discarded, as they presumably occur because a locus is found in repetitive elements or has been duplicated. Those nonoverlapping hits with at least 280/300 matching bases and an expectation  greater than five times that of any other hit to the same sequence were thus retained, giving a dataset of 5,537 sequences.(AC)Figure S1From little structure of any kind (A) to complicated aperiodic clustering (C). Plots are as described in (1.7 MB TIF).Click here for additional data file.Figure S2Plots are as described in (3.0 MB TIF).Click here for additional data file.Figure S3Figure depicts equivalent patterns of asymmetry in AT dinucleotide frequencies for the commonest cassette type, (T/A), around microsatellites in unique sequence DNA (A), LINE/L1 elements (B), and SINE/Alu elements (C). Plotting conventions are the same as for (1.4 MB TIF).Click here for additional data file.Figure S4Block position is relative to the central microsatellite (not shown).2–3 microsatellites .(A and B) Proportion of exact matches  and average number of matches, excluding exact matches (± standard error), with block position around (AC)4+ microsatellites (n = 356). Average proportion of exact matches and number of matches, calculated separately for 5′ and 3′ blocks around (AC)2–3 microsatellites, are shown by a black line in (A) and (C), and (B) and (D), respectively. Average percentage match rather than average match is plotted in (B) and (D) because overlapping blocks were truncated to exclude overlapping regions from the analysis, with the result that not all blocks contained 20 bases.(C and D) As (A and B) but for (AC)(2.9 MB TIF).Click here for additional data file.Figure S52–3 microsatellite loci (A) and over (AC)4+ microsatellite loci (B). Shown ± standard error. The microsatellite at base position 0 is not shown. Expectation, calculated separately for 5′ and 3′ sequences around (AC)2–3 microsatellites, is shown by a black line in both plots.The proportion of mismatches occurring at a given base in a flanking sequence are averaged over (AC)(3.4 MB TIF).Click here for additional data file."}
{"text": "Out of 130 ovarian cancer patients the DNA index of cells from ovarian carcinoma was studied in 56 cases in which cytospin preparations showed the presence of atypical cells. In 24 patients the population had a diploid DNA index (1.0) and in the others the DNA index ranged from 1.2 to 2.0 (tetraploid). No hypodiploid or hypertetraploid populations were detected. Repeated samples from the same patients did not show any significant differences and primary culture did not alter the DNA index. In contrast, cell cycle phase distribution differed greatly from sample to sample, as also the ratio between DNA diploid and DNA aneuploid populations. Primary culture was successful in 57% of the tumours, with a higher percentage of success in DNA aneuploid tumours. After primary culture the ratio between DNA aneuploid cells and DNA diploid cells increased. In relation to the histological gradings of malignancy, DNA aneuploid cells clustered in the highest grade of malignancy. The mean S-phase for tumours with a DNA index of 1.0 was 3.5 and 14.1% for those with DNA index greater than 1. Ovarian carcinomas show a large difference in DNA index between patients even after primary culture."}
{"text": "By Ronald J. TrentAmsterdam:Elsevier, 2005. 320 pp. ISBN: 0-12-699057-3, $79.95Molecular Medicine: An Introductory Text, like the previous editions, continues to provide a contemporary and succinct overview of DNA in medicine. Chapter topics include a history of molecular medicine, DNA, RNA, genes, and chromosomes; Mendelian genetic traits; complex genetic traits; genomics, proteomics, and bioinformatics; genetic and cellular therapies; reproduction and development; infectious diseases; forensic medicine and science; and ethical, legal, and social issues. The new chapters added to this edition represent the developments in genomics, proteomics, and bioinformatics.The third edition of Molecular Medicine valuable because the author pulls together a comprehensive assessment, highlighting Internet-based resources that greatly assist readers in further exploring the depths and details of the related topics.Many books describe DNA in medicine, from monographs to multiple-author heavy volumes, but the uniqueness of this single-author introductory textbook of molecular medicine is that it simplifies the seemingly complex concepts of DNA in medicine so that readers can find an easy entry into the world of molecular medicine. This certainly helps medical students and biomedical researcher trainees to grasp the fundamentals of the field. This book also contains many clinical examples that make the reading more relevant to practice and raise the level of interest. Senior scientists and health care professionals will also find This introductory textbook presents a well-balanced incorporation of the basic concepts, applicable clinical examples, advances in molecular biology and their impact on medicine, and gaps that will be filled by further developments in DNA biology and medicine. Emphasizing the role of DNA in molecular medicine, the book provides an interesting history of the development in DNA knowledge, covering the early discovery of DNA structure to the completion of human genome project. Detailed relationships among DNA structure, biology, and medicine highlight the role of DNA in medicine, which is further enhanced by an explanation of traditional and complex genetic traits. The incorporation of genomics, proteomics, and bioinformatics adds a new dimension. The author’s presentation of the principles of using DNA information for genetic and cellular therapies and their applications for reproduction and development, infectious diseases, forensic medicine, and basic science provides a more practical view of DNA in medicine. Finally, the discussion of ethical and legal issues, of concern to the public, brings health care professionals and laboratory scientists an awareness of the social implications of molecular medicine.The author’s definition of molecular medicine in this book—used to describe the role that knowledge of DNA is having on medical practice—may mislead readers to assume that molecular medicine has to do only with the manipulation of DNA. Posttranslational modification of proteins is a critical part of molecular medicine. Although the author has covered this aspect within the context of the book, the definition should have considered the independent role of proteins in molecular medicine as a basis for the text.This book can be recommended to medical students, health care providers, and laboratory researchers as an entry-level textbook, introductory reference, and fundamental resource for knowledge of molecular medicine. It would easily open the door for all medical professionals to molecular medicine, its past, present, and future."}
{"text": "Ascaris lumbricoides, Schistosoma japonicum and Trichuris trichiura were 30.9%, 15.7% and 47%, respectively. Hookworm infection prevalence was low (0.7%). A significant association was observed between A. lumbricoides and T. trichiura infection, and also between S. japonicum and T. trichiura infection. Variance components analysis was undertaken to investigate the aggregation of S. japonicum and the soil-transmitted helminths, A. lumbricoides and T. trichiura. While A. lumbricoides was found to aggregate only at a household level, T. trichiura was shown to cluster predominantly in families. Both genetic and household effects were found to be important in determining the risk of infection with S. japonicum. Variance components analysis for A. lumbricoides/T. trichiura co-infections indicated a significant domestic environmental effect, attributable for 32.7% of the co-infection risk. Aggregation of S. japonicum/T. trichiura co-infection was also observed at a household level. The risk of infection with multiple helminth species, although mainly environmentally influenced, was also shown to have significant involvement of genetic and household components. The results of this study indicate that a shared household is a major contributing risk factor for helminth co-infections and emphasises the need for increased standards of sanitation and hygiene to prevent parasite transmission. Further, the results suggest that susceptibility to one helminth infection is not completely independent of another, and that there exist common genetic factors underlying infection with multiple helminth species.Human helminthiases are common in China, especially in rural areas where sanitation conditions are poor. Co- and multiple infections with helminths are strikingly frequent. A cross-sectional parasitological and questionnaire survey was carried out in a population of 3205 individuals belonging to 498 families from five villages in the Poyang Lake region, Jiangxi Province, China, to assess their helminth infection status and to collect information on risk factors for infection. The prevalences for  Indeed,ng China . Althougng China  indicatemic foci . Given tthe norm .A. lumbricoides in Nepal  but were still included as dummy relatives in order to link families further. In-laws, who were not biologically related to anyone else in a family (i.e. had no children), were not included, and thus were not used in the household analysis. When inconsistencies were identified between the questionnaires of relatives (i.e. a wrong PID code of a relative), the information was checked, where possible, or omitted from the final data analysis. The final data comprised a total of 3205 individuals, belonging to a total of 498 families which ranged in size from three to 188 and spanned as many as four generations.2.3S. japonicum and STHs  eggs and 10% of slides were re-read for quality control by a senior microscopist ; minor infection (5–12 EPG); moderate infection (13–60 EPG); high infection (61–132 EPG) and highest infection (133+ EPG).All individuals from the study population were asked to provide two stool samples for parasitological examination using the Kato–Katz thick smear technique . The secoscopist . Positivpulation ; as a re2.4P < 0.001). Variance components analysis is based on a regression model whereby the phenotype is assumed to be normally distributed. If this assumption is violated, the standard errors of the parameters are underestimated and can result in type I errors, thus leading to incorrect conclusions on the model of best fit. As a result, only the binary phenotype (infected versus uninfected) and not EPG was used for S. japonicum. Aggregation of multiple helminth infections was investigated using the number of infections with any parasite as the phenotype. For this purpose a variable was generated, with values ranging from 0 (having no infection) to 3 (harbouring three parasite species), and was treated as a continuous variable with a normal distribution . If the phenotype under investigation is dichotomous, and therefore cannot be normally distributed, a multifactorial threshold model is used that assumes: (i) that there are several factors that are involved in the disorder in question; (ii) that the effect of each individual factor is small but that the effects of each factor are additive; and (iii) once the additive effect of these factors pass a critical threshold, an individual becomes affected. This implies that the underlying liability of affection is normally distributed but once the threshold is reached, one becomes affected and the phenotype is dichotomous.All collected parasitological and questionnaire data were double-entered into an Access database and cross-checked. Investigation of prevalence estimates, parasite distributions and associations was carried out in SPSS 13.0. SOLAR, a Unix-based software program, which was also used to carry out the variance components analysis , the modBriefly, in a variance components analysis, the total residual variance can be expressed as the sum of the additive genetic P < 0.05) identified in the bivariate logistic regression were included in the models as well as administration village (village effects) to account for any potential geographical differences between the study sites. The software implements a maximum likelihood method which provides a log-likelihood estimate for each model. As all models are nested on the household/polygenic model, they can then be compared with one another using a likelihood ratio test (LRT) which calculates a chi square value to be twice the difference between the likelihoods with one degree of freedom. The significance of the household and genetic effects can thus be tested individually (versus the sporadic model) and in the presence of each other (versus household/polygenic model). The model of best fit was selected as the model with most significant effects (<0.05).Here, all significant covariates  who were involved in the project. Study participants identified as stool egg-positive for schistosomiasis were treated with 40 mg/kg of praziquantel, the current dosage recommended by the WHO. Subjects infected with STHs were advised to seek treatment from a local doctor or hospital.33.1A. lumbricoides and T. trichiura ranged from 0% to 38.8% (mean 30.9%), and from 2.4% to 58.1% (mean 47%), respectively, whereas the S. japonicum prevalence ranged from 12.2% to 20.9% (mean 15.7%). Hookworm infection prevalence was low (0.7%), and was not considered in further analysis. The difference in helminth prevalence between the administrative villages was highly significant . Of the total population, 61.7% harboured at least one helminth species and 27.8% were identified as harbouring at least two parasite species. Again, differences between the mean number of helminth species harboured by individuals among villages were highly significant  although the overall distribution of the number of parasite species among individuals was normal.Among the five administrative villages, the observed prevalences for S. japonicum. Individuals with low S. japonicum infection intensity were shown to have a higher mean number of parasite species. When a higher intensity of infection was experienced, the number of other helminth species decreased. The mean number of parasite species increased again in those with highest egg counts. These differences between the egg count groups were not significant .3.2S. japonicum and A. lumbricoides infections. However, there was a significant (P = 0.03) increase in risk of T. trichiura infection in an individual infected with S. japonicum  = 1.03–1.60). Further, there was a strong association between A. lumbricoides infection and T. trichiura infection .There were no associations observed between S. japonicum compared with older age groups. Gender, water contact and the number of past treatments were highly significant risk factors for S. japonicum infection. There was no age or gender association with A. lumbricoides infection; however, there was a significant association with water contact. Age and water contact were significant risk factors for T. trichiura infection. The highest T. trichiura prevalence (63.4%) was observed in the 5–14 years age group.3.3A. lumbricoides indicating that 31.7% of risk of infection was attributable to domestic environment. No clustering of infection was observed at a familial level. T. trichiura infection was best described by the polygenic model; heritability was strong (29.9%) and significant (P < 0.05). Significant heritability was also estimated for S. japonicum infection (23.7%) as well as a strong household component (24.7%). The Kullback–Leibler R2 value estimated for all models was low, indicating that the covariates did not account for much of the variation in the models.Four models were tested for each individual helminth infection: environmental, household, polygenic and general (household and polygenic). All models were compared with each other using the LRT. The favoured models for each infection, with corresponding parameter estimates, are provided in 3.4A. lumbricoides and T. trichiura infection, a variance components analysis was undertaken to determine the factors underlying risk of having both infections concurrently (P < 0.05) and accounted for 32.7% of the risk of being infected with both parasites.Given the strong association observed between urrently . HousehoS. japonicum and T. trichiura infection was also investigated. Again a household model was favoured with a strong effect size of 43.16% .The variance components analysis estimated a heritability of 16.32% and a household effect of 8.8% for multiple helminthic infections. When both the household model and the polygenic model were compared to the general model , both ge4S. japonicum infections, males were more likely to be infected than females  was not shown to be high in any of the models and thus had little impact on the final parameter estimates. The variance components analysis for A. lumbricoides infection indicated household as a major contributing risk factor. In contrast to a previous study by T. trichiura infection although there was no aggregation at the household level. These findings are similar to those of S. japonicum infection as well as a genetic involvement. Heritability remained high at 23.7% and was a significant risk factor; household was found to account for 24.7% of the risk of infection. These results are comparable with those of studies on S. mansoni . This again may be explained, in part, by the commonality in the mode of transmission but, more notably, it could be a reflection of the socio-economic status of a particular household and the sanitation and hygiene conditions prevailing. Although data on household-specific risk factors were not collected (and merit further investigation) the results suggest that while mass chemotherapy is fundamental in the control of geo-helminths, an emphasis on the development of sanitation and clean water facilities, improved health and hygiene awareness, and more targeted treatment of those living in poor conditions is crucial to prevent transmission. Studies where sanitation and health awareness in a population have been improved and followed over time have shown marked decreases in prevalence that had been previously unattainable through chemotherapy alone  and to identify possible gene–gene and gene–environment interactions involved in single and multiple infection events. As such, this genetic analysis may result in the possible identification of new drug targets and more targeted treatment of individuals susceptible to infection with parasitic helminths."}
{"text": "Coxiella burnetii, the etiological agent of Q fever, is based on a semi-synthetic, second-generation tetracycline, doxycycline. Here, we report on the comparison of the proteomes of a C. burnetii reference strain either cultured under control conditions or under tetracycline stress conditions. Using the MS-driven combined fractional diagonal chromatography proteomics technique, out of the 531 proteins identified, 5 and 19 proteins were found significantly up- and down-regulated respectively, under tetracycline stress. Although the predicted cellular functions of these regulated proteins did not point to known tetracycline resistance mechanisms, our data clearly reveal the plasticity of the proteome of C. burnetii to battle tetracycline stress. Finally, we raise several plausible hypotheses that could further lead to more focused experiments on studying tetracycline resistance in C. burnetii and thus reduced treatment failures of Q fever.The recommended antibiotic regimen against  Coxiella burnetii, until recently considered to be an obligatory intracellular bacterium C. burnetii infection can result in outcomes ranging from asymptomatic seroconversion to death, the most typical clinical manifestations of acute Q fever are fever (91%), severe headaches (51%), myalgias (37%), arthralgias (27%) and cough (34%) Treatment of both acute as well as chronic Q fever is based on doxycycline, a semi-synthetic and second-generation tetracycline invented and clinically developed in the early 1960s C. burnetii strains resistant to doxycycline (MIC of 8 mg/mL) have been isolated from patients with Q fever endocarditis C. burnetii was recovered from cardiac valve tissue removed from a patient with Q fever endocarditis despite 4 years of antibiotic therapy with tetracycline Doxycycline in combination with hydroxychloroquine is the main therapy for treating patients with chronic Q fever. However, the optimal duration of therapy for chronic Q fever is unknown and ranges from 18 months to life-long antibiotic administration Tetracyclines are broad-spectrum agents and exhibit antibiotic activity against a wide range of microorganisms. Their favorable antimicrobial properties and the absence of major, adverse side-effects have led to their extensive use in treating infected humans and animals. Tetracyclines inhibit bacterial protein synthesis by preventing the association of aminoacyl-tRNA with the bacterial ribosome Resistance to tetracyclines mainly occurs through five mechanisms: production of ribosomal protection proteins (RPPs), active efflux of tetracycline from the cell, decreased drug permeability, mutation of the antibiotic target and their enzymatic degradation. Of note is that the first two mechanisms currently predominate in clinical settings C. burnetii (CbuG_Q212) either propagated in the presence of doxycycline or propagated without any antibiotic present. The CbuG_Q212 strain was particularly chosen since it is considered as a prototype C. burnetii chronic disease isolate in vivo conditions and might have increased possibilities to develop drug resistance. The COFRADIC proteomics technology  was used here, which is a so-called gel-free proteomics technology based on the principle of diagonal chromatography Coxiella burnetii can implement to resist protein synthesis inhibition by tetracyclines..Here, we compared the proteomes of the reference strain The COFRADIC technology used here enriches for methionine-containing peptides out of protease-digested proteomes with the aim to reduce the sample complexity before the actual LC-MS/MS analysis, and thereby, attempts to increase the overall proteome coverage The Rover tool developed by the Gevaert lab was used to compare the proteomes of both bacterial samples in silico analysis of these 24 proteins.In total, 24 proteins were found significantly regulated upon doxycycline treatment; 5 proteins were up-regulated, whereas 19 proteins were down-regulated as a result of doxycycline treatment. PSORTb v.3.0 was further used to allocate the identified proteins to the cytoplasm, the cytoplasmic membrane, the periplasmic space, the outer membrane or to extracellular sites. 5 proteins were annotated to reside in the cytoplasm, 3 in the cytoplasmic membrane, 1 was in the periplasmic space, but the majority (15) had no known localization. The regulated proteins are involved in energy metabolism (2/24), transport and binding (4/24), protein fate (2/24), amino acid biosynthesis (2/24), biosynthesis of cofactors (1/24), purine and pyrimide (1/24), DNA metabolism (1/24) and detoxification (1/24). Finally, the overrepresentation of proteins with unknown function (10/24) is of particular note .C. burnetii changed upon tetracycline stress, with the goal of providing insights into as of yet unexplored mechanisms of tetracycline resistance, which might eventually point to protein(s) that could be considered for targeting when developing novel drugs. Comparison of the proteome profiles of the two C. burnetii samples indeed pointed to several proteins with regulated expression under tetracycline stress conditions, and these proteins are discussed in the following sections.The aim of the present study was to analyze how the proteome profile of 5 proteins were found to be up-regulated upon doxycycline treatment. The predicted cellular functions of these proteins were protein and peptide secretion and trafficking (Q83CL5), energy metabolism, pentose phosphate pathway (Q83DM4), biosynthesis of pantothenate and coenzyme A (Q83EA2), aspartate biosynthesis (Q83A62) and DNA replication (Q83C00).E. coli, which spans the membrane twice C. burnetii to survive under tetracycline stress conditions. Furthermore, the functional relation of this protein with secreted and membrane proteins suggests a possible role for additional activation of such proteins by SPI and thus a more active mode of excretion of the antibiotic. Type I SPases can be irreversibly inhibited by certain penem compounds that acylate the serine residue in the active site. Since SPase activity was found to be essential for cell viability, SPases are considered to be potential targets for developing novel antibacterial agents C. burnetii SPI as a possible target for inhibition by penem derivatives.Signal peptidase I (SPI) (Q83CL5) belongs to a group of serine proteases Staphylococcus aureus strains has been reported, its increase was not associated with a possible mechanism of bacterial resistance against antibiotics Transaldolase (Q83DM4) functions in the pentose phosphate pathway and is almost ubiquitously found in the three domains of life . It provides cells with important redox cofactors such as NADPH, building blocks for nucleotide and nucleic acid biosynthesis The DNA polymerase III alpha subunit (Q83C00) might also be associated with a molecular mechanism of antibiotic resistance. This protein is considered to be the main mediator of pathogen survival through induction of mutagenicity. It contributes to the creation of mutants that are able to address the host immune system and administered antibiotics http://cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi). Coenzyme A has been implicated in resistance mechanisms against aminoglycoside-based antibiotics (AGs). AGs bind to the 30S subunit of the prokaryotic ribosome and interrupt bacterial protein synthesis, a mode-of-action that is similar to that of tetracycline. Coenzyme A is directly involved in the enzymatic covalent modification of AGs, which causes a strong decrease in the AG's binding affinity to the prokaryotic ribosome C. burnetii's coenzyme A to tetracycline resistance.3-methyl-2-oxobutanoate hydroxymethyltransferase (Q83EA2) participates in the biosynthesis of pantothenate and coenzyme A (see http://cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi). Despite extensive literature search however, no connection between methionine biosynthesis and antibiotic resistance mechanisms was found.Methyltetrahydropteroyltriglutamate–homocysteine methyltransferase (Q83A62) was also over-expressed upon doxycycline treatment. This enzyme is involved in methionine biosynthesis , aspartate biosynthesis (P24703), detoxification (Q83AQ8), threonine catabolic process (Q83F39), purine ribonucleoside salvage (Q83FC4), transport and binding  and unknown function . One example of the latter is ScvA (B5QSC0), a highly basic DNA binding protein specific for the small cell variant (SCV) of C. burnetii infection was compared with an acute one, and found to be only present in the acute strain Down-regulation of the phosphoenolpyruvate-protein phosphotransferase (Q83BF9) is of particular interest. This protein was identified in a previous study in which a persistent model of 2+) is a cofactor in a variety of biological processes and many bacteria transport iron through Feo systems (ferrous iron transport). It has been suggested that bacterial iron metabolism is relevant for the activity of antibiotics and that mutations in proteins important for this metabolism might challenge bacterial antibiotic susceptibility Pseudomonas, whereas its over-expression accelerates antibiotics-induced cell death C. burnetii down-regulate iron transport to avoid cytokine activation.Levels of the ferrous iron transport protein B (Q83AW2) were also found to be down-regulated. Iron  found down-regulated upon doxycycline treatment . Ribosomes are clearly of utmost importance for controlling protein expression. The pY protein has been studied in Escherichia coli without an apparent role in the fidelity of protein synthesis, suggesting a role in tetracycline resistance Of further note is that the vast majority of the proteins with predicted role in translation and protein biosynthesis (69 out of 77 proteins identified) had protein ratio values that place them at the border of statistically significant over-expressed proteins upon doxycycline treatment. Furthermore, stretching the limits for statistical significance a bit, of the additional 158 proteins with ratio values up to 1.1, the predicted cellular function of 52 of them was translation and protein biosynthesis. This observation, coupled to the mode of action of tetracyclines leads us to suspect the existence of a mechanism protective for bacterial ribosomes, a well-known tetracycline resistance mechanism. Tetracycline resistance by expressing ribosomal protection proteins (RPPs) is achieved by weakening the interaction of tetracycline and the ribosome with subsequent antibiotic release ++-driven multidrug efflux pump (Q83DC6) have been identified in our study. In silico analysis of these proteins identified 11 and 12 transmembrane helices, respectively (http://www.cbs.dtu.dk/services/TMHMM-2.0/), and thus, these proteins could be capable of secreting tetracycline.As mentioned before, active efflux of tetracycline from bacteria is the second most predominant tetracycline resistance mechanism in clinical settings. This efflux mainly occurs via integral membrane transporters that belong to the major facilitator transporter superfamily (MFS) C. burnetii strain C. burnetii, when referring to antibiotic resistance, one may not simply limit the potential mechanisms of resistance to those already known from other studies. Instead, we must consider that resistance to an antibiotic is not exclusively the result of a mutated gene, but rather a multifaceted process as indicated by the proteome rearrangements observed in this study.The fact that certain antibiotics may be signaling molecules at low concentrations C. burnetii strain propagated under tetracycline stress conditions with an antibiotic-free cultured strain identified 5 up-regulated and 19 down-regulated proteins upon tetracycline stress. The predicted cellular functions of the differentially regulated proteins include secretion and trafficking, energy metabolism, amino acid biosynthesis, DNA metabolism and protein fate. Several plausible hypotheses have been formed to establish a link between these proteins and tetracycline stress conditions. Our further analysis of the identified proteins indicated the proteome potential of C. burnetii to resist tetracycline. Higher antibiotic concentrations or a proteome analysis of a tetracycline resistant C. burnetii clinical isolate could further indicate proteins that are directly implicated in tetracycline resistance mechanisms, leading to more effective drug treatment and minimization of treatment failures.Quantitative proteome comparison of a C. burnetii Q212 (CbuG_Q212) phase II Q fever reference strain was propagated in confluent African Green Monkey kidney fibroblasts  in 225 cm2 angled neck flasks . Infected cells were cultured in minimum essential medium  supplemented with 4% fetal bovine serum  at 35°C in a 5% CO2 incubator. Infection was monitored by Gimenez staining g for 15 min. The pellet was re-suspended in fresh culture medium, and the cells were disrupted by three freeze-thaw cycles (from −210°C to 37°C). After centrifugation at 1000×g for 15 min, the supernatant with the liberated bacteria from the infected cells was used to infect fresh Vero cell monolayers. The inoculum was removed after 1 h of incubation at 35°C in a 5% CO2 atmosphere, the cells were washed with culture medium in order to eliminate unphagocytosed bacteria, and fresh medium containing 0.2 mg/L of doxycycline (DOX) was added. The monolayer was incubated at 35°C in a 5% CO2 atmosphere. The supernatant was removed every three days, and fresh medium containing 0.2 mg/L of DOX was added. The same procedure was followed with constantly increasing concentrations of the antibiotic until bacteria were collected for inoculation titration test and MIC determination The antibiotics used in this study was doxycycline hydrochloride (DOX) . DOX was directly diluted into freshly prepared culture medium to the appropriate concentration. Culture media containing the antibiotics were sterilized by filtration (pore size: 0.22 µm) and stored at 4°C. DOX was shown, by shell vial assay (data not included), to be nontoxic to Vero cells to concentrations up to 128 mg/L.C. Burnetii, inoculation titration tests and Minimum Inhibitory Concentration determinations were performed as described previously 2, the MIC of DOX was determined by direct immunofluorescence as the concentration of the antibiotic at which there were no intracellular bacteria observed following a six days incubation period. Even though during MIC determination tests bacteria were initially challenged by an initial concentration well beyond the MIC mentioned in the literature for C. burnetii Q212 , negative controls (no antibiotic present) were included at all times. During C. burnetii culture under tetracycline stress conditions (15 weeks), the observed MIC for doxycycline increased from 2 mg/l to 16 mg/l (MIC Q212Dox: 16 mg/l), while bacteria propagated in the absence of antibiotic for the same time period, did not show a MIC change (MIC Q212: 2 mg/l).To determine the effect of tetracycline in the culture medium on the MIC of 2PO4, 33.5 mM K2HPO4, 100 mM KCl, 15.5 mM NaCl, pH 7.0) containing a protease inhibitor cocktail (Sigma-Aldrich) underwent five 5-min freeze-thaw cycles (from −210°C to 37°C) and the protein concentration was determined by the Bradford assay. All manipulations involving viable bacteria were performed in a Biosafety Level III laboratory. It has to be noted that upon completion of the experiments all bacteria cultured under tetracycline stress conditions were killed by incubation in 8% formaldehyde at room temperature for 24 h followed by autoclaving. Prior to disposal, bacteria were used to inoculate Vero cells to check their inactivation.Bacteria propagated with DOX (Q212Dox) and without DOX (Q212) were mass cultured in parallel and under identical conditions for 10 days (with the exception of doxycycline present in Q212Dox) and finally isolated from their host cells using renographin  density gradient ultracentrifugation as described 12C3-propionate whereas the Q212Dox proteome digest with 13C3-propionate. Each peptide thus had a label on its N-terminal alpha-amino group and on its C-terminal lysine epsilon-amino group, evoking a difference of 6 Da between light and heavy peptides. Samples were mixed and analyzed by LC-MS/MS on an Ultimate 3000 HPLC system  in-line connected to a LTQ Orbitrap XL mass spectrometer . Instrument settings for LC-MS/MS analysis and generation of MS/MS peak lists were as described Coxiella burnetii database downloaded from Uniprot on June 17th, 2009 . Lys-C/P was set as the used protease with one missed cleavage allowed, and the mass tolerance on the precursor ion was set to ±10 ppm and on fragment ions to ±0.5 Da. S-carbamidomethylation of cysteine and oxidation of methionine (to its sulfoxide) were set as fixed modifications. In addition, Mascot's C13 setting was set to 1. The light and heavy labels were defined in Mascot Distiller's quantitation method. Peptide quantifications were carried out using the Mascot Distiller Quantitation Toolbox (version 2.2.1). The quantification method details were as follows: constrain search: yes, protein ratio type: average, report detail: yes, minimum peptides: 1, protocol: precursor, allow mass time match: yes, allow elution shift: no, all charge states: yes. Ratios for identified proteins were calculated by comparing the XIC peak areas of all matched light peptides with those of the heavy peptides, and the results were verified by visual inspection of MS spectra with the in-house developed Rover tool http:www.ebi.ac.uk/pride) under the experiment number 18640 .The methionine-COFRADIC procedure was performed on both Q212Dox and Q212 whole lysates as described http://au.expasy.org/). The purpose of this analysis was to calculate the molecular weight (MW) and isoelectric point (pI) of each protein using the software ProtParam Tool (http://au.expasy.org/tools/protparam.html) and Compute pI/Mw Tool (http://au.expasy.org/tools/pi_tool.html).Analysis of the amino acid sequences of all identified proteins was carried out using several Web-based software tools, freely accessible from the ExPASy Proteomics Server of the Swiss Institute of Bioinformatics (SIB) (Table S1C. burnetii samples. All 531 proteins were identified in both samples (i. e. C. burnetii cultured in presence of tetracycline and C. burnetii cultured with no antibiotic present) in different expression levels. Proteins are categorized according to their predicted cellular function; their ratio value, gene locus, molecular weight (MW), isoelectric point and predicted cellular function are indicated.List of proteins identified in both (DOCX)Click here for additional data file.Table S2C. burnetii when cultured in presence of tetracycline (Q212Dox) or in C. burnetii when cultured with no antibiotic (Q212).Proteins that were significantly over-expressed in (DOCX)Click here for additional data file."}
{"text": "A West Nile virus (WNV) human risk map was developed for Suffolk County, New York utilizing a case-control approach to explore the association between the risk of vector-borne WNV and habitat, landscape, virus activity, and socioeconomic variables derived from publically available datasets. Results of logistic regression modeling for the time period between 2000 and 2004 revealed that higher proportion of population with college education, increased habitat fragmentation, and proximity to WNV positive mosquito pools were strongly associated with WNV human risk. Similar to previous investigations from north-central US, this study identified middle class suburban neighborhoods as the areas with the highest WNV human risk. These results contrast with similar studies from the southern and western US, where the highest WNV risk was associated with low income areas. This discrepancy may be due to regional differences in vector ecology, urban environment, or human behavior. Geographic Information Systems (GIS) analytical tools were used to integrate the risk factors in the 2000–2004 logistic regression model generating WNV human risk map. In 2005–2010, 41 out of 46 (89%) of WNV human cases occurred either inside of (30 cases) or in close proximity (11 cases) to the WNV high risk areas predicted by the 2000–2004 model. The novel approach employed by this study may be implemented by other municipal, local, or state public health agencies to improve geographic risk estimates for vector-borne diseases based on a small number of acute human cases. Since its emergence in 1999, West Nile virus (WNV) triggered the largest recorded arbovirus outbreak in North America In the absence of a human vaccine, vector surveillance and control are the most effective tools for arboviral disease prevention on the population level Vector-borne disease modeling has emerged as a methodology. Vectors and pathogen reservoirs are often associated with environmental factors Many spatial analytic studies of WNV risk predictors utilized aggregated data based on administrative divisions such as census tracts or zip codes. The statistical power of analysis associated with this approach may be low due to greatly reduced sample size. It may thus be difficult to detect significant differences in disease risk where municipalities exhibit sporadic or highly clustered WNV human cases. Additionally, aggregated spatial scale characterization can be susceptible to ecological fallacy, lack of precision, and measurement error Our goal was to employ vector biology and knowledge of environmental and socioeconomic risk factors to predict spatial patterns of human West Nile Virus risk in Suffolk County, New York, USA on a local scale. A large number of factors relevant to vector, host, and human ecology were tested and the significant predictors used to generate a logistic model rendered geographically by GIS tools into a county-wide WNV human risk map. Similar approach can be easily adopted by state, county, or municipal public health agencies to investigate factors associated with WNV human transmission to enhance surveillance and control efforts, and to better understand WNV landscape epidemiology.2) with densely populated suburban areas, commercial and light industrial sites, agricultural areas, forested parkland, and numerous fresh and saltwater wetlands. WNV enzootic activity in birds and mosquitoes has been detected every year since the original virus introduction to North America in 1999, with sporadic epidemic outbreaks resulting in human cases.The study was conducted in Suffolk County, NY located on Long Island east of New York City . SuffolkThis study utilized a case-control design with household geographic location as the unit of analysis. Study cases included a) 19 households with acute WNV human cases in 2000–2004, and b) 81 additional households with no acute WNV human cases, but located inside the WNV hotspots delineated by SatScan spatial scan statistic The predictors of risk of WNV human transmission were derived from those reported in the literature  at all three spatial scales were tested for multicollinearity and either combined or removed until resolved.For pairwise comparisons between cases and controls, chi-square test, t-test, 2-way ANOVA, or Mann-Whitney test were used depending on data type and distribution . To minimize collinearity, variables with Pearson's correlation coefficient >0.75 were either combined or excluded from further analysis . Statistp<0.1 were used for the final parsimonious model with the lowest Akaike's Information Criterion (AIC). The statistical power analysis of the final model was performed using the algorithm specifically developed for multiple logistic regressions I statistics in ArcMap PWNV = eY/(eY+1), where e is a base of the natural logarithm. The final model and map were validated by 46 acute WNV cases in 2005–2010.The resulting set of IVs was characterized by Principal Component Analysis (PCA) to uncover grouping among IVs and to understand the data structure . The entInitially, 64 environmental and socioeconomic factors were developed from the geodatabases listed in p<0.05 resulting in 53 independent variables due to multiple spatial scales, out of which 14 redundant IVs were eliminated to reduce multicollinearity  using eigenvalue >1.0 to retain the principal components (PCs). A total of 8 PCs accounted for ∼73% of the total variance and were interpreted as follows . PC1 Ur.p<0.1 and were used for the final model created by removing the least significant IV and introducing a new IV with the goal of increasing the parsimony while preserving the overall model fit. The final reduced model contained 12 IVs classifying correctly 89% of the cases including 16 out of 19 (84%) acute WNV human cases in 2000–2004 , and 85% of controls (p = 0.181), however, the final model (AIC = 147.5) was more parsimonious than the full model (AIC = 168.1). Given the sample size n = 200, statistical significance α = 0.05, power π = 0.8, the baseline probability of the high WNV risk (PWNV = 0.5–1.0) P0 = 0.3, and the multiple correlation coefficient R2 = 0.53 estimated for the model as an average of (1 - tolerance) for all IVs, the final model's effect size was OR = 1.9 which was in line with the original goal.The same 39 IVs were entered in a logistic regression model. The full model classified correctly  93% of the cases including 16 out of 19 (84%) acute WNV human cases in 2000–2004, and 91% of the controls with a U-shape distribution around the cut value of 0.5. Out of 39 original IVs in the full model, 14 IVs were statistically significant at controls . FurtherI statistics 0.092, Z-score = 1.78, p = 0.0744) indicating negligible to weak unexplained clustering on a global scale. Visual map examination showed large residuals  in both cases and controls scattered throughout the western part of the County (data not shown). Slightly higher number of large residuals in and around Cluster 4  of the total land area in Suffolk County.To generate the WNV human risk map, the shapefiles of individual IVs were processed by Neighborhood function  to calculate statistics for each IV at the corresponding spatial extent of 0.5, 1.0, or 2.0 km. The resulting raster files see . Census 2X = 4.0, df = 1, p = 0.047. Out of 65 WNV acute human cases in 2000–2010, 47 (∼72%) occurred in high WNV risk areas, which comprised ∼33% of Suffolk County's land area This distribution of WNV human cases was significantly different from random based on the land area (21 out of 65 expected), Chi-square exact test 2X = 20.1, df = 1, p<0.001. Only 11% of all acute WNV human cases (7 out of 65) occurred at a distance exceeding 1.0 km from high risk areas delineated by the model.The final model and map were validated using 46 acute WNV human cases in 2005–2010 . The disTo be useful for disease surveillance and control program, a geographic human risk model should a) use predictors that are easily available and interpretable, b) be accurate against independent data, and c) generate outputs that can assist control decisions Socioeconomic conditions have emerged as the key determinants of WNV human risk Culex pipiens, was most prevalent in urban areas with significant vegetation cover and plentiful avian hosts The middle class suburban areas appeared to support the appropriate combination of vegetation, open space, and potential vector habitat favoring WNV transmission. Wealthier neighborhoods had more vegetation, more diverse land use, and less habitat fragmentation likely resulting in higher biological diversity potentially protective against the WNV human transmission, e.g. the avian host “dilution effect” Although these findings have clearly demonstrated the interdependence between socioeconomic and natural environments, such relationships may be multifaceted. For example, elderly population and vacant housing were negatively associated with WNV human risk in our study, contrary to the expectation Among predictors of WNV human risk in the model, habitat fragmentation  is an important factor facilitating transmission of many vector-borne diseases worldwide. The anthropogenic habitat fragmentation effects vary from increased erosion and surface water accumulation, reduced species richness, extinction of top predators with increase of prey species, enhanced host-vector interactions, and a shift to anthropophyllic feeding by vectors Habitat fragmentation by roads and WNV enzootic activity parameters (positive birds and distance to a positive mosquito pool) were grouped within Urbanized/WNV PC in our study. While proximity to a WNV positive mosquito pool was a strong human risk factor in the model as expected, WNV positive mosquito pool density was not significant in the final multiple regression model, and, moreover, was not correlated with Urbanized/WNV PC. These results supported inconsistent association of WNV positive mosquito pool density with human risk Culex and container breeding Aedes species The remaining predictors in this study's model described natural or manmade wetlands. WNV human risk association with freshwater wetlands and open water, potential mosquito larval habitat, has been well documented In addition to interpretability, predictive accuracy assessment is crucial for determining the model's utility, yet vector-borne disease model evaluation mostly focused on the past rather than predicted outcomes Apart from those limitations due to incomplete data or imperfect understanding of the epidemiological and biological processes, multiple regression analysis may encounter two important methodological caveats, namely collinearity and spatial autocorrelation. Collinearity is caused by inclusion of many highly correlated environmental and socioeconomic factors in a model leading to instability in the estimation of the partial regression coefficients. This is especially relevant to WNV risk modeling, since the complex epidemiology of the virus transmission cycle typically necessitates entering multiple factors at several spatial scales in the analysis. One plausible approach to deal with this problem is to use principal components analysis to reduce the dimensionality among the predictors Despite limitations and technical caveats, in our study, the WNV risk map developed using 2000–2004 human cases predicted the locations of the 2005–2010 human cases with sufficient operational accuracy. The map serves in conjunction with the entomological data, which did not fully accounted for the patterns of WNV human transmission risk in Suffolk County This study demonstrated the feasibility of state or local level GIS based modeling using limited epidemiological data to create risk maps for outbreak investigations, arbovirus surveillance, and scientific discovery. The risk map may be further improved by incorporating entomological and climatic data ultimately leading to a real-time risk model. However, the increased complexity of such undertaking will likely require much closer cooperation than is currently in place between local public health agencies and academic or research institutions.Table S1Environmental and socioeconomic independent variables in this study. Bivariate analysis compared cases  and controls  at 3 spatial scales. Principal component analysis grouped significant independent variables determined by bivariate analysis and assisted in the interpretation.(DOC)Click here for additional data file."}
{"text": "Despite a cold temperate climate and low human population density, the Northern Great Plains has become a persistent hot spot for human West Nile virus (WNV) disease in North America. Understanding the spatial and temporal patterns of WNV can provide insights into the epidemiological and ecological factors that influence disease emergence and persistence. We analyzed the 1,962 cases of human WNV disease that occurred in South Dakota from 2002–2012 to identify the geographic distribution, seasonal cycles, and interannual variability of disease risk. The geographic and seasonal patterns of WNV have changed since the invasion and initial epidemic in 2002–2003, with cases shifting toward the eastern portion of South Dakota and occurring earlier in the transmission season in more recent years. WNV cases were temporally autocorrelated at lags of up to six weeks and early season cumulative case numbers were correlated with seasonal totals, indicating the possibility of using these data for short-term early detection of outbreaks. Epidemiological data are likely to be most effective for early warning of WNV virus outbreaks if they are integrated with entomological surveillance and environmental monitoring to leverage the strengths and minimize the weaknesses of each information source. Emerging infectious diseases, including those that have appeared for the first time, rapidly increased in incidence, or expanded into new geographic areas, are a significant concern in the fields of human and veterinary medicine as well as wildlife conservation ,2. DiseaWNV is indigenous to Africa, Asia, Europe, and Australia and was first identified in North America in the New York City metropolitan area during the summer of 1999 . In 2002Disease mapping and exploratory spatial analysis of epidemiological data can help to identify the geographic locations of populations at risk and facilitate the development of hypotheses about the epidemiological and ecological processes that drive these patterns ,6. Time-Culex tarsalis, a highly ornithophilic and particularly efficient amplifying vector of WNV that can also serve as a bridge vector to humans [Culex tarsalis abundance was lowest in urban areas and highest in grass-dominated rural habitats [Culex tarsalis abundance in South Dakota exhibited much stronger lagged relationships with temperature than with precipitation [Since its arrival in 2002, WNV has had a significant public health impact in South Dakota and neighboring Great Plains states. From 1999–2008 South Dakota had the highest average incidence of WNV neuroinvasive disease in the United States . During o humans . Previouhabitats , and thehabitats . Seasonapitation ,17, and pitation .The overarching goal of our study was to extend this previous work by characterizing the spatio-temporal epidemiology of WNV disease in South Dakota from 2002–2012. The underlying rationale was the expectation that identifying spatial and temporal patterns of human disease occurrence will provide a clearer understanding of when and where WNV risk is the highest and whether these patterns have changed since the emergence of WNV in South Dakota. We addressed the following specific questions: (1) Where in South Dakota is WNV risk highest and has this geographic pattern changed over time? (2) During what part of the year is WNV risk highest and has this seasonal pattern changed over time? (3) What are the temporal patterns  of WNV anomalies and can they be used to predict future WNV risk? (4) Do WNV epidemics arise simultaneously throughout the state or do cases increase earlier in some geographic locations than in others?The study area encompassed the state of South Dakota, which is located in the northern Great Plains of the United States, a region historically dominated by prairie vegetation. Environmental variability across the state is strongly affected by precipitation, which generally decreases along an east-to-west gradient , referenced temporally by the reported date of disease onset, and referenced spatially by county, ZIP code, and city of residence. Each case was georeferenced using the 2010 map of ZIP code tabulation areas (ZCTAs) from the U.S. Census Bureau. A total of 23 cases  were missing ZIP codes and were assigned to a ZCTA based on the county and city of residence. In addition, 60 cases  had ZIP codes that were not present in the 2010 ZCTA map and were assigned to a 2010 ZCTA by referencing the 2000 ZCTA map or based on the county and city of residence. The 2010 population data for each ZCTA were also obtained from the U.S. Census Bureau.2 and a mean population of 2,115. The mean population density at the ZCTA level was 34 per km2, but individual ZCTAs ranged from 0.05 to 4,628 per km2 reflecting the variety of settlement patterns from dense cities to rural areas.ZIP codes were developed by the U.S. Postal Service to classify street segments, address ranges and delivery points for mail delivery. ZCTAs were created by the U.S. Census Bureau as spatial units to provide approximate mapping of ZIP code boundaries using aggregated Census blocks. However, it is difficult to map ZIP code boundaries precisely, and ZIP codes are frequently split, discontinued, added, or expanded . As a reSpatial patterns of WNV cases were displayed as dot density maps by selecting a random location for each case within its assigned ZCTA. Spatial patterns of WNV risk were mapped using annual incidence rates and were spatially smoothed by incorporating information from surrounding areas to reduce variability caused by small population sizes at the ZCTA level. For this initial assessment of WNV risk, smoothing was carried out using a simple population-weighted mean smoothing approach as suggested by Waller and Gotway . To geneSeasonal patterns of total human WNV cases were summarized based on a standardized 18-week season in which all cases prior to 1 June were included in week 1; the months of June (weeks 2–5), July (weeks 6–9), August (weeks 10–13), and September (weeks 14–17) were each divided into four weeks; and all cases after September 30th were included in week 18. Weekly cumulative totals of human WNV cases were graphed for each year and for the three time periods used in the mapping . Two-sample Kolmogorov-Smirnov tests were used to test for differences in the seasonal distribution of WNV cases among these three time periods. These tests were carried out using all human WNV cases (including both WNV fever and neuroinvasive disease), only the WNV fever cases, and only the WNV neuroinvasive disease cases.The 18 weeks of WNV data for each year were concatenated to generate a continuous time series of WNV cases from 2002–2012. This log-transformed time series was then decomposed into seasonal, trend, and remainder components with the seasonal and trend decomposition using loess (STL) procedure . In thisN = 9). We examined the changes in these correlations across the different weeks to determine the point during the season when WNV cases provided a reliable indicator of the total magnitude of WNV cases throughout the season.WNV cases were also analyzed to determine whether the numbers of cases occurring during the early part of the WNV season (June and July) were predictive of the total number of WNV cases occurring throughout the entire season. For this analysis, we summarized the cumulative total number of WNV cases occurring in each year from week 2 (the first week in June) through week 9 (the last week in July). We computed the Spearman rank correlation between each of these cumulative variables and the total number of cases occurring for 2004–2012 . For each of these years, we used a generalized additive model (GAM) to model the day of year (DOY) of each case as the dependent variable using a smoothed functions of the spatial coordinates (eastings and northings) of the ZCTA centroids as independent variables. Statistically significant relationships between DOY and one or both of the independent variables indicated that different geographic locations had different mean dates of WNV onset. For years where there was significant spatial variability in mean DOY, the models were applied across the entire state to produce a continuous map of the mean DOY of WNV cases for each ZCTA.r = 0.96, p < 0.001).The dot density maps emphasized the strong spatial and temporal heterogeneity of human WNV cases within South Dakota , Table 1r = 0.75, p < 0.001), exhibited a weaker correlation in 2004–2007 , and had the highest correlation in 2008–2012 . As with the maps of total WNV cases, the incidence of neuroinvasive disease shifted from central and western South Dakota in 2002–2003 toward the eastern portion of the state in 2004–2007 and 2008–2012.The smoothed maps of WNV incidence further highlighted changes in the geographic distribution of WNV risk over time. The maps of total WNV incidence showed a shift in the geographic patterns of risk from central and western portions of South Dakota in 2002–2003 to the eastern portion of the state in 2004–2007 and 2008–2012 . The smop < 0.001) and in 2002–2003 compared to 2008–2012 , but the difference was not statistically significant in 2004–2007 compared to 2008–2012 . Similar results were obtained when only the same tests were performed using only the WNV fever cases and only the WNV neuroinvasive disease cases.Seasonal patterns of WNV cases varied from year to year , indicating that these space-time patterns were relatively weak.There was statistically significant variability in the onset date of WNV cases in both the north-south and east-west directions in 2003, and in the east-west direction in 2005 and 2007 . There wThere has been a shift in the spatio-temporal niche of human WNV risk in South Dakota from the initial invasion and first major epidemic of WNV in 2003–2003 to the subsequent years of endemic WNV from 2004-onward. In 2002–2003, human WNV cases were broadly distributed across the state and in particular were higher in the central and western portions. Starting in 2004, cases were more concentrated in the eastern portion of the state, and this eastward shift became even more pronounced during the 2008–2012 period. In addition, there were significantly fewer cases in the early part of the WNV season (before the last week of July) and more cases in the later part of the WNV season (after the third week of August) in 2002–2003 compared to 2004 and later years. These changes suggest that the spatial and temporal patterns observed during the large 2003 epidemic have become less relevant to current ecological and epidemiological processes that drive WNV risk, and that disease maps and other inferences about human WNV risk should be based on epidemiological data from 2004 onward.Culex tarsalis, a particularly efficient WNV vector. Subsequent research showed that interannual variability in temperature and moisture during the spring and early summer was associated with spatial and temporal variability in WNV incidence across the region from 2004–2011 [Climatic variability is one possible explanation for the observed changes. A geographic analysis of the 2003 WNV epidemic in the northern Great Plains highlighted strong clustering of WNV in the Dakotas, Nebraska, and eastern Montana and Wyoming . This cl004–2011 . Thus, a004–2011 ,24. Resu2. It is characterized by a high density of seasonal and permanent wetlands and a landscape mosaic of row crops and grasslands (From 2004 onward, and particularly in 2008–2012, one of the regions of highest WNV risk within South Dakota was concentrated in eastern South Dakota within the James River Valley, which lies between the uplands of the Missouri Couteau to the West and the Prairie Couteau to the East. This broad lowland was created by the James Lobe of the Laurentide ice sheet and occupies approximately 49,000 kmasslands , and is Early detection systems are widely used to identify the onset of mosquito-borne disease outbreaks by tracking human disease cases. Forecasting techniques range from relatively simple methods based on thresholds determined from historical data , to moreTwo major limitations to the use of human surveillance data for forecasting disease outbreaks are data quality and data availability. For human WNV disease in the United States, all cases are assessed based on a consistent definition established by the Centers for Disease Control and are subject to laboratory confirmation. However, an unknown degree of spatial uncertainty is introduced because some patients may contract WNV at a location away their residence. In addition, the delay introduced as a result of the time lags between amplification of the virus in mosquito and bird populations, transmission to humans, onset of symptoms, medical diagnosis, and case confirmation mean that human cases are a lagging indicator of WNV risk. As result, WNV amplification will have already occurred and mosquito numbers and infection rates will likely already be high by the time WNV is detected in the human population. In a state like South Dakota that experiences human WNV cases every year, detection of anomalies in early season case data does have some potential to predict the magnitude of WNV in human populations during the peak transmission season. In particular, our results suggest that human surveillance data may be able to indicate the onset of a significant WNV outbreak with several weeks of lead time. This type of alert would not provide enough time to take action to prevent virus amplification, but it might still allow public health officials to take steps such as expanding adult mosquito control and issuing preventive warnings before the period of peak transmission to humans.One promising approach toward leveraging the strengths and minimizing the limitation of human case surveillance for disease outbreak detection is to use blended approaches that integrate multiple streams of surveillance data. For example, a study of WNV in Colorado combined environmental data, entomological data, and human WNV case data to develop an improved map of disease risk in the Northern Colorado Front Range . ResearcHuman WNV disease exhibits strong patterns of geographic variability, seasonal cycles, and interannual fluctuations in South Dakota. The geographic and seasonal patterns of WNV have changed since the invasion and initial epidemic in 2002–2003, with cases shifting toward the eastern portion of South Dakota and occurring earlier in the transmission season in more recent years. The underlying cause of these shifts is currently unknown, but the results suggest that more recent data on endemic WNV from 2004 onward should be used for risk mapping and assessment of seasonality for public health planning purposes. Temporal analyses indicate that there is a potential for forecasting upcoming large WNV outbreaks with lead times of up to several weeks. Although this time frame is likely insufficient for preventing virus amplification, it could still help reduce transmission to humans by providing time to ramp up emergency mosquito control and disseminate messages to the public to take personal protection measures. Ultimately, information from epidemiological surveillance should be integrated with environmental monitoring, entomological surveillance, and other sources of information to provide more effective predictions of the likelihood of future WNV outbreaks."}
{"text": "We demonstrate that this isotropic opto-magnetic effect, which can be called inverse magneto-refraction, is allowed in a material of any symmetry. Its existence is corroborated by the experimental observation of terahertz emission by spin resonances optically excited in a broad class of iron oxides with a canted spin configuration. From its strength we estimate that a sub-picosecond modification of the exchange interaction by laser pulses with fluence of about 1 mJ cm−2 acts as a pulsed effective magnetic field of 0.01 Tesla.Ultrafast non-thermal manipulation of magnetization by light relies on either indirect coupling of the electric field component of the light with spins via spin-orbit interaction or direct coupling between the magnetic field component and spins. Here we propose a scenario for coupling between the electric field of light and spins via optical modification of the exchange interaction, one of the strongest quantum effects with strength of 10 In the ultrafast manipulation of magnetization by light, it is thought that the electric field couples only indirectly to spins via spin-orbit interaction. Here, the authors demonstrate inverse magneto-refraction, a direct optical modification of the exchange interaction allowed for any material symmetry. J is the exchange integral; ith and jth adjacent magnetic ions. The antisymmetric part D and called Dzyaloshinskii–Moriya interaction, gives rise to canted antiferromagnetism2The symmetric part of the exchange interaction between spins is responsible for the very existence of magnetic orderingJ from the demagnetization dynamics. Nevertheless, a direct, truly ultrafast effect of the electric field of light on the exchange interaction must be feasible in any material. In a medium of arbitrary symmetry, such an effect may be expressed phenomenologically by introducing an isotropic term in the Hamiltonian of the two-photon interaction between the light and spinsThe ability to control the exchange interaction by light has intrigued researchers in many areas of physics, ranging from quantum computing4578109Iopt is the intensity of light; α and β are some scalar and vector coefficients, respectively. The presence of the interaction Hamiltonian (1) manifests itself as a magnetic refraction, described by an isotropic contribution to the dielectric permittivity ɛIMR∼M2 that leads to a dependence of the refractive index on the magnitude of the magnetization M15J=αIopt, to the symmetric Heisenberg exchange integral J, whereas the second term describes the intensity dependent contribution, ΔD=βIopt, to the Dzyaloshinskii–Moriya vector D. Recently the effect of isotropic magneto-refraction has been used to probe d–f exchange in EuTeTi acting on a spin Si due to the light-induced perturbation of the exchange parameterswhere where γ is the absolute value of the gyromagnetic ratio. The torque (2) is zero in materials with collinear magnetic configurations since In a broad class of transition metal oxides the magnetic order is governed by indirect exchange via ligand ions (superexchange)3, rare-earth orthoferrites RFeO3 (R stands for a rare-earth element) and hematite α-Fe2O3, are natural candidates for observing such ultrafast optical modification of the superexchange interactions. In these compounds the Fe3+ ions  form two magnetic sublattices, the spins of which are antiferromagnetically coupledD/J between the antisymmetric and symmetric exchange parameters. Thus, one could expect that an ultrafast optical perturbation of the exchange parameters could also change the ratio D/J and thereby trigger, by the torque defined in 2728303132Antiferromagnetic iron oxides possessing weak ferromagnetism, such as iron borate FeBOHere we reveal the inverse magneto-refractive effect to be responsible for ultrafast modulation of the superexchange interaction in a very broad class of canted antiferromagnets. Our findings are supported by a low-energy theory for the magnetic interactions between non-equilibrium electrons subjected to an external time-dependent electric field. We present quantitative estimates of the strength and timescale of the optical perturbation of the exchange parameters.3 and ErFeO3 which revealed the optical excitation of the high-frequency quasi-antiferromagnetic mode in these compounds along with the low-frequency quasi-ferromagnetic mode, another form of the antiferromagnetic resonance in canted antiferromagnets which involves the precession of the magnetization with no change in its lengthD/J via inverse magneto-refractive effect, in particular.Recently we reported measurements of THz emission signals in the rare-earth orthoferritesD/J, we have studied the THz emission from a single FeBO3 cut perpendicularly to the z-crystallographic axis so that it lacks significant in-plane anisotropy of both optical and magnetic properties. The magnetization lying in the plane of the sample was aligned horizontally by a constant bias magnetic field of ∼0.1 T. The sample was illuminated by ∼100-fs laser pulses with their photon energy centred at 1.55 eV. We performed time-resolved detection of the THz radiation emitted from the sample in the direction of the z axis  plane at the frequency of the quasi-ferromagnetic mode (∼100 GHz)38z-cut TmFeO3 sample, with a net magnetic moment oriented upwards, a laser-induced spin-reorientation transition should trigger the quasi-ferromagnetic mode in such a way that the Mx component of the magnetization decreases. The observed difference in the phases between the two oscillations shows that the quasi-antiferromagnetic mode is triggered in such a way that the Mx component increases, which means that the canting angle becomes larger. Such a behaviour can only be explained by assuming that the quasi-antiferromagnetic oscillations are triggered by an increase of the ratio of the exchange parameters D/J. If this conclusion is true, in the x-cut sample the initial phases of the two modes must be the same, since the spin reorientation in this sample proceeds in the opposite direction. Measurements in the vicinity of the spin-reorientation temperature in ErFeO3 cut perpendicular to the x axis confirm this conclusion , the x axis  and the y axis, . The iron borate FeBO3 sample (370-μm thick) and haematite α-Fe2O3 sample (500-μm thick) were cut perpendicularly to the z axis. The lateral size of all plates was ∼5 mm.The crystals used in the present study were grown by floating zone melting (orthoferrites) and from the gas phase (iron borate and hematite) The orthoferrite samples were 60–100-μm thick and cut perpendicularly to the −2. The electric field of the emitted THz wave was measured by the electro-optical sampling technique. The sample was held inside a closed cycle, helium cryostat .A conventional time-domain THz spectrometer was used in the measurements. The THz spectrometer was powered by a Ti:sapphire amplified laser, emitting a sequence of optical pulses  with the repetition frequency of 1 kHz. Each laser pulse was divided into a stronger pump pulse and a weaker probe pulse. The pump spot size was larger than the aperture in the sample holder (∼2 mm in diameter) to provide a quasi-uniform excitation with a fluence of ∼1 mJ cmWe use a general formalism in which magnetic interactions are obtained from a purely electronic model by introducing small time-dependent rotations of the spin quantization axes as was recently described in ref. . For thed orbital and atom 1 contributes one filled (oxygen) p orbital. The Hamiltonian consists of a local part Hloc and a time-dependent hopping term H′(t):This method is implemented for the simplest model system that exhibits the physics of superexchange, which consists of a chain of three atoms, labelled as 0, 1 and 2. Atoms 0 and 2 correspond to transition metal sites with one partially filled σ={↑,↓} at site j, and ɛd and ɛp are the orbital energies of d and p orbitals, respectively, and U is the local (Hubbard) interaction energy associated with d orbitals. H′(t) accounts for hopping between p and d orbitals; t0 is the equilibrium hopping parameter, while ϕ(t) is the time-dependent Peierls phase, which absorbs the effect of the time-dependent electric field. In the Coulomb gauge and for a spatially uniform vector potential the Peierls phase is given byHere A||(t) is the component of the vector potential parallel to the chain and a is the lattice spacing. In the chosen gauge, the electric field is related to the vector potential as U, U+ɛd−ɛp>>t0 and a total filling of four electrons. To compute the non-equilibrium functions, this cluster model is solved numerically using exact diagonalization of the time-dependent Schrödinger equation. The electric field is taken as the product of an oscillatory component and a gradually changing envelope function with a rising time in the order of ∼10 periods of oscillation. The frequency ω of the oscillating part is below the charge-transfer gap, which prevents direct charge-transfer transitions, consistently with the experimental conditions.where Further details related to the theoretical and numerical methods are discussed in How to cite this article: Mikhaylovskiy, R. V. et al. Ultrafast optical modification of exchange interactions in iron oxides. Nat. Commun. 6:8190 doi: 10.1038/ncomms9190 (2015).Supplementary Figures 1-9, Supplementary Notes 1-6 and Supplementary References"}
{"text": "Given an uncharacterized compound, can we develop a computational method to fast identify which ATC class or classes it belongs to? The information thus obtained will timely help adjusting our focus and selection, significantly speeding up the drug development process. But this problem is by no means an easy one since some drug compounds may belong to two or more than two ATC classes. To address this problem, using the DO (Drug Ontology) approach based on the ChEBI  database, we developed a predictor called iATC-mDO. Subsequently, hybridizing it with an existing drug ATC classifier, we constructed a predictor called iATC-mHyb. It has been demonstrated by the rigorous cross-validation and from five different measuring angles that iATC-mHyb is remarkably superior to the best existing predictor in identifying the ATC classes for drug compounds. To convenience most experimental scientists, a user-friendly web-server for iATC-mHyd has been established at  Open the web-server at http://www.jci-bioinfo.cn/iATC-mHyb, the top page of iATC-mHyb will appear on the computer screen, as shown in Figure Step 2. Either type or copy/paste the formulae of query compounds into the input box at the center of Figure Step 3. Click on the Submit button to see the predicted result. For example, if using the formulae of the five compounds in the Example window as the input, one will see Figure 1) Compound-1 belongs to three different ATC-classes; i.e., classes 3, 5 and 9, which are predicted by iATC-mDO subpredictor, meaning that the compound is covered by the ChEBI database. (2) Compound-2 belongs to only one ATC-class; i.e., class 3, which is predicted by iATC-mDO subpredictor, meaning the compound is covered by the ChEBI database. (3) Compound-3 belongs to four different ATC-classes; i.e., classes 3, 4, 10 and 12, which are predicted by iATC-mDO subpredictor, meaning that the compound is covered by the ChEBI database. (4) Compound-4 belongs to three different ATC-classes; i.e., classes 4, 5 and 13, which are predicted by iATC-mISF subpredictor, meaning that the compound is not covered by the ChEBI database. (5) Compound-5 belongs to two different ATC-classes; i.e., classes 4 and 12, which are predicted by iATC-mISF subpredictor, meaning that the compound is also not covered by the ChEBI database. All these results are fully consistent with the experimental observations.Step 4. Click on the Citation button to find the key relevant papers that have been used to document the detailed development and algorithm of iATC-mHyb.Step 5. Click the Supporting Information button to download the all the “Supporting Information” files mentioned in this paper.1) how to construct or select a valid benchmark dataset to train and test the predictor; (2) how to formulate the drug compound samples with an effective mathematical expression that can truly reflect their essential correlation with the target concerned; (3) how to introduce or develop a powerful algorithm (or engine) to run the prediction; (4) how to properly conduct cross-validation tests to objectively evaluate the anticipated accuracy; (5) how to provide a web-server and user guide to make users very easily to get their desired results. Below, let us to address these point-by-point.As demonstrated in a series of recent method-developing studies , 60–65, For facilitating comparison, in this study we used the same benchmark dataset (Supporting Information S1) as used in , 4. It cm-th ATC class , and ∪ denotes the symbol for “union” in the set theory. Listed in Table where the subset ss m = 1,,3,...,14As we can see from the table, among the 3,883 drugs, 3,295 occur in one class, 370 in two classes, 110 in three classes, 37 in four classes, 27 in five classes, 44 in six classes, and none occurs in more than six classes. For such a multi-label system, let us use a more intuitive method to describe the benchmark dataset as given in Supporting Information S2, where the symbol “1” under the title of “ATC classification” means the drug concerned occurs in the corresponding class, “0” means not.Eq.1 is defined byOne of the keys in developing a powerful predictor is to formulate the samples with an effective mathematical expression that can truly reflect their intrinsic correlation with the target to be predicted . In the T is the transposition operator, α1 stands for its maximum DO similarity score with the drugs in the subset 2 for its maximum DO similarity score with the drugs in the subset 3 for that in subset where lculated , 68 fromlculated  via KEGGlculated .ftp://ftp.ebi.ac.uk/pub/databases/chebi/ontology/), and can be defined by Eq.Note that, of the 3,833 drug compounds in the benchmark dataset, only 1,144 can be found in the current ChEBI database  classifier has been adopted to predict the ATC-classes, as described below.i-th drug in the benchmark dataset Suppose the Li given byAnd its attribution in a multi-label system can be formulated as a vector whereLikewise, for a query drug or compound, we haveIts attribution label vector in the ACT system is predicted aswherem in Eq.8 is given byThe ΔDq – Di||2 is the Euclidean distance in the 14-D space  to predict the protein subcellular localization , 70, enzwhere “Hyb” means “hybridization” with the iATC-mISF predictor .One of the important procedures  in develThe metrics used to measure the prediction quality for multi-label systems are much more complicated than those for single-label systems. To make them more intuitive and easier to understand for most experimental scientists, the following five metrics were introduced by Chou : (1) “aiThe aforementioned Chou's five metrics can be formulated as {Aiming=N is the total number of the samples concerned, M is the total number of labels for the investigated system, k-th sample, k-th sample, andWhere The above approach had been effectively used to study various multi-label systems, such as those in which a protein may occur in two or more different subcellular locations –23, 76, Three cross-validation methods are often used in statistical prediction. They are: (1) independent dataset test, (2) subsampling  test, and (3) jackknife test . Of thesth sub-equation in Eq.Since Eq.http://www.jci-bioinfo.cn/iATC-mHyb along with a step-by-step guide. Moreover, the MATLAB code for the new method is also available as in Supporting Information S4, which can be directly downloaded from the web-server.A new method for predicting the ATC classes has been developed by hybridizing the drug ontology approach with the best existing ATC predictor. The new predictor has outperformed the best existing ATC predictor in all the five metrics used to examine the prediction quality of a predictor for multi-label systems, particularly in the “absolute true” rate and the “absolute false” rate, the two most difficult-to-improve indexes. To maximize the users’ convenience, a publically accessible web-server has been established at"}
{"text": "Importantly, reinfection of EBV-loss clones with EBV, but surprisingly not transduction with individual BL-associated latent viral genes, restored protection from apoptosis. Expression profiling and functional analysis of apoptosis-related proteins and transcripts in BL cells revealed that EBV inhibits the upregulation of the proapoptotic BH3-only proteins, BIM and PUMA. We conclude that latent EBV genes cooperatively enhance the survival of BL cells by suppression of the intrinsic apoptosis pathway signalling via inhibition of the potent apoptosis initiators, BIM and PUMA.While the association of Epstein–Barr virus (EBV) with Burkitt lymphoma (BL) has long been recognised, the precise role of the virus in BL pathogenesis is not fully resolved. EBV can be lost spontaneously from some BL cell lines, and these EBV-loss lymphoma cells reportedly have a survival disadvantage. Here we have generated an extensive panel of EBV-loss clones from multiple BL backgrounds and examined their phenotype comparing them to their isogenic EBV-positive counterparts. We report that, while loss of EBV from BL cells is rare, it is consistently associated with an enhanced predisposition to undergo apoptosis and reduced tumorigenicity  Typical of c-MYC-driven lymphomas, BLs proliferate rapidly but are also sensitive to apoptosis under conditions of stress.2Epstein–Barr virus (EBV)-positive Burkitt lymphoma (BL), an aggressive and difficult to treat malignancy, is endemic (eBL) in sub-Saharan Africa, where it accounts for around half of all childhood lymphomas. BL also occurs worldwide at lower incidence, and in these cases, known as sporadic BLs (spBL), EBV is found in 15–85% of tumours, varying by geographical region.et al.3). It was shown using the Eμ-Myc transgenic mouse model of human BL  that blocking apoptosis through enforced expression of BCL-2 prosurvival proteins or deletion of BH3-only proteins or BAX greatly accelerates lymphoma development.7Cellular proapoptotic BH3-only proteins  induce cell death by unleashing the proapoptotic multi-BH domain executioner BCL-2 family members, BAK and BAX, allowing them to form pores in the mitochondrial outer membrane, which commits the cells to apoptosis. BH3-only proteins can achieve this by binding and inhibiting prosurvival BCL-2 family proteins , which in the absence of an apoptotic stimulus restrain BAK and BAX, thereby preventing cellular destruction. Additionally, BIM, PUMA and tBID can directly activate BAX and BAK , two non-coding RNAs (EBER1 and EBER2), and two families of microRNAs (BART microRNAs (miR-BARTs) and miR-BHRF1s), which together drive proliferation and promote cell survival   .10 Cell lines derived from these tumours show marked resistance to apoptosis due to epigenetic silencing of the BIM promoter11 and functional inhibition of BIM, PUMA, BID and BAK by the viral BCL-2 homologue, BHRF1.12Crucially, however, most of the Latency III genes are not expressed in established BLs. Instead, in BLs, EBV exhibits more restricted forms of latency characterised by expression of EBNA1, the EBER transcripts and the miR-BARTs . Only a 14 and precise Ig-MYC chromosomal translocations.16 It is therefore unsatisfactory to introduce the virus or viral genes into EBV-negative spBL lines to study the role of EBV in eBL. Instead, efforts have focused on trying to rid EBV-positive eBLs of the virus to assess the contribution of EBV to the growth and survival of BL in an isogenic system. Treatment of EBV-positive BL cells with a dominant-negative form of EBNA1 leads to loss of EBV genomes and widespread apoptosis.20 While implying that EBV is essential for the continued survival of BL cells, this method yielded few EBV-loss clones for mechanistic studies. Hydroxyurea treatment can also eradicate EBV, but these BL clones do not show a consistent apoptosis predisposition phenotype.21 Additionally, an unusual EBV-positive spBL (Akata-BL) cell line has been reported to spontaneously lose EBV in vitro, yielding clones with impaired cell growth.22EBV-positive and -negative BLs are genetically distinct, differing in terms of their cellular mutational profiles24 The present study resolves the unmet need for a systematic approach to analyse multiple EBV-loss BL clones on several tumour backgrounds both in vitro and in vivo in a xenograft model of BL. This work has shown unequivocally that EBV in a Latency I infection can protect BL cells from apoptosis mediated by the proapoptotic BH3-only proteins, BIM and PUMA.A significant limitation of these previous studies has been the small number of tumour backgrounds and/or clones analysed. BL, like many cancers, can exhibit considerable inter- and intratumoral genetic heterogeneity.A panel of EBV-positive BL cell lines (1 spBL and 11 eBLs) were seeded at single-cell dilutions to establish more than 1800 clones. These clones were screened for EBV episome copy number by quantitative, real-time PCR (q-PCR); the results are summarised in Before investigating the phenotype of the EBV-positive clones, their EBV infection status was fully characterised. This included flow cytometry for EBERs to confirm that all cells carried the virus, as well as immunoblotting and q-PCR for 45 EBV transcripts to ensure that the clones retained Latency I gene expression  .in vivo, isogenic EBV-positive and EBV-loss clones derived from three eBL backgrounds , were transplanted by intraperitoneal injection into female NSG mice and the animals monitored for tumour burden. Tumours arose in the spleen, ovaries and pancreas and resembled human BL in terms of pathology and histological ‘starry sky’ appearance . To broaden these assorted but limited analyses, each of the Latency I-associated genes were introduced into multiple EBV-loss clones derived from several tumours. 34 were used to express EBNA1 protein and the BART miRs in EBV-loss cells at levels equivalent to those detected in EBV-positive BLs (EcoRI J region of the Akata strain EBV genome (KC207813.1),29 was used to efficiently express physiologically high levels of EBER RNAs , is highly contradictorytive BLs . A derivBER RNAs .35 Expression of cluster 1 miR-BARTs, cluster 2 miR-BARTs or miR-BART-5 alone also conferred no protection to EBV-loss BL clones 40 and a variant of the B95.8 prototype strain termed ΔCpWp-B95.8 (B-V), which is forced to adopt a Latency I infection because of genomic deletions spanning the Latency III-associated viral promoters, Cp/Wp41 EBV. These experiments are not trivial as reinfection with EBV usually gives rise to the extensive Latency III infection rather than a restricted Latency I pattern, Cp/Wp41 . ReinfecBamHI A genomic locus, which is naturally deleted in B95.8 and its derivative, B-V.43 These data highlight the clonal variation and emphasises the need to analyse multiple clones before drawing general conclusions.Importantly, Ak-V could protect EBV-loss BL cells from apoptotic stimuli; six cell lines were fully protected, two were substantially protected and one (Akata n2) showed no protection . Reinfec46To determine which cellular genes are modulated by EBV in Latency I BL, RNA was extracted from EBV-positive and -loss clones from four BL backgrounds  and microarray gene expression analysis was undertaken. Surprisingly, no genes were significantly changed more than twofold in EBV-loss compared with EBV-positive clones across all tumours. Additionally, no differences in steady-state expression of BCL-2 family transcripts were observed . We wereversus 79% viability), with concordant cleavage of PARP and increased caspase activation , could be useful clinically in the treatment of EBV-positive BLs.To our knowledge, this is the most extensive analysis of the role of EBV in BL. The large-scale, single-cell cloning of BL cell lines and isolation of only 61 spontaneous EBV-loss clones from >1800 clones strongly indicate that EBV is highly selected for in BL. Consistent with reports that EBV’s role in BL is to protect cells from death stimuli,43 but also a number of transcripts of unknown function that appear to be highly transcribed in latent BL cells.53 Therefore, it is likely that genes encoded within the B95.8 deletion contribute to Latency I-mediated protection from apoptosis. Further development of rEBV genomes is required to decode the precise contribution of these complex transcripts to BL.Importantly, reinfection of EBV-loss clones with recombinant Ak-V could fully restore apoptosis resistance, confirming that EBV is directly responsible for the increased resistance to cell death. Interestingly, B95.8-derived rEBV, which harbours a large genomic deletion, could partially, but never fully restore apoptosis protection to BL cells. This genomic deletion spans not only a number of the miR-BARTs31 EBER RNAs were shown to confer tumorigenicity to EBV-loss Akata-BL in vivo and to enhance the growth properties of cells in vitro, in part through upregulation of BCL-2 and IL-10.54 Yet we found no evidence that BCL-2 or IL-10 were more highly expressed in EBER-expressing or EBV-positive cells compared with EBV-loss cells. Consistent with our findings, others reported that expression of EBERs alone did not restore protection from apoptosis in Akata-BL cells in vitro, but that coexpression of EBNA1 and EBERs rendered two EBV-loss Akata-BL clones more tumorigenic in vivo.25 The authors attributed this to increased EBER expression in the EBNA1-/EBER-positive EBV-loss cells. However, an alternative interpretation is that EBNA1 enhances the tumorigenicity of EBER-expressing cells by functional cooperation. Another report attributed apoptosis protection to the miR-BARTs,18 but the considerable clonal variation (between cell lines and viruses) in this study precludes firm conclusions. BART miRs have been reported to downregulate PUMA in epithelial cells36 (a finding we could recapitulate in 293 cells), but consistent with other groups,56 this downregulation was not apparent in B-lineage-derived cell lines.No Latency I-encoded gene product alone  could restore apoptosis resistance to EBV-loss BLs. This indicates a requirement for cooperation between multiple genes, including transcripts from the B95.8 deletion. There is much debate in the literature in this regard. EBNA1 is essential for virus genome maintenance and also reported to function in cell survival in certain circumstances.3 In addition to the upregulation of BIM and PUMA proteins after apoptosis induction in EBV-loss cells, increased expression of BIM and PUMA transcripts was also observed compared with EBV-positive cells, although this did not reach statistical significance. Consistent with cooperation between multiple EBV genes, we therefore hypothesise that the repression of BIM and PUMA by EBV occurs at both the protein and RNA levels and involves multiple molecular mechanisms.Our work implicates the BH3-only proteins BIM and PUMA as the key cellular genes regulated by EBV in Latency I BL. These proteins are critical initiators of the intrinsic apoptotic pathway, able to bind and inhibit all BCL-2 prosurvival proteins and also capable of directly activating the executioners of apoptosis, BAX and BAK.56 and therefore likely inhibit their translation. Additionally, EBERs have been shown to activate AKT/PI3K signalling,57 which is important for BL cell survival.58 Interestingly, tonic AKT/PI3K signalling has been reported to suppress induction of PUMA and BIM, by both transcriptional and post-translational mechanisms, and consequently suppress apoptosis in leukaemia cells following growth factor withdrawal.59 Furthermore, EBNA1 is able to deplete cells of SMAD2,61 which can lead to inhibition of PUMA expression through the TGF-β pathway.63 A recent study showed that both RNA and DNA viruses, typified by Semliki Forest virus and herpes simplex 1 virus, respectively, control intrinsic apoptosis via regulation of PUMA.64 Therefore, it is likely that Latency I EBV genes co-operate to regulate PUMA and BIM both directly and indirectly and that multilateral regulation of prodeath BH3s is critical and conserved in human viruses.BART miRs are predicted to bind multiple sites in the 3′-UTRs of BIM and PUMA65 Therefore, EBV infection of a preneoplastic cell bearing a c-MYC chromosomal translocation would be predicted to accelerate tumour development by blocking BIM and PUMA upregulation. Our work opens up the possibility to incorporate BH3-mimetic drugs  into the treatment regimen for EBV-positive BL to help improve patient survival, which remains woeful in certain patient groups.The blocking of BIM and PUMA upregulation by EBV has important implications for BL pathogenesis as it has been shown that deregulated c-MYC expression can sensitise cells to apoptosis and that this predisposition must be overcome for BL to develop.9 When referring to BL-derived subclones, the prefix ‘P’ indicates an EBV-positive clone, whereas ‘n’ denotes a clone that has lost the virus. BL cell lines were maintained in RPMI-1640 supplemented with 10% FCS , 6 mM glutamine, 1 mM pyruvate, 50 μM α-thioglycerol, 20 nM bathocupronine disulphonic acid and 8 μg/ml gentamycin . All cells were routinely grown at 37 °C in a humidified atmosphere containing 5% CO2.Akata-BL was a kind gift from Prof. Kenzo Takada, Rael-BL and the lymphoblastoid cell line (LCL), Raji-BL, X50-7, were kind gifts from Prof. George Klein. All other BL cell lines were established by Prof. Alan Rickinson and co-workers. Jurkat clone E6.1 was from the European Collection of Cell Cultures, HEK-293 cells were obtained from the American Tissue Culture Collection (product number: ATCC CRL-1573) and 293FT cells (R700-07) were from Life Technologies . Clonal cell lines were established by single-cell cloning carried out as described previously.4/cm2 in the presence of inducers or inhibitors of apoptosis, including: ionomycin ; etoposide ; Fas-activating CH11 antibodies  ; roscovitine ; staurosporine  , Danvers, MA, USA); IgM crosslinking antibodies (5 μg/ml) ; the human cytokine, IL-10 (50–500 ng/ml) ; Q-VD.OPh (20 μM)  and/or a vehicle-only control (DMSO) as stated, unless otherwise specified in figure legends. Where inducible lentiviruses were used, gene of interest expression was induced by the addition of 1 μg/ml doxycycline 24 h before the experiment set-up. All assays were carried out in triplicate and on at least three occasions. Data are expressed as mean and standard deviation of independent triplicates unless otherwise specified. Cell viability was determined by dual staining of cells with Annexin-V-APC and PI or PI staining alone and analysed by flow cytometry. Surface CD95 staining was determined using the DX2-PE-CY7  as per the manufacturer’s instructions. A minimum of 10 000 events was recorded for all samples and data were analysed using the FlowJo Software  or Accuri C6 Software .BL cells were seeded at 9x10β-actin ; caspase-3, -7 and -9 ; DEDD2/FLAME3 ; PUMA ; BIM ; CFLAR ; CASP8AP2 ; BID ; BAD ; BCL-2 ); BCL-X ; MCL-1 ; cIAP1 , Livin ; Survivin ; XIAP ; NOXA ; BAK ; and BAX . Protein size analysis and densitometry were carried out using the Chemidoc MP system equipped with the ImageLab v.5.2 Software . Size markers were SeeBlue Plus 2 (Life Technologies) and densitometry calculations were normalised to endogenous control proteins, calregulin or β-actin.Proteins in whole-cell lysates were separated by SDS-PAGE and transferred to PVDF membranes that were probed using antibodies raised against: EBNA1 (AMo serum); EBNA2 (PE2); LMP1 (CS1-4); BHRF1 (5B11); Cg-Prkdcscidil2rgtm1Wjl/SzJ) mice were given intraperitoneal or subcutaneous injections of BL cells diluted in PBS. Cell numbers used were 1 × 106 for Kem-BL, Awia-BL and Mutu-BL clones or 1x107 for Akata-BL. Mice were monitored for signs of tumour growth and killed when their tumour burden reached 1 cm3. Lymphoma cell suspensions made from explanted tumour tissues were shown to be >99% positive for human CD19 FACS  to confirm that tumours were derived from xenotransplanted BL cells.Mice were kept in specified pathogen-fee animal areas at the Walter and Eliza Hall Institute of Medical Research (WEHI). All experiments involving mice were conducted in accordance with the requirements of the WEHI Animal Ethics Committee. Eight-week-old, female NSG  in the presence of 32P-labelled dCTP . Probe hybridisation and washes were carried out using the XpressHyb System  according to the manufacturer’s instructions. Exposures were carried out at −80 °C for 2–14 days.RNA was isolated using Trizol reagent, separated on precast urea gels (both Life Technologies) and blotted onto nylon membranes. Radio-labelled DNA probes were generated by PCR amplification of the ACTB and GAPDH. Absolute quantitation of EBV transcripts and housekeeping controls were quantitated by real-time PCR relative to a DNA plasmid standard to enable absolute copy numbers to be calculated. This method and all the relevant primer-probe sets have been described elsewhere.53 Stem-loop primed cDNA was prepared with miR-specific primers and TaqMan microRNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s guidelines and miR expression was quantified using commercially available TaqMan primer/probe mixes (Life Technologies). EBV genome load was calculated by DNA q-PCR. Briefly, DNA was extracted using the GenElute DNA Kit (Sigma) according to the manufacturer’s instructions and 5 ng DNA was used per q-PCR assay. Genome load was calculated by quantitating copies of the EBV polymerase gene (BALF5) normalised to β-2-microglobulin (β2M), assuming two genomic copies of β2M per cell. Each measurement was made in triplicate.Total RNA was prepared using miRVana RNA Extraction Kit (Life Technologies) according to the manufacturer’s instructions. Cellular apoptosis-related gene expression was quantified using TaqMan Low Density Array human apoptosis panel cards . Random hexamer-primed cDNA was prepared using the qScript cDNA Supermix . All experiments were carried out in triplicate and samples were run in triplicate. Data were analysed using Data Assist v.3.01 (Life Technologies) using the ΔΔCt method, and then normalised to the mean of two endogenous controls, S-variant proteins were expressed from an F-UTG-derived lentivirus, which constitutively expresses GFP and drives expression of the gene of interest from a dox-inducible, polII, Trex promoter, as described previously.67 Recombinant Akata virus (Ak-V)-producing cells were a kind gift from Prof. Kenzo Takada and the CpWp-KO B95.8 virus (B-V) was developed by Dr. Rosemary Tierney. Detailed descriptions of all recombinant EBVs and expression constructs are available in The EBNA1 protein, clusters of BART miRs and BIM68 or from Ak-V-infected BL cells by crosslinking surface IgG40 as described previously. Before EBV infection, EBV-loss BL clones were transduced with an expression construct expressing the EBV receptor, human CD21, to enhance the infection efficiency.69Lentivirus containing supernatants were produced by transfecting 293FT cells (Life Technologies) with the expression constructs of interest alongside the envelope plasmid, pMD2.G, and the packaging plasmid, psPax2, using Lipofectamine 2000 (Life Technologies). Infectious virus stocks produced from B-V BAC-carrying 293 clones by BZLF1/BALF4 transfections-BH3 variant functional assays, reinfection studies, q-PCR and immunoblot gene expression analyses the significance of differences between cell lines were calculated using an unpaired, two-tailed, Student’s T-test. For apoptosis assays to characterise the effect of EBV loss, unpaired, two-tailed Student’s T-tests were carried out on clones from each background. Additionally, the difference in cell survival between all EBV-positive clones and all EBV-loss clones across different BL backgrounds was analysed by two-way ANOVA. Survival comparisons of mouse cohorts were calculated using log-rank (Mantel–Cox) analysis. T-tests, Mantel–Cox and ANOVA analyses were carried out in the GraphPad Prism 5.0 Software . Differences were considered significant where the P-value fell below 0.05 and were classified as follows: *P<0.05, **P<0.01, ***P<0.001 and nsP>0.05 (not significant).For BIM"}
{"text": "Despite anti-VEGF therapy, some patients develop chronic diabetic macular edema. The objective of this study was to evaluate anatomic and visual outcomes of switching patients with chronic DME from intravitreal bevacizumab or ranibizumab to intravitreal aflibercept injection.In this retrospective observational case series, 11 eyes with recalcitrant diabetic macular edema (DME) were evaluated 6 months prior to and 6 months following initial intravitreal aflibercept injection (IAI). Recalcitrant DME was defined as having a thickened retina (≥350 μm) on spectral domain optical coherence tomography (SD-OCT) with persistent cystic changes  over 6 months prior to intravitreal aflibercept switch despite aggressive treatment for DME during this time.One hundred and forty-seven patients in total were treated with IAI during this time, and of these, 31 patients were treated with IAI for DME. 18 eyes had less than 4 treatments within the 6 months prior to switch to IAI, 6 patients had a central retinal thickness (CRT) on SD-OCT of less than 350 μm at time of switch to IAI, and 2 patients had a greater than 15% decline in CRT on SD-OCT over the 6 months prior to switch to IAI. A total of 11 patients were included in the study. Over the 6 months prior to switch, the mean change in central retinal thickness was +18.6% and over the 6 months following switch to aflibercept the mean change in central retinal thickness was −27.1%. Switching to a regimen of at least 3 intravitreal aflibercept injections over 6 months resulted in some anatomic improvement and improvement or stabilization of Snellen visual acuity in all eligible patients.In patients with recalcitrant diabetic macular edema, switching to intravitreal aflibercept resulted in improved a 25% or more decrease in central retinal thickness in 81% (9/11) patients at 6-month follow-up. Sixty-three percent (7/11) had improvement in Snellen visual acuity after switching to intravitreal aflibercept injection, suggesting some reversibility of functional damage. Diabetic macular edema (DME) is the leading cause of central vision loss in people with diabetes and is characterized by edema and thickening of the macula . From thIntravitreal ranibizumab  became the first VEGF inhibitor FDA-approved for the treatment of DME in 2012 following the RIDE and RISE trials, which showed significant superiority of both 0.3 and 0.5 mg ranibizumab groups over sham injection in improving visual acuity and decreasing central retinal thickness in patients with DME , BOLT 77, and a In July 2014, the FDA approved intravitreal aflibercept , a highly specific fusion protein with high VEGF-binding affinity, for the treatment of DME. This approval followed the VIVID and VISTA clinical trials, which showed superiority in anatomic and visual outcomes in patients treated with intravitreal aflibercept in comparison to laser treated controls. Patients in these studies required a 3-month washout period for patients previously treated with anti-VEGF agents .In March 2015, Protocol T of the Diabetic Retinopathy Clinical Research Network (DRCR.net) published results of a study comparing the relative efficacy of intravitreal bevacizumab, ranibizumab, and aflibercept in treatment of DME. They found that all three anti-VEGF treatments improved vision in center-involving DME, with the relative effect depending on baseline vision. In cases of mild vision loss (20/40 or better), there was no significant difference between the three treatment groups. However, in cases with poorer baseline visual acuity (20/50 or worse), aflibercept was significantly more effective than both ranibizumab and bevacizumab in improving vision. Although Protocol T provides data on the comparative effectiveness across the 3 anti-VEGF agents, this study was not designed to inform on the relative efficacy when switching agents, and excluded eyes that received an anti-VEGF injection within the preceding 12 months [Since FDA approval of aflibercept for the treatment of DME and results from Protocol T, many ophthalmologists are switching patients from ranibizumab or bevacizumab to aflibercept, especially those with minimal or incomplete response to the former treatments. In the current study, we evaluated the outcome of switching to aflibercept (IAI) in patients with DME considered to be “recalcitrant” to therapy. Patients were deemed recalcitrant if they had minimal decrease in central retinal thickness (CRT) on spectral domain optical coherence tomography (SD-OCT) despite aggressive treatment during the prior 6 months.A retrospective, observational case series was performed to study the outcome of switching to intravitreal aflibercept in eleven eyes with DME who were considered to be sub-optimal responders to standard of care and were considered to be “recalcitrant” to current therapy. The Institutional Review Board at Tufts Medical Center approved the study protocol for human subjects. The study was complaint with the Health Insurance Portability and Accountability Act of 1996 and adhered to the tenets of the Declaration of Helsinki.Intravitreal injection logs for all patients treated with intravitreal aflibercept 2.0 mg (0.05 mL) at two clinical sites of the Vitreoretinal Service at Tufts Medical Center Department between March 2014 and July 2015 were reviewed. Electronic medical records were then reviewed to identify possible participants that were treated with intravitreal aflibercept injection for DME.Inclusion criteria included ability to provide written informed consent, age 18 years or older, adequately clear media for SD-OCT imaging, and Snellen visual acuity of 20/40 to 20/300 in the study eye. Anatomic criteria for inclusion required CRT ≥ 350 μm on SD-OCT and recalcitrant DME was defined as persistent cystic change with ≤15% decrease in CRT over the 6 months prior to IAI switch despite having at least 4 total treatments for DME, with at least 3 of these treatments being intravitreal anti-VEGF injections (excluding IAI). Treatments for DME prior to IAI switch included intravitreal bevacizumab (IVB) and ranibizumab (IVR), intravitreal triamcinolone acetonide (IVTA), sub-Tenon’s triamcinolone acetonide (STTA), dexamethasone intravitreal implant , and laser photocoagulation. If both eyes of a patient met entry criteria, then the worse-seeing eye was included in the study. If patients were treated with previous corticosteroids, baseline intraocular pressure had to be 21 mm Hg or less either with or without pressure reducing drops. Those patients who were previously treated with corticosteroids also had to have adequate clarity of media to allow adequate SD-OCT image quality. Patients were excluded if they had previously received intravitreal aflibercept in the study eye or a history of systemic anti-VEGF therapy. Inclusion and exclusion criteria are outlined in Table Intravitreal injections were carried out using the same standard procedure in all patients. Dosing of intravitreal aflibercept was administered on a monthly as needed (PRN) regimen following the initial dose of aflibercept. The PRN treatment algorithm for DME entailed treatment with intravitreal aflibercept and monthly monitoring with SD-OCT. If at any point the central macula had no fluid at all, further aflibercept injections were withheld and the patient was monitored on a monthly basis. If new or increase in fluid occurred from the previous visit secondary to worsening of diabetic macular edema, re-treatment with intravitreal aflibercept was performed.If the macula continued to have fluid after a minimum of 3 aflibercept injections and the macula achieved a 15% or more reduction in CRT compared to baseline on SD-OCT, then treatment could be withheld. If the macula continued to have fluid after a minimum of 3 aflibercept injections and the macula achieved <15% reduction in CRT compared to baseline of SD-OCT, aflibercept was continued or an alternative therapy was considered (corticosteroids or laser). If alternative therapy was utilized, the patient was not included in this analysis.Patient charts were reviewed until 6 months after the first aflibercept injection. At each visit, best-corrected distance visual acuity was measured using a standardized Snellen chart, a comprehensive eye examination was performed including intraocular pressure measurement via applanation, and OCT imaging was obtained using Spectralis OCT  or Cirrus HD-OCT .The occurrence of any severe postoperative complications, including infection, inflammation, retinal detachment, thromboembolic events, or death within the first 6 months after the first aflibercept injection was recorded.The injection logs were reviewed for all patients receiving IAI between March 1, 2014 and July 1, 2015. One hundred and forty-seven patients in total were treated with IAI during this time, and of these, 31 patients were treated with IAI for DME. Twenty patients were excluded from the study for the following reasons: 18 eyes had less than 4 treatments within the 6 months prior to switch to IAI, 6 patients had a CRT on SD-OCT of less than 350 μm at time of switch to IAI, and 2 patients had a greater than 15% decline in CRT on SD-OCT over the 6 months prior to switch to IAI. A total of 11 patients with DME meeting inclusion criteria for the study were evaluated. The mean patient age was 65 years old (range 47–83 years). There were 7 men (64%) and 4 women (36%), and 5 (45%) right eyes and 6 (55%) left eyes included in the study. The self-reported mean hemoglobin A1c was 7.2 (range 6.1–10) and the mean duration of diabetes was 15 years (range 5–34 years) at the time of enrollment in the study. Baseline characteristics are outlined in Table The average duration of treatment duration for DME prior to inclusion in the study was 32 months (range 8–77 months). The mean total number of treatments given prior to inclusion in the study was 13 (range 5–30) and included intravitreal bevacizumab, intravitreal ranibizumab, intravitreal triamcinolone acetonide, sub-tenon triamcinolone acetonide, dexamethasone intravitreal implant, and focal laser. The anti-VEGF injections given prior to inclusion in the study included ranibizumab exclusively in 4 patients (36%), bevacizumab exclusively in 2 (18%) patients, and a combination of ranibizumab and bevacizumab in 5 (46%) patients. Other treatments included intravitreal triamcinolone acetonide , sub-tenon triamcinolone acetonide , focal laser , and dexamethasone intravitreal implant .The average number of treatments for DME administered to the cohort during the 6 months before switch to IAI was 4.7 (range 4–6), with an average of 4.3 (range 3–6) being anti-VEGF injections. Eight patients (73%) were treated with ranibizumab exclusively, 2 patients (18%) were treated with bevacizumab exclusively, and 1 patient (9%) was treated with a combination of ranibizumab and bevacizumab. Other treatments included intravitreal triamcinolone acetonide  and focal laser . No patients received panretinal photocoagulation during this time.During the 6 months after switch to IAI, an average of 4.7 (range 4–6) IAI were given. All patients had 6-month follow-up after switching to IAI and this visit was used for outcome analysis. Treatment characteristics are outlined in Table The average CRT as measured on SD-OCT at 6 months prior to switch to IAI was 428 μm (range 299–690). The mean change in CRT during the 6 months prior to switch to IAI was an 18.6% increase. The average CRT at time of switch to IAI was 487 μm (range 363–685). At 6 months following switch to IAI, the average CRT was 326 μm (range 262–454). The mean change in CRT during the 6 months after switch to IAI was a 27.1% decrease. Anatomic and Snellen visual acuity outcomes before and after switch to IAI are outlined in Table At 6 months after switch to IAI, 9 of 11 patients (81%) had a 15% or more decrease in CRT, meeting the primary endpoint. At 6 months after switch to IAI, 9 of 11 patients (81%) had a 25% or more decrease in CRT and 1 of 11 patients (9%) had a 50% or more decrease in CRT. Eight of 11 patients (73%) had a CRT of less than 350 μm. Representative SD-OCT data before and after switch to intravitreal aflibercept is shown in Fig. Six months prior to switch to IAI, the Snellen visual acuity ranged from 20/25 to 20/100. Over the 6 months prior to switch to IAI, the Snellen visual acuity declined in 8 patients (73%), improved in 2 patients (18%), and remained unchanged in 1 patient (9%). At time of switch to IAI, the Snellen visual acuity ranged from 20/40 to 20/200. Six months after switch to IAI, there was some improvement in Snellen visual acuity in 7 patients (63%) and remained stable in 4 patients (36%). No patients had decline in Snellen visual acuity after switch to IAI.After inclusion in the study, 52 anti-VEGF injections were given prior to switch, and 52 aflibercept injections were given after the switch. No ocular adverse events, including endophthalmitis, uveitis, retinal tears or detachment, or elevated intraocular pressure necessitating treatment were observed. There were no systemic adverse events such as thromboembolic phenomena  during the study.Inflammatory changes in the retinal environment are involved in the pathogenesis of DME, including vascular leakage, leukostasis, ischemia, and release of pro-inflammatory mediators such as VEGF, interleukins 6 and 8, and TNF-alpha . The retIn clinical practice, some patients have chronic fluid, despite aggressive treatment with intravitreal injections of bevacizumab or ranibizumab. It is important to know if there is benefit to switching incomplete or non-responders to an alternative anti-VEGF agent, rather than continuing ranibizumab or bevacizumab injections, switching to corticosteroids, or combining anti-VEGF and corticosteroid treatment. The current study used stringent criteria identify patients with recalcitrant DME to evaluate if switching to IAI would be of benefit. In this small study, switching to IAI resulted in some anatomic improvement in all eligible patients, and 81% of patients met the pre-specified primary endpoint of 15% or greater decrease in CRT.Our results are consistent with two previously reported cases series. Wood, Karth, Moshfeghi, and Leng evaluated short-term outcomes of switching to IAI in 14 eyes with persistent fluid from DME despite at least 3 monthly ranibizumab or bevacizumab. There was no minimum central retinal thickness requirement for inclusion and follow-up occurred after a single IAI. At 1 month after injection, 79% of patients had anatomic improvement on SD-OCT .Rahimy et al. evaluated 50 eyes treated with at least 4 consecutive injections with ranibizumab or bevacizumab prior IAI switch, and two or more IAI after switch. Included patients had minimum central retinal thickness of 300 μm and follow-up occurred at month 2 or 3 after switch to IAI. At most recent follow-up, 56% had anatomic improvement on SD-OCT, while 24% had complete resolution of fluid .The greater response to aflibercept in patients with chronic DME recalcitrant to treatment with bevacizumab and ranibizumab may be due to a number of factors, and may help explain the results seen in Protocol T. Aflibercept has been shown to have significantly higher binding affinity to VEGF-A compared to ranibizumab and bevacizumab. Also, aflibercept binds to VEGF-B and placental growth factor (PlGF), unlike ranibizumab and bevacizumab . These cIt is unclear both from inconsistencies in the published literature and clinically as to what parameters should be used to define recalcitrant DME . The current study used pre-defined and very stringent criteria for classifying patients as having recalcitrant DME, and included visual acuity, central retinal thickness, and treatment parameters. Recalcitrant patients were identified as those with substantially thickened retinas (>/350 μm CRT) with minimal improvement in CRT (<15% across 6 months prior to switch) despite aggressive therapy with at least four treatments for DME (3 of which were VEGFs other than aflibercept). These eyes were not improving anatomically prior to switch despite frequent anti-VEGF therapy. Switching to a pre-defined minimal use of aflibercept resulted in some anatomic improvement in all eligible patients. All patients were followed for at least 6 months, with the majority of patients gained some visual acuity after switching to aflibercept, suggesting some reversibility of functional damage.The major limitations of this study include the retrospective design and the relatively small number of participants, precluding the ability to apply statistical analyses. Further studies are needed on a larger scale but this small, retrospective study suggests that switching to aflibercept can be beneficial for recalcitrant DME patients as defined in this study.In conclusion, in patients with recalcitrant DME, defined as having thickened retinas (≥350 μm) with minimal decrease in CRT (<15% over 6 months prior to switch) despite aggressive treatment , switching to intravitreal aflibercept may result in improved anatomic and visual outcomes at 6-month follow-up."}
{"text": "Background: Most human breast cancer cell lines currently in use were developed and are cultured under ambient (21%) oxygen conditions. While this is convenient in practical terms, higher ambient oxygen could increase oxygen radical production, potentially modulating signaling pathways. We have derived and grown a series of four human breast cancer cell lines under 5% oxygen, and have compared their properties to those of established breast cancer lines growing under ambient oxygen.Methods: Cell lines were characterized in terms of appearance, cellular DNA content, mutation spectrum, hormone receptor status, pathway utilization and drug sensitivity.Results: Three of the four lines  were triple negative , with NZBR1 also over-expressing EGFR. NZBR3 was HER2+ and ER+ and also over-expressed EGFR. Cell lines grown in 5% oxygen showed increased expression of the hypoxia-inducible factor 1 (HIF-1) target gene carbonic anhydrase 9 (CA9) and decreased levels of ROS. As determined by protein phosphorylation, NZBR1 showed low AKT pathway utilization while NZBR2 and NZBR4 showed low p70S6K and rpS6 pathway utilization. The lines were characterized for sensitivity to 7-hydroxytamoxifen, doxorubicin, paclitaxel, the PI3K inhibitor BEZ235 and the HER inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. In some cases they were compared to established breast cancer lines. Of particular note was the high sensitivity of NZBR3 to HER inhibitors. The spectrum of mutations in the NZBR lines was generally similar to that found in commonly used breast cancer cell lines but TP53 mutations were absent and mutations in EVI2B, LRP1B, and PMS2, which have not been reported in other breast cancer lines, were detected. The results suggest that the properties of cell lines developed under low oxygen conditions (5% O2) are similar to those of commonly used breast cancer cell lines. Although reduced ROS production and increased HIF-1 activity under 5% oxygen can potentially influence experimental outcomes, no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5 vs. 21% oxygen. This is considerably higher than that %. This i tissues , 4. In od damage , 7 and m range 1–% as a co2, 5% CO2, 90% N2) to mimic physiologically oxygen levels in tumors. The cell lines of ATCC provenance were cultured in an atmosphere of 5% CO2 in air at 37°C.Culture conditions have been described , 10 for For estrogen response, MCF-7 and NZBR3 cell lines were cultured in estrogen deprived media , and penicillin/streptomycin  for the 2 days before 17β-estradiol treatment. Estrogen-deprived medium was used for all assays related to estrogen stimulation.As described previously , tumor t2, 5% CO2, and 90% N2 in a Tri-Gas Forma incubator. Tissue culture plates or flasks had previously been coated with a thin layer of agarose to prevent the growth of fibroblasts . Material containing larger aggregates was pipetted into tubes, collected by low-speed centrifugation to remove blood cells, necrotic material, and debris , and then washed twice  in growth medium. Preparations were monitored by phase contrast microscopy, and cytospins of cell suspensions were stained by hematoxylin/eosin and examined by a pathologist to ensure that they contained tumor cells. Cultures were set up in growth medium supplemented with 5% FBS under an atmosphere of 5% OEarly passage cell lines (< passage 33) used in this study were developed in this laboratory. All cell lines were tested negative for mycoplasma contamination.Everolimus, afatinib, dacomitinib, lapatinib and ARRY-380 was purchased from Selleck Chemicals . 17β-estradiol, tamoxifen, paclitaxel and doxorubicin were purchased from Sigma . NVP-BEZ235 , 12 was The NZBR cell lines were typed by short tandem repeat profiling by DNA Diagnostics  Table . The com3H-thymidine (0.04 μCi per well) was added to each well and cultures were incubated for 6 h; cells were harvested on glass fiber filters using an automated TomTec harvester. Filters were incubated with Betaplate Scint and thymidine incorporation measured in a Trilux/Betaplate counter. Effects of inhibitors on the incorporation of 3H-thymidine into DNA were determined relative to the control (non-drug-treated) cells.As described previously , 13, 14,For the growth response to 17β-estradiol in 5 and 21% oxygen culture conditions, cells were seeded at 2,500 per well in 96 well plates in the presence or absence of 50 nM 17β-estradiol for 3 days. For the growth response to doxorubicin in 5 and 21% oxygen culture conditions, cells were seeded at 1,000 per well in 96 well plates in the presence of varying concentrations of inhibitors for 5 days.5) were harvested, incubated with 20 μM DCFDA in medium for 60 min in the dark at 37°C. Cells were analyzed in a BD Accuri™ Flow Cytometer.Intracellular ROS were detected with the cell-permeable fluorescent probe 2',7'- dichlorofluorescein diacetate (DCFDA) (ABCAM) according to the manufacturer's instruction. Cells (1 × 10CA9), which is the most well established target of HIF, quantitative RT-PCR (qRT-PCR) was performed using gene-specific primers (Table HPRT1 RNA expression. The 2∧(–delta delta CT) method was used to analyze the relative changes in gene expression.As described in detail , oligo-drs Table  and SybrAs described , 10, 16,For whole exome sequencing (WES), 250 ng of genomic DNA from each cell line was sheared using the EpiShear™ Multi-Sample Sonicator (Active Motif). The quantity and fragment size of the sheared DNA were assessed on a Tapestation 2200 (Agilent) with the high sensitivity D1000 tape. Sheared DNA (100 ng) was used for the preparation of the whole exome libraries (WELs). WELs were prepared using the SureSelect XT2 (SSXT2) reagent kit and the SureSelect Clinical Research Exome V2 exome enrichment kit following the manufacturer's instructions (Agilent Technologies). The WELs were sequenced on a NextSeq500  to obtain around 40 to 44 million paired end reads  per exome.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were aligned to the human reference genome (hg19) with BWA (bwa 0.7.12)  . The res 0.7.12) . Mpileupgenome hg with BWA 0.7.12) . The var 0.7.12)  followed 0.7.12) . Variant 0.7.12) .T-tests or Mann-Whitney Rank Sum Tests was used for comparison of groups. Correlation analysis was performed with Spearman Rank Order correlation coefficient (R) and statistical significance (P) using SigmaPlot . Values of P < 0.05 were considered to be statistically significant.The cell lines were characterized by cellular DNA content, hormone receptor expression and tamoxifen sensitivity. The lines were all aneuploid and three of the four lines  were triple-negative with no expression of estrogen receptor, progesterone receptor and HER2 Table . The ER+3H]-thymidine incorporation assay was used to assess the effect of estrogen on cell proliferation when the cells were cultured under 5 and 21% oxygen conditions. Both MCF-7 and NZBR3 cell lines displayed significant growth stimulation by estrogen and MCF-7 cells showed increased response to estrogen as compared to NZBR3. However, the two oxygen concentrations did not have distinguishable effects on the growth response to estrogen , Cyclin D1 (CCND1) and Trefoil Factor 1 (TTF1)  . Both GRn Figure .T- test, p < 0.05)  in cells cultured under 5 and 21% oxygen concentrations. A significant increase in ROS concentrations was observed in cells cultured in 21% oxygen (pairwise ) Figure .CA9) -regulated genes, including carbonic anhydrase IX (CA9) . Cell lin Figure .The phosphorylation status of AKT, p70S6K, rpS6, and ERK in the NZBR lines was examined Figure . NZBR1 sWe have shown that estrogen receptor positive (ER+) breast cancer cell lines generally are more sensitive to everolimus than are receptor negative lines . However3H-thymidine incorporation assay. The ER+ HER2+ NZBR3 cells showed high sensitivity to everolimus (IC50 11.5 nM), yet the triple-negative NZBR2 with low mTOR pathway utilization also showed increased everolimus sensitivity (IC50 1.1 nM)  between NZBR2 and the other three cell lines in BEZ235 sensitivity were also observed.Two out of the three triple-negative NZBR cell lines were resistant to everolimus, with IC50 values of over 500 nM Figure  in a 3-d) Figure . SignifiThe sensitivities of the NZBR cell lines, and the ER+ MCF-7, HER2+ SKBR3 and triple-negative MDA-MB-231 cell lines to the therapeutic agents doxorubicin (a topoisomerase II poison) and paclitaxel (a microtubule poison) were tested Figure . The ER+T-test, p > 0.05) in sensitivity to doxorubicin was observed when the cell lines were cultured in 5% vs. 21% oxygen . As expected, drug resistance was observed in the HER2- cell lines; however, the HER2+ cell line ZR75.1 also showed similar resistance Figure . HER2+ NThe effects of ARRY-380 on HER2, AKT, and ERK activation were examined in four HER2+ breast cancer cell lines, which showed a range of sensitivities to ARRY-380. After overnight exposure to 100 nM or 1000 nM of ARRY-380, we observed no effect on total HER2, AKT and ERK expression in the cell lines. ARRY-380 inhibited HER2 phosphorylation in all lines, and ERK phosphorylation in cell lines with detectable phosphorylation Figure . Of inteAKT1, ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, FANCC, MEN1, MRE11A, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, ALB2, PIK3CA, PMS1, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCCC2) were selected from the hereditary breast cancer panel of AmbryGenetics, GeneDx, and University of Washington. The somatic mutations gene list  was selected from somatic mutations in the cancer (COSMIC) database. Several sequence variants that result in missense or nonsense mutations were detected in genes associated with breast cancer  gene lies within an intron of the NF1 (Neurofibromatosis type 1) gene and is transcribed in the opposite direction to the NF1 gene (NF1 and EVI2B RNA expression levels in relation to those of the estrogen receptor gene ESR1 in the genome-wide RNA transcript profile from TCGA (breast invasive carcinoma gene expression) using the RNAseq data set . A weak but significant positive correlation was observed in the expression of ESR1 and NF1  while EVI2B and ESR1 expression were negatively correlated . No significant correlation of NF1 and EVI2B was observed .Most mutated genes found in the NZBR cell lines had also been found to be mutated in other breast cancer cell lines Table . The muts. Table . Since tNF1 gene , we comp2) is generally similar to that of commercially available breast cancer cell lines. It is of interest that the hormone receptor status of the new lines covers the main classes of breast cancer, with three of the four having triple negative receptor status. Proliferation of the ER+ NZBR3 cell line was sensitive to estrogen stimulation, and expression of the estrogen responsive genes GREB1 and TFF1 was increased after 24 h of estrogen exposure   of a cell line cohort (TP53 mutations is lower in cell lines derived under low oxygen conditions.The genomic mutation frequency in the breast cancer cell lines is significantly higher than that in breast carcinoma 2) Table , but thees Table . The avae cohort . HoweverThe triple negative line NZBR1 is of interest because of the relative absence of gene mutations in the genome sequence. One possible explanation is that this cell line possesses a CpG island methylator phenotype (CIMP) –16 that BRCA2 mutation as well as an AKT1 mutation. The latter differs from the hot spot mutation AKT1E12K that confers sensitivity to the dual mTOR/PI3K inhibitor BEZ235 , mismatch repair system component homolog 2 (PMS2), MutL homolog 1 (MLH1) and Ataxia Telangiectasia Mutated serine/threonine kinase (ATM). BRCA2, a tumor suppressor that is essential for the repair of double-strand DNA breaks by homologous recombination, is associated with risk of developing breast cancer , EGFR (L858R), EGFR (L858R/T790M) and HER2 with IC50s of 0.5, 0.4, 10, and 14 nM respectively in cell-free assays. Dacomitinib is a potent, irreversible pan-ErbB inhibitor, mostly of EGFR with an IC50 value of 6 nM in a cell-free assay. ARRY-380 is a potent and selective HER2 inhibitor with IC50 of 8 nM, equipotent against truncated p95-HER2, 500-fold more selective for HER2 over EGFR . ARRY-380 is under clinical investigation for treating HER2+ metastatic breast cancer patients. Since ARRY-380 is HER2 specific, it has the potential to block HER2 signaling without causing the toxicities of EGFR inhibition . In comp2) is generally similar to that of commonly used breast cancer cell lines developed under 21% oxygen conditions. Although no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5% vs. 21% oxygen, reduced ROS and upregulation of the hypoxia response, as indicated by CA9 expression, were observed in cells cultured under 5% oxygen conditions. Therefore we cannot exclude the possibility that culture of cells under 5 vs. 21% oxygen conditions could have consequences for the effectiveness of drug treatment in cell lines as predictors of therapy responses in patients.In summary, our results suggest that the behavior of cell lines developed under low oxygen conditions (5% OEthics approval and consent to participate: This study was carried out in accordance with the recommendations of New Zealand Health and Disability Ethics Committee guidelines–ref AKL/2000/184/AM06, New Zealand Health and Disability Ethics Committee. The protocol was approved by the Northern A Health and Disability Ethics Committee. All subjects gave written informed consent in accordance with the Declaration of Helsinki.EYL and BCB designed this study. EYL, CF-P, WRJ, EM, PMK, DCS and MEA-A carried out the study. EYL, CF-P, SKB, RJB, DCS and MEA-A assisted with the data analysis. EYL drafted the manuscript. RJB, GJF, DCS, CF-P, MEA-A, SKB and BCB assisted with the manuscript preparation. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "Although hatching is perhaps the most abrupt and profound metabolic challenge that a chicken must undergo; there have been no attempts to functionally map the metabolic pathways induced in liver during the embryo-to-hatchling transition. Furthermore, we know very little about the metabolic and regulatory factors that regulate lipid metabolism in late embryos or newly-hatched chicks. In the present study, we examined hepatic transcriptomes of 12 embryos and 12 hatchling chicks during the peri-hatch period—or the metabolic switch from chorioallantoic to pulmonary respiration.THRSPA. Using pairwise comparisons of embryo and hatchling ages, we found 1272 genes that were differentially expressed between embryos and hatchling chicks, including 24 transcription factors and 284 genes that regulate lipid metabolism. The three most differentially-expressed transcripts found in liver of embryos were MOGAT1, DIO3 and PDK4, whereas THRSPA, FASN and DIO2 were highest in hatchlings. An unusual finding was the “ectopic” and extremely high differentially expression of seven feather keratin transcripts in liver of 16 day embryos, which coincides with engorgement of liver with yolk lipids. Gene interaction networks show several transcription factors, transcriptional co-activators/co-inhibitors and their downstream genes that exert a ‘ying-yang’ action on lipid metabolism during the embryo-to-hatching transition. These upstream regulators include ligand-activated transcription factors, sirtuins and Kruppel-like factors.Initial hierarchical clustering revealed two distinct, albeit opposing, patterns of hepatic gene expression. Cluster A genes are largely lipolytic and highly expressed in embryos. While, Cluster B genes are lipogenic/thermogenic and mainly controlled by the lipogenic transcription factor ectothermy (embryo) to endothermy (hatchling) in the chicken. Several transcription factors and their coactivators/co-inhibitors appear to exert opposing actions on lipid metabolism, leading to the predominance of lipolysis in embryos and lipogenesis in hatchlings. Our analysis of hepatic transcriptomes has enabled discovery of opposing, interconnected and interdependent transcriptional regulators that provide precise ying-yang or homeorhetic regulation of lipid metabolism during the critical embryo-to-hatchling transition.Our genome-wide transcriptional analysis has greatly expanded the hepatic repertoire of regulatory and metabolic genes involved in the embryo-to-hatchling transition. New knowledge was gained on interactive transcriptional networks and metabolic pathways that enable the abrupt switch from The online version of this article (10.1186/s12864-018-5080-4) contains supplementary material, which is available to authorized users. The developing mammalian embryo is completely dependent upon its mother and her placenta to supply nutrients, exchange respiratory gases, and to remove nitrogenous waste products. Quite the opposite is true among amniotes , which must undergo independent embryonic development in a pre-formed and completely enclosed system—the cleidoic egg. In chickens, the principal source of energy for late embryonic growth is derived from yolk lipids stored in the egg by the hen. The embryo exhibits exponential growth between days 13 to 18 of embryonic development (E13 to E18), when the total energy required for growth comes from β-oxidation of fatty acids derived from yolk lipids . The finDIO3) at E20, whereas the activity of the activating deiodinases (DIO1 and DIO2) increases in E20 embryos during hatching and afterwards in the hatchling. The pro-thyroid hormone (T4) is readily deactivated to reverse rT3 by DIO3 in liver of E16 and E18 embryos; then with onset of pulmonary respiration, the activity of (DIO1) and (DIO2) increases in liver to meet the ensuing energy demands of hatching and endothermy. The embryo must switch from chorio-allantoic to pulmonary respiration and instantly meet the high metabolic demands of an endotherm—the hatchling chick. Therefore, it is essential to understand the transcriptional networks that integrate and control function of the endocrine system to meet the dynamic metabolic demands of growth imposed upon the liver.The chicken embryo develops as an aquatic ectotherm with respiratory gas exchange via the chorioallantoic membrane. On the last day of embryonation (E21), the fully-developed embryo must suddenly convert to pulmonary respiration after pipping through the eggshell. During the peri-hatch period, the precocious chick becomes an endotherm with a very high and self-sustaining metabolic rate maintained by a fully-functional thermoregulatory system. The thyroid axis plays a major role in regulating metabolism and energy expenditure. Hence, the activity and importance of the thyroid axis has been extensively studied during the peri-hatch period of the chicken –18. The see Fig. THRSPΑ, SCD, FASN, ME1, HMGCS, and EVOL6).Our first glimpse of the liver transcriptome during the chicken’s peri-hatch period came from an earlier low-resolution transcriptional scan using the original chicken liver 3.2 K) microarray . This in K microaectothermy (embryo) to endothermy (hatchling) in the chicken.The present high-resolution genome-wide transcriptional analysis of the liver during the peri-hatch period has greatly expanded our hepatic repertoire of regulatory and metabolic genes involved in the embryo-to-hatchling transition. Several transcription factors were identified that appear to exert opposing actions, which lead to the predominance of lipolysis in embryos or lipogenesis in hatchlings. Furthermore, we have gained new knowledge on interactive transcriptional networks and metabolic pathways that enable the abrupt switch from Fertile eggs from Ross x Ross broiler (meat-type) chickens were purchased from a commercial hatchery . The eggs were incubated in an automated incubator  at 39 °C and 95% relative humidity. At E20, eggs containing viable embryos were transferred to hatching trays to determine the approximate time of hatch . Chicks hatched within a 4 h period on the 21st day of incubation were wing-banded and held in the incubator for an additional 4 h. The remaining one day-old (D1) chicks were vaccinated against Marek’s disease virus and transferred to a heated battery brooder  maintained at 33 °C. The chicks received a commercial starter ration  and water ad libitum throughout the experiment. After continuous light for the first two days, the birds were maintained on a 20 h light: 4 h dark (20 L:4D) photoperiod. All protocols used in this study were in accordance with the United States Department of Agriculture guidelines on the use of agricultural animals in research and approved by the University of Delaware Animal Care and Use Committee.The experimental design used for the present transcriptional profiling with Affymetrix Chicken Genome arrays was similar to the design utilized in our original 3.2 K microarray analysis of liver in peri-hatch chickens . Four maTotal RNA was purified from each liver sample using an RNeasy Mini kit with on column DNase I treatment, as recommended by the manufacturer’s . Quantitation of total RNA was done using a NanoDrop spectrophotometer  and the RNA was diluted to a standard concentration (250 ng/μL). RNA quality was assessed using an Agilent 2100 Bioanalyzer and an Agilent RNA 6000 Nano Kit . Liver RNA samples (1 μL each) were analyzed on a RNA 6000 Nano Chip. The ribosomal RNA ratio (28S/18S) was assessed for RNA integrity, where with a RNA Integrity Number (RIN) of 9 to 10 indicates RNA of high quality for use in amplification of RNA for the microarray analysis and for verification by qRT-PCR analysis. Each liver RNA sample was divided into two identical aliquots and stored frozen at − 85 °C until use in the transcriptional analyses below.Microarray-based transcriptome profiling was carried out using 24 Affymetrix GeneChip™ Chicken Genome Arrays and one-cycle target labeling protocol according to the manufacturer’s (Affymetrix) protocols. The experimental design for hybridization of the 24 chicken genome arrays was based on four replicate hybridization days, where all six ages were represented by one of four biological replicates  per age group  of the microarray data was completed using GeneSpring GX software . Raw cell files from the Affymetrix GeneChip Chicken Genome Arrays were imported into GeneSpring software and unfiltered differentially-expressed genes  identified for a preliminary hierarchical and k-means cluster analysis  and a log2 ratio ± 0.75 cutoff. This gene expression level threshold of log2 ratio ± 0.75 (or a 1.68-fold difference) has proven useful for statistical analysis of microarray data in model plants  control expression of multiple metabolic enzymes , kinases , a phosphatase (LPIN1), and transporters (STARD4 and SLC6A6). The gene network in Fig. PPARG, PPARGC1A, NR0B1, NCOR1and ZBTB20) with key lipogenic metabolic enzymes , growth factors (IGF1), binding proteins  and the adiponectin receptor (ADIPOR2). Other direct targets of PPARG shown in this gene network were NDRG1, PLIN2, PER3 and VNN1.Two gene interaction networks from the D1 vs. D9 contrast were functionally annotated by IPA as involved in “Lipid Metabolism” Fig. . As showTHRSPA, PPARG, PPARD and KLF15), where PPARG and PPARD share seven target genes . The TF PPARG and several of its direct targets are expressed at higher levels in liver of D9 hatchlings, including ADIPOR2, FBXO9, ELOVL6, ELOVL2, ELOVL1, HSD17B12, PEX11A, HECTD2, VNN1 and FDPS. The fasting-induced transcriptional factor KLF15 and four direct gene targets  are expressed higher in liver of fasted D1 hatchlings. The Ingenuity Upstream Regulator Analysis , since 21 of its target genes are down-regulated (green symbols) AR-DEGs in this contrast. Based on known relationships in the Ingenuity Knowledge Base, Ingenuity predicts that inhibited PPARG would block the expected activation (blunt orange lines) of CDK1, CPT1A and LPL. Ingenuity predicts that KLF15 would be activated, which would lead to activation (up-regulation) of seven direct targets  as indicated by the orange arrows. Interestingly, KLF15, a key transcriptional regulator of gluconeogenesis, was highly expressed in liver of fasted D1 hatchling chicks, as revealed by three pairwise contrasts .The final gene interaction network Fig.  was alsosis Fig.  identifiCOX7A2L) during the embryo-to-hatchling transition were examined by qRT-PCR analysis  show higher log-based expression in embryos and a sharp decline after hatching. Surprisingly, hepatic expression of DIO1 was relatively stable, except in E18 embryos where DIO1 was slightly elevated. The invariant gene (COX7A2L) was used by geNorm software for normalization of the qRT-PCR expression levels. The expression patterns of eight lipogenic genes during the embryo-to-hatching transition are depicted in Fig. THRSPA and SCD was log-scale and increased exponentially, reaching a plateau in liver of fed hatchlings between D3 and D9. Similar log-scale expression patterns were found for fiv other metabolic genes , while HMGCL and HMGCS2 only showed a slight increase in transcript abundance in D9 hatchlings.Hepatic expression patterns of seven DEGs that were expressed higher in embryos and an invariant gene ]. The deactivating deiodinase DIO2 and the adipokine ADIPOQ showed a progressive increase in hepatic expression from E16 to a peak in E20 embryos. Although DIO2 levels were lower in D1 and D3 hatchlings, a peak in abundance was reached in D9 hatchlings. The qRT-PCR analysis . Five hepatic genes  had higher expression levels, albeit with a lower amplitude, in D3 and D9 hatchlings. We were surprised to find that hepatic expression of DIO1 was rather stable across embryonic (E16-E20) and hatchling (D1-D9) ages. Furthermore, DIO1 was not detected as a DEG by genome array analysis or qRT-PCR analysis; therefore, DIO1 was considered an invariant gene and not included in the Pearson’s Correlation Analysis.Figure sis Fig. , bottom sis Fig. , top disr = 0.929; 11 degrees of freedom) indicates a highly significant (P ≤ 0.01) correlation between gene expression levels obtained from both microarray and qRT-PCR analyses  which interact with each other and their respective target genes in late embryos as they utilize yolk lipids and prepare for hatching on E21. Upon feeding, hatchling chicks abruptly increase expression of several ligand-activated transcription factors  which in turn control numerous down-stream metabolic enzymes, transporters and kinases/phosphatases to support greater rates of energy expenditure for lipogenesis, adipogenesis and somatic growth. Hierarchical clustering revealed two opposing patterns of gene expression in liver during the peri-hatch period, where Cluster A had higher expression in embryos, which sharply declined after hatching  provides a detailed view of the innate choreography of major transcriptional responses involved in the abrupt switch from a lipid-laden ectotherm (embryo) to a free-living endotherm (hatchling chick). We have identified several transcription factors  for rapid growth and metabolism in hatchling chicks  were up-regulated in liver of D9 hatchlings with direct interactions with SREBF2 and ACACA, which encodes the rate limiting enzyme in fatty acid synthesis. Apparently, both THRSP and MID1IP1 are glucose-responsive genes, which have overlapping roles in regulating expression of several lipogenic genes  had 19-times greater abundance in liver of riboflavin-rescued embryos than riboflavin-deficient embryos between E13 and E15. Thus, three independent chicken microarray platforms and multiple expressed-sequence tag (EST) sequences in GenBank clearly support abundant “ectopic” expression of FKER in liver of E15 and E16 embryos from both egg-type (Leghorn) [FKER in the lipid-laden liver of late embryos and the relationship between hepatic expression and cutaneous expression of FKER in embryos will require further definitive study.Interestingly, d GGA27) –67. Fiven rescue . We propatocytes . Furtherabolites . Examinarop milk . The unir THRSPA . The pre embryos , which cGPL1742) . This cDLeghorn)  and, prePPARA, PPARGC1A, NR1H4 and SIRT1, SERTAD2, KLF11, KLF13 and KLF15) and scores of metabolic enzymes, transporters, phosphatases and kinases. The metabolites derived from these processes interact with nuclear receptors, G-coupled protein receptors (GCPRs), transmembrane receptors and extracellular factors. Highly-expressed hepatic DIO3 inactivates thyroid hormones in the embryo, while its expression sharply declines in hatchling chicks. Although hepatic expression of DIO1 was rather constant during the peri-hatch period, another thyroid hormone-activating enzyme DIO2 reached peak abundance at two ages, E20 and D9. Upon pipping through the egg shell, the hatching chick immediately converts to the pro-hormone T4 into metabolically-active T3, which supports achievement of thermodynamic freedom in hatchlings and synergetic activation of THRSPA, the major lipogenic transcription factor in chickens. Furthermore, T3 bound to its receptor (THRB) forms heterodimers with other nuclear receptors, which then control programmed expression of major lipogenic enzymes . In addition to THRSPA, other transcriptional regulators  also interact and contribute to the sharp increase in lipogenesis observed in liver of D3 and D9 hatchlings.The present analysis of the embryo-to-hatchling transition in meat-type chickens provides the first detailed view of the choreography of innate and dynamic transcriptional responses made by embryos and newly hatched chicks during the critical metabolic jump from lipid-laden ectotherm to free-living self-sufficient endotherm, respectively. The metabolism of late (E16-E20) embryos is dominated by β-oxidation of yolk lipids, glycolysis and gluconeogenesis, which utilizes the residual albumen and other proteins in allantoic fluid ingested by the embryo just prior to hatching. These metabolic processes are precisely controlled by multiple and interacting transcription factors  chickens. We have proposed that hepatic expression of FKER in e15-e16 embryos could be an adaptive response to the coincident and massive accumulation of yolk lipids. This idea is supported by the exceptional expression of multiple β-keratin transcripts, including FKER, in the lactating crop sac of the pigeon [THRSPA. Nevertheless, the biological function of FKER expressed in liver of e15-e16 embryos and the relationship between hepatic FKER and cutaneous FKER remain to be elucidated by definitive study. The present study provides new insight into dynamic interaction of multiple transcription factors and their direct target genes that provide homeorhetric regulation of metabolism during the abrupt embryo-to-hatchling transition of the domestic chicken, Gallus gallus.Our present observation of the extraordinary expression of multiple e pigeon , which aAdditional file 1:Experimental design of microarray hybridizations. A Microsoft Excel file containing a single work sheet “Array Hybridization Design” which describes the hybridization scheme used for the 24 Affymetrix Chicken Genome Arrays. (XLSX 12 kb)Additional file 2:Primers used for qRT-PCR analysis. A Microsoft Excel file containing a single work sheet “qRT-PCR Primer Information”. This table provides the chicken gene symbol, GenBank ID number, 5′-3′ sequence for forward and reverse primers, and amplicon size (bp) for each gene used for qRT-PCR analysis. (XLSX 12 kb)Additional file 3:P ≤ 0.05; ±0.75 cutoff) for each contrast. (XLSX 905 kb)Annotated gene list for 14 pairwise contrasts of liver transcriptomes in embryos and hatchlings across six ages during the peri-hatch period (E16-D9). The last worksheet provides the list of 1005 commonly-shared AR-DEGs identified by the Venn diagram shown in Fig. Additional file 4:P ≤ 0.05) that AR-DEGs belong to a particular canonical pathway or biological function accrued in the Ingenuity Knowledge Base. (XLSX 53 kb)Canonical and biological pathways identified by the contrast of all embryos vs. all hatchlings. The AR-DEGs from the E vs. H contrast were assigned by Ingenuity Pathway Analysis (IPA) to canonical and functional and pathways. Fisher’s Exact Test is used in IPA to determine probability  of each AR-DEG belonging to a particular canonical pathway or biological function. (XLSX 36 kb)Canonical and biological pathways identified by IPA in the E18 vs. E20 contrast. The AR-DEGs from the E18 vs. E20 contrast were assigned by IPA to overrepresented canonical and functional pathways. IPA uses Fisher’s Exact Test to determine probability  of each AR-DEG belonging to a particular canonical pathway or biological function. (XLSX 44 kb)Canonical-biological pathways identified by IPA in the contrast of E18 embryos vs. D3 hatchlings. The AR-DEGs identified in the E18 vs. D3 contrast were assigned by IPA to overrepresented canonical and functional and pathways. IPA uses Fisher’s Exact Test to determine probability  of each AR-DEG belonging to a particular canonical pathway or biological function. Each worksheet provides a list of AR-DEGs assigned to particular canonical pathways or biological functions, annotated with gene symbol, Entrez name, log2 ratio, cellular compartment, type and Entrez protein ID. (XLSX 31 kb)Canonical and biological pathways found by IPA in the D1 vs. D9 hatchling contrast. The AR-DEGs identified in the D1 vs. D9 contrast were assigned by IPA to overrepresented canonical and functional and pathways. IPA uses Fisher’s Exact Test to determine probability  indicates a highly significant correlation between gene expression levels obtained from both microarray and qRT-PCR analyses. (XLSX 16 kb)Pearson’s correlation analysis of gene expression levels of 15 “candidate” DEGs determined by genome microarray and verified by qRT-PCR analyses. A Microsoft Excel file containing the average normalized expression levels (log2 ratios) of 15 candidate DEGs as determined by genome microarray analysis and verified by qRT-PCR analysis. The Pearson’s correlation coefficient ("}
{"text": "Slow tool servo (STS) diamond turning is a well-developed technique for freeform optics machining. Due to low machining efficiency, fluctuations in side-feeding motion and redundant control points for large aperture optics, this paper reports a novel adaptive tool path generation (ATPG) for STS diamond turning. In ATPG, the sampling intervals both in feeding and cutting direction are independently controlled according to interpolation error and cutting residual tolerance. A smooth curve is approximated to the side-feeding motion for reducing the fluctuations in feeding direction. Comparison of surface generation of typical freeform surfaces with ATPG and commercial software DiffSys is conducted both theoretically and experimentally. The result demonstrates that the ATPG can effectively reduce the volume of control points, decrease the vibration of side-feeding motion and improve machining efficiency while surface quality is well maintained for large aperture freeform optics. Most currently conventional optical devices from 2D optical design methods are usually rotationally symmetric. While systems with such optics perform well in numerous applications, the requirement of high energy efficiency or aberration correction often cannot be satisfied with the same optics because of its inherent symmetry limitations on geometry. In order to meet those kinds of demand, 3D optical design methods have been developed, such as the simultaneous multiple surface (SMS) 3D method , supportOver the past decades, much research work has been conducted on tool path generation for diamond turning of freeform optics, including angle sampling strategy, spiral path and tool radius compensation. Fang et al.  used nonx-axis) when it follows the c-axis. Due to the limitation of the dynamic response of the servo axis, this will lead to the following error and vibration.During the conventional multi-axis computerized numerical control (CNC) milling of freeform surfaces, it is crucial to adapt the side-feeding motion and forward sampling of tool motions to surface shape variations to achieve uniform surface quality with maximum efficiency . Koren eGenerally, the following problems exist in tool path generation algorithm for slow tool servo diamond turning of large aperture freeform optics:Current angle sampling strategies are still constant-angle and constant arc-length methods ,16, whicThe cutting residual is caused by the tool radius envelope along the feeding direction, which is regarded as surface roughness. Kwok et al.  and Yu eCurrent tool path generation methods do not consider the dynamic performance of machine tools. In traditional machining of rotational symmetrical surfaces, the feed axis and spindle are independent, so the dynamic performance of machine tool is not considered. However, when a freeform surface is machined by STS diamond turning, the feed axis and the spindle should maintain a strict spatial position relationship. Therefore, an optimal tool path is very important. Although the ATS method proposed by Zhu  can prodIn this paper, a novel path generation of STS diamond turning for large aperture freeform optics; namely, adaptive tool path generation (ATPG) is proposed. Firstly, the principle of the novel generation method is introduced and the path generation algorithm is presented in detail with analysis of the motion characteristics of machine tools. The results show that, on the premise of satisfying the machining accuracy, the proposed algorithm can reduce the volume of control points, and improve the dynamic characteristics of machine tools and machining efficiency. Finally, both theoretical analysis and experimental comparison of the algorithm are conducted.z-axes of machine tools (STS).The principle of diamond tool turning freeform optics is that the diamond tool contours the complex surface along the spiral path as shown in Lθ between the two consecutive control points as well as the local surface profile. Generally, most diamond machine tools offer PVT spline interpolation for STS, which creates a path known as a cubic Hermite Spline. Therefore, for a given interpolation tolerance εf, the algorithm adaptively adjusts the angle interval Lθ according to the error of Hermite interpolation to ensure that the error is within the range , so that the error is homogeneously distributed in the whole cutting direction.There are two intrinsic errors in STS machining, namely interpolation error and cutting residual as shown in 1 and θ2 of the same radius is different, so the calculated feedrate f1 and f2 on the same radius are different, which leads to the position of diamond tool changing with spindle angle in the process of feeding. As a result, micro-fluctuations are generated in the process of machining, which would deteriorate the surface quality of machining, just as shown in In the feeding direction, in order to improve the machining efficiency, the feedrate should be adjusted with surface curvature. Because the curvature of a freeform surface not only changes with radius, but also changes with polar angles, as shown in Although the feedrate in ATPG is slightly smaller than ATS, it is still larger than the traditional settings. The advantages of this setting are that it not only improves the machining efficiency, but also suppresses the micro-fluctuation caused by surface shape. Although this will result in a slight heterogeneous distribution of interpolation error at different angles on the same radius, it is almost negligible, and the advantage is to increase the stability of the machine tool.In the traditional STS diamond turning, the machined surfaces have to be evaluated after the machining process which gives a high risk of the machined surface failing to meet the contour accuracy. The surface generation algorithm is not only the generation of a diamond tool path, but also a predictive method to analyze the contour accuracy by using different cutting strategies.Due to the geometrical complexity of freeform surfaces, it is necessary to check tool interference, and the sectional curve method  is used z = f, which can be also expressed as z = g by coordinate transformation.The coordinate system of the workpiece for STS machining is shown in iN-th revolution of the spiral path is ρi at the angle θi = 2πi, so the corresponding curvature at this revolution can be defined as , and ϕ(4)(∙) denotes the 4th derivative.PVT mode provides an excellent contouring capability because it takes the interpolated commanded path exactly through the control points. It generates a path known as a Hermite spline. Hence, according to the error of piecewise cubic Hermite interpolation, the relationship between the interpolation error tolerance εucted by :(10)εf≤LLθ and θk+1 be derived from Equations (10) and (11) by numerical calculation.The following relation is also established as:kN+1 for kP+1 can be obtained by:The corresponding revolution kP+1 can be obtained by:The other parameters ρ for kP+1 can be obtained by combining the equations from Equation (8) to Equation (14). By conducting the iterative steps with respect to N1 = 0, the whole toolpath can be adaptively resampled.Thus, the coordinate of point xz-direction compensation , and the interpolation errors are homogeneously distributed over the entire surface. For DiffSys which adopts the constant-angle methods, the surface quality of central region is better than that of the outer region, and in order to ensure the allowable interpolation error on the outer region, a large number of sampling points are needed on the outer region of the surface, which results in redundant control points on the center region. In this case, the number of control points in ATPG  was only about 64% of that in DiffSys (6061). Therefore, the machining time in DiffSys would be much longer than that in ATPG to have similar machining accuracy.We computed the intermediate “way-points” for each axis for each point along the spline path by Hermite spline interpolation. After subtracting the desired surface, error maps of the theoretically generated surface for ATPG and DiffSys are illustrated in f = 8.9 μm/rev, which was based on the surface finish specification and the maximum slope of the part, and the tool radius compensation in DiffSys was set along the z-direction as well.The astigmatic (AST) surface was machined for investigating the performances. Considering the testing ability of the interferometer, the aperture of the workpiece was 50 mm, the amplitude was set as 4.8 μm, respectively. To have a comparison, the tool path was generated by both ATPG and DiffSys, and DiffSys follows the constant-angle strategy that was based on the interpolation tolerance and the minimum radius of curvature of the part. The feedrate in DiffSys was set as In order to validate the performance of ATPG for large aperture optical surfaces, an off-axis paraboloid (OAP) with an aperture of 213 mm was also machined, where the vertex curvature radius R was 915 mm and off-axis magnitude was 915 mm. We compared the programs generated both by ATPG and Diffsys for the same setting rule.® 250 ultra, Keene, NH, USA). The configurations of this experiment were shown in The cutting experiments were performed on a CNC ultra-precision lathe  was employed to measure the machined surface form error. The AST surface was tested directly by the interferometer and the OAP was tested though optical properties of quadric surfaces as shown in After subtracting the design surface, the form errors for the two machined surfaces (AST) are respectively shown in The machined OAP surfaces were measured through optical properties of quadric surfaces as shown in Generally speaking, more control points mean higher interpolation accuracy. However, as the number of points increases, the number of points is no longer the bottleneck for machining accuracy. Furthermore, because the new tool path makes the axis speed variation very small, the machine tool can adapt to a higher spindle speed. Hence, the comparison verifies the high machining efficiency of the ATPG for achieving the same surface quality. It is noted that the PV value of the form error and roughness is higher than the preset theoretical one. The reason is that, besides the accuracy of interpolation, factors that determine the surface form and roughness still include machine tool vibration, tool wear, lubrication and clamping deformation, etc.With consideration of machine tool motion characteristics, an adaptive tool path generation is investigated, including establishing the functional relationship between spiral radius and revolution number with respect to cutting residual, smooth curve fitting and solving cutter contact points in the cutting direction with the consideration of interpolation error and tool radius compensation.A theoretical investigation for ATPG is conducted. The results indicate that the smooth curve produced by the spiral radius and revolution number can effectively suppress vibration of side-feeding motion, which can make the machining operated at a higher spindle speed.A comparison investigation on surface generation in ATPG and DiffSys was experimentally conducted on typical freeform surfaces. By adopting ATPG, the volume of tool path control points and machining time are effectively reduced, while surface form error as well as roughness is maintained. Therefore, the proposed ATPG significantly increases the machining efficiency for large aperture freeform optics.This research proposes a novel adaptive tool path generation (ATPG) for freeform optics to improve machining efficiency. The main conclusions are as follows:"}
{"text": "Compound sinusoidal grid surface with nanometric finish plays a significant role in modern systems and precision calibrator, which can make the systems smaller, the system structure more simple, reduce the cost, and promote the performance of the systems, but it is difficult to design and fabricate by traditional methods. In this paper, a compound freeform surface constructed by a paraboloidal base surface and sinusoidal grid feature surface is designed and machined by slow tool servo (STS) assisted with single point diamond turning (SPDT). A novel combination of the constant angle and constant arc-length method is presented to optimize the cutting tool path. The machining error prediction model is analyzed for fabricating the compound sinusoidal grid surface. A compound sinusoidal grid surface with 0.03 mm amplitude and period of 4 is designed and cutting process is simulated by use of MATLAB software, machining experiment is done on ultra-precision machine tool, the surface profile and topography are measured by Taylor Hobson and Keyence VR-3200, respectively. After dealing with the measurement data of compound freeform surface, form accuracy 4.25 μm in Peak Village value (PV), and surface roughness 89 nm in Ra are obtained for the machined surface. From the theoretical analysis and experimental results, it can be seen that the proposed method is a reasonable choice for fabricating the compound sinusoidal grid surface. When compared with traditional elements, compound freeform surface elements can obviously make the systems smaller, make the system structure much simpler, reduce the cost, and promote the performance of the systems, and it has drawn more attention in recent years ,2. The ca) on binderless tungsten carbide [Nowadays, many researches have concentrated on how to design and manufacture a single simple piece of optical surface, such as spherical or aspherical surface, off-axis surface, and simple micro-structure surface. Ying Cheng et al. manufactured an off-axis optical reflective integrator by ultra-precision machining, the advancement in this study is to reduce the stepwise discontinuity among the facets to make it applicable for turning, and the off-axis optical reflective integrator is more simple in structure and smaller in size when compared with existing refractive integrators, and simulations and experiments show that above 90% of illumination uniformity can be achieved using this new design . Xiaodon carbide . Shijun  carbide . Szymon  carbide . Jayant  carbide . S. Wojc carbide . From th carbide .By analyzing the previous research work, it can been seen that most of research work concentrated on manufacturing single freeform surfaces, few researches investigated on machining compound freeform surfaces. In this paper, a compound freeform surface is designed by combining a paraboloidal base surface and a sinusoidal grid feature surface, optimization of cutting tool path, tool nose radius compensation, and error analysis are done in Compound freeform surface is different from traditional freeform surface in generating, traditional surface is confirmed by an equation or a series of equations, compound freeform surface is obtained by combining double or more freeform surfaces, and From the above illustration of freeform surface, we can reach a conclusion that compound freeform surfaces compose of a base surface and other one feature surface or more feature surfaces, which compares with the traditional single freeform surface. In view of the complex composition of compound freeform surface, the tool path will be more complicated and more challenges will be faced. In this paper, authors select the compound sinusoidal grid surface to be fabricated, which consists of a paraboloidal base surface and a sinusoidal grid feature surface.Compound sinusoidal grid surface include two features, paraboloidal surface and sinusoidal grid surface. In this section, a base surface paraboloidal surface model will be established, the equation of which is described, as follows:This section mainly describes the process of paraboloidal surface establishing, which is the first step to build compound sinusoidal grid surface.From the previous sections, we can know that a compound freeform surface is composed of two or more features; this section will present the second feature surface that is named sinusoidal grid surface, which can be expressed as Equation (2).Base surface and feature surface have been established up to now, and the following research will concentrate on integral compound freeform surface generation. According to According to Equation (3), the calculated compound sinusoidal grid surface consequence is shown in As displayed in In this paper, a compound freeform surface on a cylindrical end face was manufactured, there are two cutting path generation methods including constant angle and constant arc-length methods. As is depicted in For the sake of achieving high accuracy of the compound freeform surface, it is critical to choose an appropriate cutting path. As for the constant angle tool path that is displayed in S cutting path is that each arc between double adjacent cutting points is a constant value, as shown in When compared with constant angle tool path, the main characteristic of constant arc-length ΔDepending on the above analysis, there is a high accuracy at the central region with constant angle cutting path, and the surface accuracy will be superior in outer area with constant arc-length cutting path, so this paper proposed a method that combines the constant angle and the constant arc-length means that the central region of compound sinusoidal grid surface adopts constant angle cutting path and the outer area employs a constant arc-length cutting path. The recombination result of constant angle and constant arc-length method is displayed as As is shown in r, clearance angle α, which is shown as The previous works have determined the compound sinusoidal grid surface, cutting path, and tool contact points. Ultra-precision machining also requires a suitable diamond tool with optimal parameters including tool nose radius Tool geometry parameters have a great influence on the machined surface feature, thus a series of fit tool geometry parameters is essential to guarantee the high accuracy of compound freeform surface finish. The subsequent search is about to concentrate on tool geometry analysis.a shows the way that obtains the surface radial curve for tool nose radius optimum, The first study is about tool nose radius optimum, because the desired compound sinusoidal grid surface is concave-convex severely, and the corresponding cutting path is concave-convex. It will do harm to the compound freeform surface finish accuracy with an unfit tool nose radius. Figure 11Clearance angle is another indispensable factor for optimum of tool geometry, it has a considerable impact on machined surface, as shown in After confirming the advanced double elements, wrap angle is also critical to contribute to surface accuracy, geometry of wrap angle and relation between diamond tool and cutting path are illustrated as Wrap angle The above sections have investigated tool geometry in detail, this part will explain the tool nose radius compensation. Depending on the actually machining processes, diamond tool nose is not a point in fact, but a circular arc, as for that, it is imperative to make tool nose radius compensation to obtain tool location points, the geometry of compensation is presented as The subsequent procedure is to solve the direction of tool nose radius offset, which is expressed as the following equation.The subsequent procedure is to solve the direction of tool nose radius offset, which is expressed as the following equation.r is tool nose radius. All the preparatory works have been accomplished, the last step is tool location points The desired surface compound sinusoidal grid surface can be fabricated on the basis of advanced study, however, there is deviation between the ideal surface and the surface manufactured in reality, including two types of surface errors, named cutting residual error and cutting linearization error. The following section is to analyze them.Cutting residual error is analyzed in the first place, which is caused by the cutting path along feed direction, and it is dominant by tool nose radius and feed rate, schematic diagram of cutting residual error is illustrated in H is cutting residual error, r is tool nose radius and f is feed rate.R is the radius of curvature at the point between adjacent tool trajectory.The cutting residual error including three situations has been explained in above section, and we can restrain the cutting residual error by optimize tool nose radius and feed rate parameters to meet the requirement of accuracy based on Equations (9)–(11), while the tool nose radius is confirmed, feed rate is the critical parameter to ensure the desired surface accuracy. Therefore, in the light of Equations (9)–(11) which can be used to do inverse computation to obtain the optimized feed rate, the corresponding equations of (9)–(11) are as follows.Based on Equation (12), the required feed rate can be expressed as the following:In addition to the cutting residual error, the subsequent section is to analyze cutting linearization error that is the other machining error that is generated in the cutting direction in the course of machining, which is due to the distinction between the ideal surface profile and the linear cutting trajectory, it relies mainly upon the distance between adjacent points along the cutting direction. h is the demand cutting linearization error.From the schematic diagram illustration of cutting linearization error, which uses the approximate concept that an arc between adjacent points is approximate to an ideal arc, therefore, the cutting linearization error is obtained, as follows, based on the approximate concept and the schematic diagram of cutting linearization error.r1 = r2 = 1000, A compound sinusoidal grid surface expressed by Equation (3) with Before manufacturing the workpiece, the critical procedure is to center tool by the UltraSet Optical Tool Setter (USOTS) and to balance the spindle that ensures the machining precision of the desired surface. The applied cutting path shown in In order to verify the effectivity of the proposed method, the compound freeform surface roughness and form accuracy were measurement and evaluated with a precision form measurement system Taylor Hobson  and a high magnification microscope Keyence VR-3200 , respectively.The machining activity was conducted using Nanoform250 ultra-precision machine tool. The actual roughness measurement of the compound sinusoidal grid surface is done in a Taylor Hobson. The surface topography of the compound sinusoidal grid surface measured by Keyence VR-3200 is shown in From the above experiments results, we can know that a compound sinusoidal grid surface is successfully fabricated with well surface finish, which is based on former critical procedures. Above all, the compound freeform surface recombination theory must be acquainted as you expect to machine compound surfaces, because a component with well machined surface needs sufficient theoretical knowledge in order to sustain. Then, an appropriate fabrication method is another vital element, SPDT with STS used in this paper is suitable and highly efficient when compared with the integrated solution (combining fast tool servo with slow tool servo) and laser direct writing method or other solutions, the experiment results have demonstrated that STS is well enough to manufacture compound freeform surfaces. The cutting tool path generation is another indispensable part, most of the research teams acquired failing or defective surfaces on account of inadequate preparation about cutting tool path. A cutting tool path of combining constant angle with constant arc-length path is adopted to process compound sinusoidal grid surface, which contains all the benefits of the two sorts of cutting paths. Finally, a machining error prediction model is necessary to forecast the coming error for designed compound freeform surface, a majority of teams fabricated components by means of trying cutting to guarantee desired surface precision, which is attached with a number of uncertain factors. When compared with the published literature, Xiaodong Zhang et al. studied the mathematic and optical model to propose a grid machining method for the fabrication of compound eye lens. They presented an off-centering machining configuration to avoid the shape distortion that is caused by tool un-alignment. Based on the proposed methods, a compound freeform surface that was constructed by sphere base surface and compound eye lens was successfully machined, but they did not show the surface accuracy and just indicated that the method presented is suitable for the compound eye lens with curve substrate. As far as the author is concerned, there are more and more application fields for compound surfaces with the development of society and technology, and the demand for surface precision will be also greatly improved, so more researches should be done to focus on how to design and machine some compound freeform surfaces.In this paper, the following conclusions can be obtained. Firstly, a new recombination method of compound freeform surfaces is presented that demonstrates the method to achieve a compound freeform surface. Secondly, SPDT assisted with STS is presented in order to successfully manufacture the compound sinusoidal grid surface. Thirdly, a cutting tool path that recombines the constant angle cutting path and the constant arc-length cutting path is proposed to manufacture the compound freeform surface, which can ensure the surface precision. Finally, a fabrication error prediction model is established for forecasting the machining deviation in order to obtain the optimum cutting path parameters, which avoids trying cutting to achieve an ideal surface finish when compared with the other academic research. By cutting an Al6061 workpiece and measuring the machined surface, it is found that the roughness value and PV value of the machined compound freeform surface can be 89 nm and 4.25 μm, respectively. Obviously, the theoretical analysis and experimental results demonstrate that the proposed methods are feasible to machine a compound freeform surface and can improve the machining precision."}
{"text": "Combined stereotactic body radiotherapy (SBRT) for lung lesions and conventional radiotherapy (CRT) for nodal areas may be more effective than CRT alone in patients with locally advanced lung cancer.This study included 21 patients with small primary lung tumors distant from the regional nodal areas. The SBRT dose was 40–60 Gy in 4 fractions. CRT doses were 66 Gy in 30 fractions for non-small cell lung cancer and 52.5 Gy in 25 fractions for small cell lung cancer.The median follow-up duration was 12 months, and the median survival was 13 months. The 1 year overall survival, local recurrence-free survival, and distant metastasis-free survival rates were 60.5, 84.8, and 62.1%, respectively. Two patients experienced in-field local recurrence combined with out-field regional recurrence and/or distant failure. The major recurrence pattern was distant failure . Three patients aged ≥79 years experienced grade ≥ 3 acute radiation pneumonitis, and one also had idiopathic interstitial pneumonia.The combination of SBRT for the lung lesion and CRT for the nodal region seems to be effective and safe for lung malignancies. However, patients older in age and/or with underlying pulmonary disease require stricter lung dose constraints. Definitive concurrent chemoradiotherapy (CCRT) has been recommended as the standard treatment for unresectable or medically inoperable stage II–III non-small cell lung cancer (NSCLC), based on the findings of randomized prospective trials –3. DefinAs the primary lung lesion is located adjacent to the mediastinum and hilar lymph node (LN) region in most lung cancer patients, similar fractionation schemes are generally used for primary lung tumors and metastatic LNs. Some patients, however, are diagnosed with an isolated primary lesion, relatively small in size and distant from the nodal area Fig. . AlthougStereotactic body radiotherapy (SBRT) consists of the administration of one or a few fractions of radiation (with high doses in each fraction), and is expected to achieve improved local control. Several reports indicate the effectiveness of SBRT in the management of patients with early-stage NSCLC, with 2–3 year local control rates of approximately 90% in patients with T1–2 disease –8. In adThis strategy might also reduce toxicities, owing to the reduction in PTV margins and the characteristics of SBRT including high targeting accuracy and rapid dose falloff. Because the SPACE trial found that the rates of pulmonary and esophageal toxicities were lower with SBRT than with CRT , our cenThe records of patients diagnosed with lung malignancy and treated with S + CRT at our hospital between March 2009 and July 2017 were retrospectively reviewed. All patients who received S + CRT had a small isolated primary lesion located distant from the nodal area  reflecting respiratory motion were acquired. The gross tumor volume (GTV) of the lung was delineated at the end-exhale phases using the lung setting . The internal target volume (ITV) was contoured using maximum intensity projection images, whereas the PTV was a 5 mm expansion of the ITV. The SBRT dose  was administered in four fractions, as determined by the radiation oncologist. After SBRT, all patients underwent CT  for three-dimensional CRT planning. The GTV at the LN was contoured using the mediastinal setting , and the PTV margins were maintained as 7 mm radially and 10 mm longitudinally. The clinical target volumes were also delineated for some patients to cover the regional nodal areas in the same axial sections; however, smaller PTV margins  were considered in these patients. In patients treated with induction chemotherapy, the post-chemotherapy volume was defined as the GTV. Standard radiotherapy consisted of 66 Gy (2.2 Gy/fraction) for NSCLC and 52.5 Gy (2.1 Gy/fraction) for SCLC.The organs at risk (OAR) included healthy lung tissues, the esophagus, the spinal cord, and the heart. The normal organ constraints included a maximal dose for the spinal cord of < 50 Gy, a mean lung dose (MLD) of < 20 Gy, a volume of the lung receiving at least 20 Gy (V20) of < 30%, a mean esophagus dose of < 35 Gy, and heart doses of V60 < 1/3, V45 < 2/3, and V40 < 100%.Chest X-rays (CXR) were obtained weekly to monitor changes in tumor volume and any acute toxicities. Treatment was verified by weekly kV imaging guidance, using set-up correction based on carina and bony anatomy.The main concurrent chemotherapy regimen for NSCLC consisted of weekly doses of paclitaxel combined with cisplatin or carboplatin. Patients with very large tumors and a high risk of radiation-related toxicities were initially treated with induction chemotherapy for volume reduction, with a regimen consisting of two cycles of gemcitabine plus cisplatin in sequential schedules every 3 weeks. Chemotherapy for SCLC consisted of intravenous etoposide and cisplatin administered every 3 weeks for four cycles, with radiotherapy initiated along with the third cycle of chemotherapy. Patients with a poor performance status or poor lung function were recommended to undergo radiotherapy alone.All patients underwent weekly complete blood counts and CXR. Patients were routinely followed up by a radiation oncologist and/or medical oncologist, with chest CT and/or CXR performed 1 month after treatment, every 3 months during the first 2 years, and every 6 months thereafter until 5 years after treatment.The primary outcome was 1 year OS rate. Secondary endpoints included 1 year local recurrence-free survival (LRFS) and distant metastasis-free survival (DMFS) rates and toxicities. Toxicities were evaluated using the Common Terminology Criteria for Adverse Events version 4.03. Adverse events that occurred during treatment and within 3 months after CRT were defined as acute toxicities.Follow-up durations were calculated from the date that treatment was discontinued. OS was calculated using the Kaplan-Meier method from the date of treatment discontinuation until death. Clinical and dosimetric factors of patients with and without grade ≥ 3 acute radiation pneumonitis (RP) were compared using Mann-Whitney U-tests. All statistical analyses were performed using SPSS version 21.0.n = 4), IIIA (n = 8), and IIIB (n = 9). All patients had forced expiratory volume in 1 s (FEV1) ≥40% of normal, making them eligible for the NPC 95–01 study [A total of 21 patients were included; their demographic and clinical characteristics are shown in Table n = 6), including idiopathic interstitial pneumonia (IIP) (n = 1), chronic obstructive pulmonary disease (COPD) (n = 1), and patient refusal (n = 1).The median SBRT dose was 54 Gy , which was administered in 4 fractions . Eight patients who received 60 Gy over 4 fractions underwent treatment twice a week, whereas the others were treated daily. Seven patients were treated with SBRT using three-dimensional conformal RT (3D-CRT), whereas 13 received volumetric arc therapy. Non-isocentric technique using a Cyberknife was applied to one patient. Median time interval between the start of SBRT and the start of CRT was 8 days . The median CRT dose was 60 Gy , and the median dose per fraction was 2 Gy . Only one patient received 6 Gy fractions, which is strictly defined as hypofractionated radiotherapy, rather than CRT. Nevertheless, because this study focused on the difference in fractionation regimens administered to the lung and nodal area, this patient was included. Four patients received intensity modulated RT, and the others received 3D-CRT. Nine patients did not receive any chemotherapy due to old age (≥75 years) and comorbidities . Median survival was 13 months . The 1 year OS rate was 60.5% Fig. , whereasTwo patients experienced in-field local recurrence, combined with out-field regional and/or distant failure. The locations of the in-field recurrences were the mediastinal and low cervical nodal areas. Both patients had NSCLC, with one having recurrence after lobectomy. Neither received any chemotherapy. Five patients exhibited out-field regional failure , with three simultaneously diagnosed with distant recurrence. All out-field regional recurrences occurred in nodal areas, not in lung fields, and all of these patients had NSCLC. Four of these patients received definitive treatment, whereas one received salvage treatment. Three did not receive any chemotherapy. The major recurrence pattern was distant failure, which occurred in nine patients . Of these nine patients, only one had SCLC, two received salvage treatment, and two did not receive chemotherapy.Nine patients experienced acute radiation esophagitis, including eight with grade 2 disease and one with grade 3 disease. Except for one patient who finally progressed to grade 3 late radiation esophagitis, the other patients were successfully managed medically. Eight patients experienced acute RP, including five with grade 2, one with grade 3, and two with grade 4 (life-threatening) disease. The characteristics of the three patients with acute RP of grade ≥ 3 are summarized in Table One patient who initially had IIP showed poor lung function with a DLco of 37%; this patient was treated with hypofractionated radiotherapy to the nodal area (60 Gy over 10 fractions). Nevertheless, as the PTV of the nodal area was very small (27.7 cc), more conservative dose constraints were applied. Dosimetric parameters showed that his MLD was 7.1 Gy and his V20 was 9.1% (Table p = 0.011). Other demographic and clinical factors, including sex, performance status, smoking history, pre-treatment pulmonary function test results, aim of radiotherapy , and combined chemotherapy, were not significantly associated with grade ≥ 3 RP. Due to the small number of patients enrolled, we could not reliably determine the dosimetric parameters, such as MLD, V5, V20, mean ipsilateral lung dose, and PTV size, predictive of grade ≥ 3 RP. Two patients without a history of acute RP experienced late RP of grade 2. None of the patients experienced any grade ≥ 2 hematologic toxicity.The only significant factor associated with grade ≥ 3 RP was older age  and OS  in the SBRT arm [Only two of our patients experienced in-field recurrences at nodal areas alone. A previous study investigated the patterns of loco-regional failure in locally advanced NSCLC patients who received definitive CCRT (66 Gy in 24 fractions) . There wSBRT arm . AlthougSBRT arm . This fiAttempts have been made to administer SBRT to patients with locally advanced cancers. For example, a prospective, single-institution study evaluated the feasibility of CRT (59.4 Gy in 33 fractions) followed by SBRT (20 Gy in 2 fractions or 19.5 Gy in 3 fractions) in patients with stage II–III NSCLC and residual disease on positron emission tomography . Over a Patients administered S + CRT are likely to experience the abscopal effect of SBRT . Becausep = 0.006) and a lower rate of RP  in the SBRT than in the CRT group [Previous studies of patients with NSCLC treated with 63–66 Gy in fractions of 1.8–2 Gy reported grade ≥ 3 pulmonary toxicities presenting as acute complications in 4–5% of patients and late toxicities in 11% . AcceptaRT group , our patOne patient in the present study with grade 4 RP had IIP and a DLco of only 37%. A more pronounced decrease in lung function has been reported in individuals with an initial DLco of ≤50% . Lung coThis study had several limitations, including its retrospective design, the small number of patients, and the heterogeneity of the enrolled patients. Although CCRT was the standard treatment, 43% of these patients did not receive any chemotherapy. Therefore, the results of the present study are not representative of standard populations. The potential numbers of candidates for S + CRT are limited, because relatively few patients present with primary lung lesions distinct from nodal areas.This retrospective analysis of lung cancer patients treated with S + CRT showed promising local control and survival rates, as well as acceptable toxicities. Stricter lung dose constraints are need for older aged patients and those with underlying pulmonary diseases. Additional studies, in larger numbers of patients and with stricter enrollment criteria, are required to confirm these findings."}
{"text": "UV exposure is associated with oxidative stress and is the primary factor in skin photoaging. UV-induced reactive oxygen species (ROS) cause the up-regulation of metalloproteinase (MMPs) and the degradation of dermal collagen and elastic fibers. Garlic and its components have been reported to exert antioxidative effects. The present study investigated the protective effect of garlic on UV-induced photoaging and MMPs regulation in hairless mice. Garlic was supplemented in the diet, and Skh-1 hairless mice were exposed to UV irradiation five days/week for eight weeks. Mice were divided into four groups; Non-UV, UV-irradiated control, UV+1% garlic powder diet group, and UV+2% garlic powder diet group. Chronic UV irradiation induced rough wrinkling of the skin with hyperkeratosis, and administration of garlic diminished the coarse wrinkle formation. UV-induced dorsal skin and epidermal thickness were also ameliorated by garlic supplementation. ROS generation, skin and serum malondialdehyde levels were significantly increased by UV exposure and were ameliorated by garlic administration although the effects were not dose-dependent. Antioxidant enzymes such as superoxide dismutase and catalase activities in skin tissues were markedly reduced by UV irradiation and garlic treatment increased these enzyme activities. UV-induced MMP-1 and MMP-2 protein levels were suppressed by garlic administration. Furthermore, garlic supplementation prevented the UV-induced increase of MMP-1 mRNA expression and the UV-induced decrease of procollagen mRNA expression. These results suggest that garlic may be effective for preventing skin photoaging accelerated by UV irradiation through the antioxidative system and MMP regulation. Wrinkleteration .Excessive exposure to UV irradiation generates reactive oxygen species (ROS) in the skin , and oveROS lead to the activation of transcription factors which causes up-regulation of metalloproteinase (MMPs) and degradation of dermal collagen and elastic fibers . MMP-1 aGarlic has long been used widely not only as a flavoring agent but also as a folk medication and is one of the most well-known herbal medicines worldwide. Numerous therapeutic effects of garlic and its constituents such as antioxidant, anti-microbial, anti-atherosclerotic, anti-diabetic, anti-mutagenic, anti-carcinogenic and immune-modulation activities have been reported ,13,14,15The changes of body weight and serum biochemical indicators in the hairless mice were determined to examine the animal toxicity of garlic powder. Mean body weights of all experimental groups were similarly increased throughout the period of study. There were no significant differences in body weight and food intake between the groups during the eight weeks of the experimental periods (data not shown), suggesting that body weight gain and food consumption were not affected by UV irradiation and garlic supplementation.The hepatotoxicity of garlic was determined by measuring the serum aspartate transaminase (AST) and alanine transaminase(ALT) concentrations . UV irraThe effect of garlic on UV-induced wrinkle formation was observed by visual assessment of close-up photos in the dorsal skins of hairless mice. Chronic UV irradiation induced deep, irregular wrinkling of the skin with hyperkeratosis, and marked reduction in UV-induced wrinkle formation was observed in the UV+G-1% and UV+G-2% groups compared with the UV group A.Chronic UV irradiation induces skin inflammation, which causes excessive epidermal proliferation and skin thickening . The effp < 0.05). The results again showed that 1% garlic supplementation exhibited better protection from UV-induced skin damage.Sections of dorsal skin from hairless mice were stained with Hematoxylin and eosin (H & E) or the Verhoeff van-Gieson method to determine epidermal hyperplasia and elastic fiber, respectively. Epidermal thickness, as a histological feature of photoaged skin, can be used as one of the quantitative parameters to reflect UV-induced skin damage, since thickened epidermis can contribute to skin roughness . Based oElastic fibers are important components of the skin and are responsible for skin elasticity. To visualize the changes in elastic fibers in the dermal areas, histological sections of the skin were subjected to Verhoeff’s staining. As shown in Oxidative stress is a primary factor in the photoaging process. UV increases levels of hydrogen peroxides and other reactive oxygen species in skin and decreases the levels of antioxidant enzymes ,6. IncreMalondialdehyde (MDA) is the aldehydic decomposition product of lipid oxidation, which is the major thiobarbituric acid reactive substance (TBARS). The lipid peroxide levels in skin and serum were determined by measuring the TBARS concentration. As shown in Serum SOD and catalase activities were significantly reduced by UV irradiation, while dietary garlic supplementation significantly increased these enzyme activities compared with the UV-irradiated control group . No signMMPs  have been known to play important roles in the degradation of skin collagen and elastin. To investigate the mechanism underlying the protective effect of garlic on UV-induced photoaging, expressions of MMP-1, MMP-2, and procollagen type 1 were analyzed . The resType 1 collagen is the most abundant collagen in skin connective tissue as well as the human body, and newly synthesized procollagen type 1 is secreted into the dermal extracellular space, and has an anti-wrinkle effect in UV-irradiated skin. Also, a decrease in procollagen expression caused by repeated UV irradiation has been considered to be a cause of photoaging . TherefoS-allylcysteine and allixin, suppress LDL oxidation in vitro. Furthermore, short-term supplementation of garlic in human subjects has demonstrated an increased resistance of LDL to oxidation as well as blood antioxidant parameters [One of the most important biochemical properties of garlic is its antioxidant potential. Garlic or garlic oil supplementation prevented the increase in oxidative stress associated with gentamicin-induced nephrotoxicity or nicotine-induced lipid peroxidation in rats ,20. Oxidrameters ,14.Chronic exposure to UV irradiation results in photoaging of skin including various characteristic changes, such as DNA damage ,21,22, hSkin consists of three layers: the epidermis, the dermis, and the subcutis. The outer layer, the epidermis, is composed of keratinocytes, melanocytes, and langerhans cells. This layer serves as a barrier to protect the body by synthesis of the protein keratin. The dermis is made up of collagen fiber, elastin fiber, and the derma matrix, which are produced by fibroblasts . The epiet al. [Photo-oxidative damage caused by chronic UV exposure is the leading cause of extrinsic aging of the skin. In normal cells, antioxidative defense systems, including SOD, catalase, and glutathione peroxidase, have evolved to quench ROS ,6,24. Hoet al.  demonstret al. . In the in vivo [Photoaging is characterized by the degradation of collagen and the accumulation of abnormal elastin in the dermis, and several MMPs have been implicated in this process ,9,29. Inin vivo . Collagein vivo . SimilarS-allylcysteine (SAC), and vinyl dithiines [Garlic contains unique organosulfur compounds, which provide its characteristic flavor/odor and most of its potent biological activity. The main sulfur compound in both raw garlic and garlic powder is alliin, and allicin was found to be a major constituent of solvent extracted garlic . Allicinithiines .S-alk(en)yl-l-cysteine sulfoxides have been reported [et al. reported that topical treatment of S-allyl cysteine, the most abundant component of aged garlic, exhibits antioxidative, anti-wrinkle activity, and down-regulates MMP-3, -9, and -12 protein expression [et al. [Antioxidative effects of sulfur compounds such as allicin, allyl disulfide, allyl cycteine, alliumthiosulfinates and reported ,33,34. Kpression . Howeverpression . Recentl [et al.  reportedN-acetyl-S-(2-carboxypropyl)-cysteine, N-acetylcysteine and hexahydrohippuric acid, have been detected in human urine after ingestion of garlic [Bioavailability of active ingredients in garlic is likely essential, and it was suggested that the metabolites of allicin, rather than the allicin itself, are responsible for the health effects of garlic. SAC is one of the water-soluble organosulfur compounds in garlic. SAC absorbed rapidly and showed almost 100% bioavailability after oral administration . Also, tf garlic . At presf garlic . Howeverw/w), and 1% garlic supplementation exhibited better or similar effect to 2% garlic supplementation, suggesting that 1% garlic supplementation reached the peak effective dose. The 1% garlic supplementation in the present study was 250 mg/kg/day garlic powder (food intake was about 5 g/day/mice) and was equivalent to approximately 4.5 g~5.7 g garlic powder/day for humans on the bases of energy requirements . Pitana et al. [The doses of garlic supplemented in the present study were 1% and 2% of the diet , purchased from Seosan  in July 2009, were peeled, vacuum dried, and powdered.Fresh garlic (w/w) in commercial mouse diet . Body weight and food intake were regularly measured throughout the study. The animal protocol used in this study was reviewed and approved by the Kyunghee University Institutional Animal Care and Use Committee (KUIACU).Six-week-old female albino hairless mice (Skh:HR-1) were obtained from Central Lab Animals, Inc. . The animals were housed under controlled temperature (23 ± 2 °C), humidity (55% ± 5%) and light (12 h light/dark cycles). All animals had free access to water and commercial pellet food. After an acclimation period (one week), mice were randomly divided into four groups of eight animals: Non-UV, UV-irradiated control, UV+G-1% (UV+1% garlic powder diet group), UV+G-2% (UV+2% garlic powder diet group). Garlic powder was supplemented  at a distance of 20 cm from the UV light source provided by an array of UVA and UVB  lamp with a dose output ratio at 1:4.2 for the first week, with an increase to 55 mJ/cm2 at second week, 63 mJ/cm2 at third week, 68 mJ/cm2 at fourth week, 76 mJ/cm2 at fifth week, 81 mJ/cm2 at sixth week, and 89 mJ/cm2 at seventh to eighth week for total dose of 2850 mJ/cm2. The non-irradiated control group was treated identically with the lamp power off. The integrated UV irradiation was measured with a UV meter . The animals were sacrificed three days after the final irradiation to allow the recovery from the acute UV effects. The blood collected via cardiac puncture was allowed to clot, and the serum was obtained by centrifugation at 3000× g for 30 min. Aliquots of serum samples were stored at −20 °C until analyzed.The mice were exposed to UV irradiation five days per week starting with 50 mJ/cmSkin thickness was assessed by measuring skin-fold thickness. Briefly, midline dorsal skin of mouse was lifted up at the neck and base of the tail by pinching and skin-fold thickness was measured mid-way between the shoulders and hips using a caliper . The graders were blinded to the groups of the mice.The hepatotoxicity of garlic powder was determined by measuring the activities of alanine transaminase (ALT) and aspartate transaminase (AST) using commercial kits .Morphologic changes of mouse skin surface were observed using a digital microscope .For histopathological assessment, the dorsal skin tissue was fixed in 10% neutral buffered formaldehyde solution, embedded in paraffin and sliced into 5 μm sections. H & E staining was performed to assess epidermal hyperplasia which was measured by epidermal thickness. Verhoeff’s staining was performed using a Verhoeff Van Gieson elastin staining kit  to examine elastic fibers.g for 20 min at 4 °C, and total supernatant was used for the analysis.Skin samples were harvested from the sacrificed mice and the skin tissue (0.4 g) was homogenized  with Ultra Turrax  in nine volumes of cold normal saline (4 °C) to obtain the skin tissue homogenate. The un-dissolved pellet was removed by centrifugation at 3000× A fluorometric assay was used to determine ROS level. Non-fluorescent 2’,7’-dichloro fluorescin diacetate (DCFDA) is oxidized to the highly fluorescent 2′,7′-dichlorofluorescin (DCF) in the presence of esterase and ROS, including lipid peroxides . Brieflyw/v TCA and 0.375% w/v TBA in 0.25 N HCl) reagent was added and heated for 15 min, flocculent precipitate was removed by centrifugation at 1000× g for 10 min and absorbance was measured at a 535 nm against a blank containing no tissue homogenate. The MDA concentration was calculated using an extinction coefficient of 1.56 × 105/m·cm.MDA has been identified as the product of lipid peroxidation. MDA levels in the skin and serum were estimated by the thiobarbituric acid assay. To 1 mL of skin tissue homogenate or serum sample, 2 mL TCA-TBA-HCl . Catalase activity was measured according to the method of Aebi . One uniThe production of MMP-1 and MMP-2 were determined in skin homogenate supernatant using commercially available ELISA kits .® Protect Mini kit  to measure the mRNA expression of procollagen I and MMP-1. Complementary DNA was synthesized from mRNA using QuantiTect® Reverse Transcription kit (Qiagen). Real-time PCR was performed using Quanti Tect TM SYBR® Green PCR kit (Qiagen) according to the manufacturer's protocol. The cDNA was amplified for 45 cycles of denaturation (95 °C for 30 s), annealing (58 °C for 30 s), and extension (72 °C for 45 s) using the following primers: GAPDH forward 5′-CAT GGC CTT CCG TGT TCC TA-3′, reverse 5′-GCG GCACGT CAG ATC CA-3′; MMP-1 forward 5′-TTG CCC AGAGAA AAG CTT CAG-3′, reverse 5′-TAG CAG CCC AGA GAAGCA ACA-3′; procollagen type I forward 5′-GGACCT TGG AAG CCT TGG GGA CC-3′, reverse 5′-GGA ATT CGA AACCAC CGG CGT CGA AGG A-3′. Analyses were performed using Rotor-Gene Q® (Qiagen) and gene expression values were calculated based on the comparative ΔΔCT method.Dorsal skin tissues were homogenized with buffer RLT  in ice bath. Total RNA was extracted from dorsal skin tissue lysate using RNeasyp < 0.05.Data were presented as mean ± SD. One-way ANOVA was used to determine treatment effects. Differences among means were inspected using Tukey test and were considered to be significant at All together, the findings of this study indicated that dietary supplementation of garlic (1%) is effective in wrinkle improvement by alleviating oxidative stress and dermal extracellular matrix damage, resulting in the improvement of dermal collagen and elastic fiber status in the hairless mice photoaged model. In conclusion, this study suggests a high possibility for the practical use of garlic as an anti-wrinkle agent."}
{"text": "GATA4) and Friend of GATA 2 protein  form a heterodimer complex that has been shown to influence transcription of genes in a number of developmental systems. Recent evidence has also shown these genes play a role in gonadal sexual differentiation in humans. Previously we identified four variants in GATA4 and an unexpectedly large number of variants in ZFPM2 in a cohort of individuals with 46,XY Differences/Disorders of Sex Development (DSD) .GATA‐binding protein 4  and variant protein–protein interactions.Here, we review variant curation and test the functional activity of GATA4 and ZFPM2 variants we identified are benign in their contribution to 46,XY DSD. Indeed, only one variant, in the conserved N‐terminal zinc finger of GATA4, was considered pathogenic, with functional analysis confirming differences in its ability to regulate Sox9 and AMH and in protein interaction with ZFPM2.Our findings support that the majority of GATA4 and ZFPM2/FOG2 are not causative.Our study helps define the genetic factors contributing to 46,XY DSD and suggests that the majority of variants we identified in  GATA4 and FOG2. Variant curation and functional analysis revealed the majority of these variants are not likely to be causative.We identified a number of 46,XY DSD individuals with variants in  AVSDAtrial ventricular septal defectsCHDcongenital heart diseaseDIH3diaphragmatic hernia 3DSDdifferences/disorders of sex developmentHEK293human embryonic kidney 293OMIMonline mendelian inheritance in manSNVsingle nucleotide variantSRXY946XY sex reversal 9TADtransactivation domainTOFtetralogy of fallot1GATA4 family member has been shown to be involved in development of the heart, pancreas, liver, foregut as well as in the genital ridge of both sexes  were first identified in patients with congenital heart disease (CHD) including Atrial ventricular septal defects (AVSD)  are an evolutionally conserved family that plays various roles in embryonic development. The GATA members have two zinc finger domains that are required for recognition and affinity for DNA and interaction with other transcription factors  directly interacts with GATA4, by forming a heterodimer that represses expression of GATA4 target genes  and a heterozygous change c.779G > A (p.R260Q) . Here we have re‐curated these GATA4 and ZFPM2 variants using updated tools, and have tested their molecular activity in the context of gonadal signaling using several in vitro assays.While screening 279 46,XY DSD individuals for variants in genes known to cause DSD, we previously identified four variants in 22.1GATA4 (MIM# 600576) (four variants in seven patients), detailed in Table GATA4 variants, five presented with hypospadias . Case 5 in addition to hypospadias presented with multiple congenital anomalies including imperforate anus and dysmorphic facial features , c.1180C > A (p.Pro394Thr), and c.1220C > A (p.Pro407Gln), in association with Atrio ventricular septal defect   was located in the N‐terminal Zn‐finger  were located in the C‐terminal TAD  variants in 10 patients occurring in a wide spectrum of 46,XY DSD phenotypes ranging from males with hypospadias , ambiguous genitalia  to 46,XY individuals presenting with female external genitalia  , diaphragmatic hernia , or 46, XY DSD . Interestingly, two of the patients (case 8 and 9) each had a novel variant and an identical second hit in a more commonly observed variant  that has been reported in ClinVar with numerous ZFPM2‐associated conditions. In these cases it is not known whether the variants effect the same or different alleles  of the ZFPM2 amino acid changes occur in annotated functional domains. The ZFPM2 (FOG2) protein has eight Zn‐finger domains, and an N‐terminal region (1–247) that is required for GATA4‐mediated repression of target genes  were reclassified as likely benign or benign. Primarily, the global MAF or Popmax filtering AF (95% CI) in GnomAD was higher than the expected frequency of the condition  was curated as pathogenic. The variant is present in a highly conserved domain without benign variation and absent from population databases. Additional pathogenic supporting evidence was confirmed by familial segregation of the variant in the affected maternal uncle and genotype–phenotype correlation . While three missense  and one in frame deletion (p.L607del) were re‐classified as variants of unknown significance (VUS) due to conflicting or lack of evidence to support classification. Similarly, to individuals with GATA4 variants, limited patient information was available for curation  (benign classification) and case 4 had a well‐described pathogenic variant in the ligand binding domain of the androgen receptor (AR:NM_000044.4:exon7:c.2599G > A (p.V867M))  and case 16: NR5A1:NM_004959.4:c.251G > A (p.R84H)   in NR5A1 activation of mTesco (p < .0001)  using the previously described deleterious variant GATA4 p.G221R, as a positive control  with wild‐type ZFPM2 had a loss of repression of NR5A1 mediated activation of mTesco (comparable to NR5A1/ZFPM2 wild‐type levels without GATA4) (p < .0001)  had a 50% reduction in activity. The other GATA4 variants show activity comparable to wild‐type  of interest was overexpressed in combination with the wild‐type binding partner (ZFPM2) or vice versa in HEK293 cells. The complex was immunoprecipitated using the tag antibody of the wild‐type protein and variant interaction was assessed using the native antibody on Western blot. Wild‐type GATA4 and ZFPM2 protein complex was detected  showed reduced interaction . Zinc finger variants p.G221R and p.W228C variants were almost undetectable  were from distinct ethnic backgrounds such as Pakistan, Cambodia and Indonesia. GnomAD has limited population data for these geographical subsets. Due to the small numbers available from South East Asian countries (like Cambodia and Indonesia), these populations were likely included in the “other” category  which was considered pathogenic. Multiple lines of evidence contributed to pathogenicity including, confirmation of inheritance in an affected family member, genotype–phenotype correlation with a published study  were found in three of cases presented here .These results confirm the outcome of our re‐curation, where the remaining Taken together, our study highlights the importance of periodically reassessing DSD gene variants with up to date curation evidence, and disease/tissue‐specific functional assays, particularly when they have been identified as pathogenic in association with another condition. We believe that an integrative approach between the clinical and research setting is essential to further advance our understanding of the genetic basis of DSD.44.1Collaborating clinicians recruited patients for the study, informed consent was obtained and EDTA bloods were collected. Approval for this study was obtained from the Human Ethics Committee of the Faculty of Medicine at the Royal Children's Hospital (application HREC 22073). DNA was extracted by an independent laboratory such as Victorian Clinical Genetics Service (VCGS) or by other hospital providers.4.2https://www.mutalyzer.nl/). Variants of lower quality were verified by Sanger sequencing (details not shown). The sequencing data for each patient is available from the Sequencing Read Archive (SRA) using reference numbers SRP092281 and project PRJNA350857     and GATA4  were created by site‐directed mutagenesis  according to the manufacturer's instructions  and pCMV6‐Entry‐hZFPM2  both with a C‐terminal Myc‐DDK tag. Prior to introducing mutations to the pCMV6‐Entry‐hGATA4 the vector was modified to remove the Myc‐DDK tag and introduce a C‐terminal HA tag. Sanger sequencing using universal vector primers was used to confirm the introduction of the correct variant.The variant overexpression vectors for 4.5mTesco) and human proximal promoter of AMH  were setup using HEK293‐T human embryonic kidney cells and Lipofectamine‐2000 as the transfection reagent. Cells were setup in 96‐well plates, co‐transfected with promoter vector (75 ng pGL4‐mTesco or 100 ng pGL4‐hAMH) and 5 ng of Renilla (pRL‐TK) as a marker of transfection efficiency. For the mTesco assay: transcription factors were also transfected with or without 40 ng of wild‐type pCMV6‐Entry‐hNR5A1 ; 40 ng of pCMV6‐GATA4 wild‐type  or mutant vectors; and 40 ng pCMV6‐ZFPM2 wild‐type  or mutant vector. Total DNA for each mTesco assay was 200 ng, pCMV‐empty was used to adjust the total DNA for each well. In the case of the hAMH assay: 45 ng wild‐type pcDNA‐WT1 KTS−/−; with or without) 15 ng pCMV‐GATA4 (wild‐type or mutant); and 15 ng pCMV‐ZFPM2 (wild‐type or mutant), were used at a respective ratio of 3:1:1 (WT1:GATA4:ZFPM2). Total DNA for each hAMH assay was 180 ng, pCMV‐empty was used to adjust the total DNA for each well. Assays were lysed 24 hr post‐transfection and luciferase activity was measured using the dual‐luciferase reporter assay  on an Infinite M200 Pro plate reader (Tecan). Each data point represents the average ratio of firefly to renilla luciferase for each condition (performed in triplicate), normalized to the empty vector control (fold change relative to the negative control). The standard error of the mean is shown for three to four independent experiments that were run in technical triplicate.Luciferase transactivation assays for mouse enhancer of Tesco (pGL4‐4.6GATA4 (wild‐type or mutant) over expression, rabbit polyclonal anti‐GATA4 antibody at 1:200 ; and for ZFPM2 (wild‐type or mutant) over expression, mouse monoclonal anti‐FOG2 (H5) at 1:1000 . The next day, cells were washed several times with PBS before incubating with the following secondary antibodies in 1% BSA in PBS: for GATA4 overexpression staining secondary antibodies donkey anti‐rabbit Alexa‐488  and for ZFPM2 overexpression donkey anti‐mouse Alexa‐488 . Nuclear counterstaining with DAPI (blue) was also performed. Images were acquired on a Zeiss AXIO Imager M1 for each overexpressed gene the variants were captured using the same settings as for the wild‐type image.Protein for immunofluorescent imaging was prepared by seeding HEK293‐T cells on 8‐well chamber slides, 200 ng of individual overexpression constructs was transfected with Lipofectamine 2000 (Invitrogen). After 24 hr post‐transfection cells were processed for staining by removing media, briefly washing cells with ice‐cold PBS, fixing cells for 10 min with 4% PFA, 10 min permeabilization with 1% triton‐X‐100 in PBS, and blocking with 2% BSA in PBS. Cells were then incubated overnight with primary antibody in 1% BSA PBS as follows: for 5 HEK293‐T cells in a 24‐well plate and transfecting 800 ng pCMV6‐Entry‐GATA4 (NM_002052.4)  or pCMV6‐Entry‐ZFPM2 (NM_012082.3) overexpression construct  (wild‐type or mutant) with Lipofectamine 2000 (Invitrogen). Protein was harvested 24 hr post‐transfection by removing media and washing the cells with ice‐cold PBS, and lysed using Pierce IP lysis buffer  with protease inhibitors . Protein was quantified using the Pierce BCA protein assay kit , along with BSA protein standards. Five micrograms of total protein was run on a 10% Bis‐Tris gel with MOPS buffer (GATA4), or 4%–12% Bis‐Tris with MOPS buffer (Invitrogen) (ZFPM2), transferred to PVDF membrane, blocked using 5% skim milk powder/TBST and incubated with rabbit polyclonal anti‐GATA4 antibody ; or mouse monoclonal anti‐FOG2 (H5)  overnight at 4°C. After washing with TBST, swine anti‐rabbit HRP (1:10000 DAKO P0399) was incubated at room temperature for 2 hr. After blot washing the Amersham ECL Prime Western blotting detection reagent was used and visualized with the GE Healthcare Life Sciences ImageQuant LAS 4000. Blots were washed and then incubated with loading control—anti‐alpha Tubulin (HRP)  or anti‐GAPDH (HRP) , washed and detected as mentioned above.Protein analysis using Western blot was prepared by seeding 4 × 104.7GATA4 and ZFPM2 proteins was detected as follows: HEK293‐T cells were seeded at 1.6 × 106 in 6‐well plates, transfected with 1.5 ug of pCMV6‐GATA (HA‐tag)  containing the human cDNA ORF of GATA4 (NM_002052.4) (wild‐type or variant); and 1.5 ug pCMV6‐Entry‐hZFPM2 (myc‐DDK tag)  containing the human cDNA ORF of ZFPM2 (NM_012082.3) (wild‐type or variant) with Lipofectamine 2000. After 24 hr, cells were washed and harvested in 500 ul of Pierce IP lysis solution with protease inhibitors , protein lysates were quantitated using the Pierce protein assay kit. For each IP reaction 500 ug of protein lysate  was incubated with 1.5 ul rabbit anti‐HA  (when testing for ZFPM2 protein interaction) or 1.5 ul rabbit anti‐FLAG  (when testing for GATA4 protein interaction) overnight on a suspension mixer with inversion at 4°C degrees. The following day, 20 ul A/G plus agarose beads  was directly added to each IP reaction and incubated for 4 hr on a suspension mixer at 4°C degrees. The IP reaction was then centrifuged at 587g for 5 min at 4°C degrees, supernatant carefully removed and the pellet washed 4–5 times with 500 ul PBS, in the same manner as mentioned above. The IP protein was then resuspended directly in 40 ul 2x SDS protein loading buffer , boiled at 95–100°C for 5 min to denature the protein and release it from the beads. Western blot analysis of the co‐immunoprecipitation was setup as described above: for each IP reaction 15 ul was run per lane; while the 5% of the input sample was run on a separate gel as a control. When testing for GATA4 mutant protein interactions, anti‐GATA4 was used to probe the IP reactions and anti‐ZFPM2 was used to probe the input (as mentioned above). When testing for ZFPM2 mutant protein interactions, anti‐ZFPM2 was used to probe the IP reactions and anti‐GATA4 was used to probe the input (as mentioned above).Interaction of Dr Davis reports having received honoraria from Besins Healthcare and Pfizer Australia and has been a consultant to Mayne Pharmaceuticals, Lawley Pharmaceuticals and Que Oncology. All other authors declare no conflict of interest.The manuscript was compiled and written by J.vdB, K.A and A.S. Patient database, sample preparation, sequencing and analysis was performed by J.vdB, G.R, S.E and K.A. In vitro and functional studies were performed by J.vdB, while supervision and project guidance were provided by K.A and A.S. Management and recruitment of patients involved in the study was coordinated by S.F, S.R, S.G, P.B, C.K, S.K, A.J, C.S, J.R, I.A, S.D, M.A, and V.H. All authors were involved in critical review of the manuscript. Click here for additional data file."}
{"text": "Parkinson's disease is a progressive neurodegenerative disease which causes health problem that affects more patients in the past few years. To be able to offer appropriate care, epidemiological analyses are crucial at the national level and its comparison with the international situation.The demographic description of reported patients with parkinsonism  according to the International Classification of Diseases (ICD-10) from the national health registries.Retrospective analysis of data available from the National Health Information System–NHIS and the National Registry of Reimbursed Health Services (NRRHS). Analyzed epidemiological data are intending to determine the regional and specific prevalence of Parkinsonism in the Czech Republic. The International Classification of Diseases diagnoses (ICD-10) of G20 (Parkinson’s disease—PD) and G23.1, G23.2, G23.3 , and G31.8  from the period of 2012 to 2018 were included into the analysis.We identified 78 453 unique patients from national registries in the period 2012 to 2018. Diagnoses of G20, G23.1, G23.2, and G31.8 were registered as the principal diagnoses in 76.6% of all individual patients.We have found a growing number of patients coded with ICD-10 of dg. G20, G23.1, G23.2, G23.3, or G31.8 . We have proven regional differences in the prevalence of Parkinson´s diagnoses. Therefore we assume most likely also differences in the care of patients with PD based on the availability of specialty care centers. Parkinson's disease (PD) is a progressive neurodegenerative disease that affects mainly the function of basal ganglia. Common PD motor signs include bradykinesia, rigidity, tremor, and postural instability. Incidence and prevalence of PD appear to increase over the recent years . PD signThe prevalence of the disease ranges from 1 to 3 per 1000 in unselected populations, and it affects 1% of the population above 60 years . AccordiÚstav zdravotnických informací a statistiky České republiky) from 2012 report 26 680 patients . The popThe demographic description of reported patients with parkinsonism  according to the International Classification of Diseases (ICD-10) from the national health registries.We performed a retrospective analysis of data available from NHIS , respectively NRRHS  with respect to the STROND checklist. In both registries, the data from health care providers about the type and amount of care are collected. We analyzed epidemiological data intending to determine the regional and specific prevalence of Parkinsonism in the Czech Republic. The International Classification of Diseases (ICD-10) diagnoses of G20 (Parkinson’s disease) and G23.1, G23.2, G23.3 , and G31.8  from the period of 2012 to 2018 were included into the analysis. Atypical parkinsonian syndromes were included due to overlap of clinical signs, at least during the initial stages of Parkinsonism. We selected the period till 2018, as the last previous data on national prevalence were published in 2018. The diagnosis G23.3 (according to ICD-10) was not found in the analyzed data, so we excluded this diagnose from our further evaluation. The standardized annual prevalence of the patients with parkinsonism  has been counted. The analyzes of age and gender distribution, age in the date of diagnosis, and regional distribution of patients within the Czech Republic districts in the analyzed sample.No patients were involved in the design of the study. For the research purposes analyses, there is not special ethical approval needed according to the Czech law if they are analyzing by the Institute of Health Information and Statistics and not by the third party.We identified 205 490 records of patients with PD from national registries in the period from 2012 to 2018  of these patients were followed under ICD-10 diagnostic codes of atypical Parkinsonism . According to available published data, the reported prevalence of atypical Parkinsonism is 0.4% (400 cases per 100 000 populations) in dementia with Lewy bodies (G31.8), multisystem atrophy with dominant parkinsonian features , and progressive supranuclear palsy  in 5 to 10 cases per 100 000 of the general population, increasing to 7 to 8 per 100 000 inhabitants in elderly . The ratAccording to data available from health registries (NHIS), we have shown that most patients diagnosed and treated with PD were in the age group of 70–84 years . Age at the time of PD diagnosis was 70–84 years, which differs from available EU data , 18. EurWe are aware of several limitations of this study. Both unintentional and intentional coder errors, such as misspecification, and up-coding, as potential sources of errors, were described . As we hThe importance of the availability of movement disorder centers is underscored by data showing that nearly half (47%) of PD diagnoses are incorrect when performed in the primary care setting. Specialists without expertise in movement disorders have an error rate of approximately 25%, while movement disorder specialists made mistakes in only 6% to 8% of cases , 27.According to available epidemiological data from the Czech health care registries, we were able to perform an analysis of population data concerning ICD-10 codes coding Parkinsonism and related disorders. We also analyzed the regional prevalence of these diagnoses in respective regions of the Czech Republic. We have found a growing number of records coded with ICD-10 of dg. G20, G23.1, G23.2, G23.3, or G31.8 . We have proven regional differences in the prevalence of diagnoses. Therefore we assume most likely also differences in the care of patients with PD based on the availability of specialty care centers. The big issue for the future in the Czech Republic is the implementation of the standardized processes of care based on nationally accepted evidence-based guidelines. 27 Nov 2020PONE-D-20-31004Representation of Parkinson's disease and atypical Parkinson's syndromes in the Czech Republic - A nationwide retrospective studyPLOS ONEDear Dr. Búřil,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. 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Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this manuscript, the authors reported a retrospective analysis of data available from the National Health Information System – NHIS and the National Registry of Reimbursed Health Services (NRRHS) with respect to the STROND checklist to determine the prevalence and other epidemiological features of Parkinsonism in the Czech Republic. Data on parkinsonian syndromes  in different Eastern European countries are scarce and this study could be valuable by depicting epidemiological features of these diseases in understudied populations. However, some changes should be reconsidered.Abstract:The aim of the study is not well stated.The results are not well defined  diagnoses of G20 (Parkinson’s disease) and G23.1, G23.2, G23.3 , and G31.8 \"-How do the authors explain the fact the diagnosis G23.3 (according to ICD-10) was not found in the analyzed data? and why did they decide to excluded this diagnosis from further evaluation? This is a result they could discuss later on in the discussion part.-What do the authors mean by \" regional\" and \"specific\" prevalence of Parkinsonism. This should be better specified in the Methods part.-What do the authors mean by: \" Atypical parkinsonian syndromes were 36 included due to overlap of clinical signs, at least during the initial stages of Parkinsonism.\"? How does this affect their methodology? this should be better explained.-The authors should precise all the data analyzed later in the results in the methodology part.Results:-What do the authors mean by \"we identified 78 453 patients\" and then \"were registered as the principal diagnoses in 76.6 % of all individual patients\"? What does the first figure stand for? all patients with \"any disease\"? what does the percentage stand for?-The authors stated at the end of this section: \"Various genetic and social factors may influence the regional distribution of patients with Parkinsonism, as was already reported in the eastern part of our country. [13]\". This should be rather put in the discussion section and better explained.-In the sentence: \" The ratio of the parkinsonian subtype of PSP (PSP-P) to the cerebellar subtype is 2:1 to 4:1 in 66 most countries. \", the authors are rather talking about MSA and not PSP. This should be amended.-Other minor changes suggested:• The quality of figures and tables should beReviewer #2: In this manuscript, the authors attempted to determine epidemiological features of parkinsonian syndromes in the Czech Republic by a retrospective analysis of data available from the NHIS and the NRRHS. Further studies, such this one, on epidemiology of parkinsonism especially atypical parkinsonian syndromes are needed. However the authors should clarify some points, as follows:1-The aim/objectives of the study is not well stated in the abstract and later in the introduction.2- The authors should explain the exact meaning of the different codes used in the methodology part and precise all items/parameters that will be later detailed in the results3-The results are not well defined both in the abstract and in the manuscript4-The sentence \"Various genetic and social factors may influence the regional distribution of patients with Parkinsonism, as was already reported in the eastern part of our country. [13]\" should be in the discussion section.5-The quality of figures and tables could be improved.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  10 Dec 2020The aim of the study is not well stated. Reviewer #1 The aim was stated: Aim of the analyses: The main aim of our analyses was to identify the number of reported patients with Parkinson's disease (PD) – dg. G-20 (ICD-10) from the national health registries.The results are not well defined  diagnoses of G20 (Parkinson’s disease) and G23.1, G23.2, G23.3 , and G31.8 \" Reviewer #1 Accepted – the data were analysed based on the record and codes reported by clinicians to the National health registries.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0198736&type=printable). How do the authors explain the fact the diagnosis G23.3 (according to ICD-10) was not found in the analyzed data? and why did they decide to excluded this diagnosis from further evaluation? This is a result they could discuss later on in the discussion part. Reviewer #1 Accepted – the diagnosis G23.3 (according to ICD-10) was not found in the analyzed data – there were no record of the dg. G23.3 in the whole analysed dataset. The possible reason is, that there were no incidence of patients with dg. G23.3 which could relate to the problem that the clinicians are unable to diagnose it appropriately. Also published literature is confirming that the dg. G23.3 is not reported, the commonly used codes are 23.1, 23.2 and 23.9 , which means 78 453 unique patients in total . We have found a growing number of records coded with ICD-10 of dg. G20, G23.1, G23.2, or G31.8 .The authors stated at the end of this section: \"Various genetic and social factors may influence the regional distribution of patients with Parkinsonism, as was already reported in the eastern part of our country. [13]\". This should be rather put in the discussion section and better explained. Reviewer #1 Accepted - we add the information in the discussion section.In the sentence: \"The ratio of the parkinsonian subtype of PSP (PSP-P) to the cerebellar subtype is 2:1 to 4:1 in 66 most countries. \", the authors are rather talking about MSA and not PSP. This should be amended. Reviewer #1 Accepted - sorry for the misinterpretation – the text was changed. Other minor changes suggested:• The quality of figures and tables should be Reviewer #1 We could not address this comment – maybe the comment was not finished?The aim/objectives of the study is not well stated in the abstract and later in the introduction. Reviewer #2 The aim was stated: Aim of the analyses: The demographic description of reported patients with Parkinson's disease according to the International Classification of Diseases (ICD-10) from the national health registries. .The authors should explain the exact meaning of the different codes used in the methodology part and precise all items/parameters that will be later detailed in the results Reviewer #2 Accepted – double checked and added explanations.The results are not well defined both in the abstract and in the manuscript Reviewer #2 Accepted – double checked and added explanations.The sentence \"Various genetic and social factors may influence the regional distribution of patients with Parkinsonism, as was already reported in the eastern part of our country. [13]\" should be in the discussion section. Reviewer #2 Accepted - we add the information in the discussion section.The quality of figures and tables could be improved. Reviewer #2 The figures are used from the data analyses system – we have no possibility to improve the quality.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Editor Accepted – Thank you so much.At this time, please address the following queries:a) Please clarify the sources of funding  for your study. List the grants or organizations that supported your study, including funding received from your institution.b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”c) If any authors received a salary from any of your funders, please state which authors and which funders.d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Editor The authors received no specific funding for this work.http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see Jiri.Jarkovsky@uzis.cz)We will update your Data Availability statement on your behalf to reflect the information you provide. Editor There is legal restrictions on sharing data publicly – all the data form the National registries must be kept safely and authors are working with the anonymised data. For the data you can ask the head of the department of data Analyses team from the Institute of Health Information and Statistics of the Czech Republic - Dr. Jiří Jarkovský  3] in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software . For more information, see our copyright guidelines: We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:1. You may seek permission from the original copyright holder of Figure(s) [3] to publish the content specifically under the CC BY 4.0 license. http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:We recommend that you contact the original copyright holder with the Content Permission Form (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0  remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.The following resources for replacing copyrighted map figures may be helpful... Editor The figure no. 3 was produced by our author’s team. We have published this kind of figure with different type of health data has been already published without any need for copyrighted.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 30 Dec 2020PONE-D-20-31004R1Representation of Parkinson's disease and atypical Parkinson's syndromes in the Czech Republic - A nationwide retrospective studyPLOS ONEDear Dr. Búřil,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Feb 13 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Weidong LeAcademic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your \"Accept\" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: NoReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Most of the required modifications were amended. However, minor revisions are still needed:1-In the abstract and in the manuscript: in the aim of the study, \" The demographic description of reported patients with Parkinson's disease\"==> The demographic description of reported patients with parkinsonism : the aim should be in accordance with the article title.2-The authors should precise all the data analyzed later in the results in the methodology part. This was not amended.3-The quality of figures and tables should be improved.Reviewer #2: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  6 Jan 2021In the abstract and in the manuscript: in the aim of the study, \" The demographic description of reported patients with Parkinson's disease\"==> The demographic description of reported patients with parkinsonism : the aim should be in accordance with the article title. Accepted – the aim has been reworded according the reviewer´s comment.The authors should precise all the data analyzed later in the results in the methodology part. This was not amended. Accepted – the information in the methodology part has been added.The quality of figures and tables should be improved. Accepted – the figures were graphically edited and uploaded.AttachmentResponse to Reviewers_6_1_2021.docxSubmitted filename: Click here for additional data file. 18 Jan 2021Representation of Parkinson's disease and atypical Parkinson's syndromes in the Czech Republic - A nationwide retrospective studyPONE-D-20-31004R2Dear Dr. Búřil,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Weidong LeAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your \"Accept\" recommendation.Reviewer #1: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: No 21 Jan 2021PONE-D-20-31004R2 Representation of Parkinson's disease and atypical Parkinson's syndromesin the Czech Republic - A nationwide retrospective study Dear Dr. Búřil:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Weidong Le Academic EditorPLOS ONE"}
{"text": "Reproductive tract infections (RTIs) are a public health concern in Ethiopia. However, the relationship between menstrual hygiene management (MHM) and water, sanitation, and hygiene (WASH) factors to RTIs have not been well addressed. A community-based cross-sectional study was conducted from January to March 2019 among 602 systematically selected reproductive-age women aged 15–49 years in Dessie City. Data were collected using a questionnaire and a direct observation checklist. RTIs were identified by the presence during one year before data collection of one or more signs of vaginal discharge, itching/irritation or ulcers/lesions around the vulva, pain during urination and sexual intercourse, and lower abdominal pain and lower back pain. Data were analyzed using multivariable logistic regression analysis with 95%CI . The self-reported prevalence of RTIs was 11.0%(95%CI:8.5–13.7%) during one year prior to the survey. The most commonly reported symptoms of RTI were burning micturition (9.1%) and vaginal discharge (6.1%). Three-fourths 443(75.0%) of households used traditional pit latrines and the majority of the study participants 527(89.2%) did not meet the basic access requirement of 20 liters of water per capita per day. The majority 562(95.1%) of the study participants did not have multiple sexual partners during the last year and 97.8% did not practiced sexual intercourse during menstruation. The most common type of blood-absorbent material used was a sanitary pad 497(84.8%) followed by cloth 89(15.2%). Factors significantly associated with RTIs were using unclean latrines , not washing hands with soap before touching the genital area , history of symptoms of RTIs in the past year , having multiple sexual partners in the past year , changing absorbent material only once per day , and washing the genital area only once per day during menstruation . The self-reported prevalence of RTI showed that one women experienced RTI among ten reproductive-age women. Designing a women’s health policy that focuses on ensuring availability of WASH facilities and improving MHM at the community level is key for sustainably preventing RTIs. Reproductive tract infections (RTIs) are infections of the reproductive system and belong to a group of communicable diseases that are transmitted predominantly by sexual contact  but also include endogenous infections and iatropenic infections linked with unsafe abortions and poor delivery practices . A womanRTIs are among the most common causes of illness among women of reproductive age worldwide and especially in developing countries. Nevertheless, public health research, interventions, and services give very little attention to RTIs . The NatRTIs are highly prevalent in Ethiopia. For instance, in a study of 210 patients conducted at St. Paul’s Hospital in Addis Ababa between September 2015 and July 2016, the overall prevalence of bacterial vaginosis was reported at 48.6% .The impact of menstrual hygiene, which is critical for women, has been largely neglected by water, sanitation, and hygiene (WASH) sector researchers. As a result, millions of women and girls continue to be denied WASH, health, education, dignity, and gender equity . In GujaIf RTIs are left untreated or treatment is delayed, complications can result such as pelvic inflammatory disease, infertility, cervical cancer, puerperal sepsis, chronic pelvic pain, ectopic pregnancy, pregnancy loss, preterm delivery, neonatal blindness, premature membrane rupture, and low birth weight . There iA community-based cross-sectional study was conducted in Dessie City from January to March 2019. Dessie is the capital of South Wollo Zone, located about 400 km from Addis Ababa, the capital of Ethiopia. The city is located at an altitude of 2,470 to 2,550 meters. The Dessie City Administrative Health Office estimated the population of Dessie in 2018/2019 was 223,639, of which 104,800 (46.86%) were males and 118,839 (53.14%) were females  in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software . For more information, see our copyright guidelines: We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:1.    You may seek permission from the original copyright holder of Figure(s) [1] to publish the content specifically under the CC BY 4.0 license. http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:We recommend that you contact the original copyright holder with the Content Permission Form (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0  remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.The following resources for replacing copyrighted map figures may be helpful:http://viewer.nationalmap.gov/viewer/USGS National Map Viewer (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/The Gateway to Astronaut Photography of Earth (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.htmlMaps at the CIA (public domain): http://earthobservatory.nasa.gov/NASA Earth Observatory (public domain): http://landsat.visibleearth.nasa.gov/Landsat: http://eros.usgs.gov/#USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://www.naturalearthdata.com/Natural Earth (public domain): Additional Editor Comments (if provided):There are some issues that need to be addressed for a REVISED VERSION:1- In Table 1 in the age section, 20-year-olds were divided into two parts (15 to 20 and 20 to 34 years old). Why?2- What is the meaning of No formal education in Table 1? Do you mean illiterate people or people who have been educated at home?3- What is meant by Above secondary? Does it mean university education? Or people with a PhD degree? Please write a note.4- In Table 2 for Distance of latrine from thehouse Written 15> and 15-30, Is 15 in the first category or the second?[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This article is valuable and can be published, but there are some issues that need to be addressed:1- In Table 1 in the age section, 20-year-olds were divided into two parts (15 to 20 and 20 to 34 years old). Why?2- What is the meaning of No formal education in Table 1? Do you mean illiterate people or people who have been educated at home?3- What is meant by Above secondary? Does it mean university education? Or people with a PhD degree? Please write a note.4- In Table 2 for Distance of latrine from thehouse Written 15> and 15-30, Is 15 in the first category or the second?Reviewer #2: This manuscript discusses a much needed work on Menstrual Hygiene Management, Water, Sanitation and Hygiene Remains a Public Health Problem for Women’s in Urbans of South Wollo, Amhara Region, Ethiopia with some basic results. The paper is suitable for publication with following corrections:1. Add some of the most important quantitative results to the Abstract.2. In Discussion and conclusion segment, authors can include the limitations of this study and more ideas and suggestions for prevention and control. The discussion section should be detailed with citation of all the recent references. Add more information about the significance and importance of this study.3. A clear distinction of usage of the statistical tests has to be mentioned.4. Authors should carefully review the final resolution of the figures prior to publication for better understanding of results.5. Cite the source of future investigations in this study.6. Rectify grammatical errors to reach up to international standards.7. Ensure that the references and whole manuscript is as per journal format. Make certain that all the tables and figure citations are also in the standard format.8.The authors are suggested to improve the quality of the language usage and correct grammatical errors.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  28 Jul 2020Response to reviewersWhen submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfResponse: Thank you for this remark. We re-formatted the revised manuscript using the PLoS ONE format guidelines. The whole content of the manuscript, including the abstract, introduction, methods, discussion and reference are formatted using the guidelines (please see the revised version for each section).2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.Response: We provided the questionnaire in English version and original language as supporting information S I and S II, respectively. http://journals.plos.org/plosone/s/licenses-and-copyright.3. We note that [Figure(s) 1] in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software . For more information, see our copyright guidelines: We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:Response: The figure was removed in our submission. Reviewer #1This article is valuable and can be published, but there are some issues that need to be addressed:Response: Thank you for the positive comments on our paper. Your concerns are addressed below. 1- In Table 1 in the age section, 20-year-olds were divided into two parts (15 to 20 and 20 to 34 years old). Why?Response: Thank you very much for finding out such errors. The correct grouping is 15 to 19 (See Table 1). 2- What is the meaning of No formal education in Table 1? Do you mean illiterate people or people who have been educated at home?Response: Sorry for the confusion. We mean they are illiterate. Illiterate not attended both formal and informal education (See Table 1). 3- What is meant by above secondary? Does it mean university education? Or people with a PhD degree? Please write a noteResponse: Primary education means Grade 1-8; secondary education Grade 9-12, and above secondary education means that diploma, degree, masters and others. 4- In Table 2 for Distance of latrine from the house Written 15> and 15-30, Is 15 in the first category or the second?Response: 15 is the second category since for the first category we used less than 15. Reviewer #2Reviewer #2: This manuscript discusses a much needed work on Menstrual Hygiene Management, Water, Sanitation and Hygiene Remains a Public Health Problem for Women’s in Urbans of South Wollo, Amhara Region, Ethiopia with some basic results. The paper is suitable for publication with following corrections:Response: Thank you very much for the positive reflection of our paper. Corrections were made as follows. 1. Add some of the most important quantitative results to the Abstract.Response: We added some of the main quantitative results to the Abstract . 5. Cite the source of future investigations in this study.Response: The recommendations for future investigations are the author’s idea and therefore need no source. 6. Rectify grammatical errors to reach up to international standards.Response: We edited the grammatical errors (see the revised version). 7. Ensure that the references and whole manuscript is as per journal format. Make certain that all the tables and figure citations are also in the standard format.Response: We formatted them as per the journal guidelines. 8.The authors are suggested to improve the quality of the language usage and correct grammatical errors.Response: We edited language usage and grammatical errors. 3 Aug 2020Does menstrual hygiene management and water, sanitation, and hygiene predictors for reproductive tract infections among reproductive age Women in urban Areas of Ethiopia?PONE-D-20-19169R1Dear Dr. Adane (PhD),We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Hans-Uwe Dahms, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :This MS has now be revised to an extend that it becomes acceptable.Reviewers' comments:    Accept 5 Aug 2020PONE-D-20-19169R1 Does menstrual hygiene management and water, sanitation, and hygiene predictors for reproductive tract infections among reproductive age Women in urban Areas of Ethiopia? Dear Dr. Adane (PhD):I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Hans-Uwe Dahms Academic EditorPLOS ONE"}
{"text": "Visceral leishmaniasis (VL) is a common infectious disease that is endemic in Iran. This study aimed to investigate the spatial autocorrelation of VL in the northwest of Iran. In this cross-sectional study, the data of all patients were collected in 2009–2017 and analyzed by SPSS23 and Moran's and General G Index. The MaxEnt3.3.3 software was used to determine the ecological niche. A big hot spot area was identified in five counties in the northwest of Iran. More than 70% of the cases were reported from these regions, and the incidence rate increased in the northwest of Iran from 2013 to 2017. Seasonal rainfall and average daily temperature were the most important climate variables affecting the incidence of VL in this region (p < 0.05). Therefore, it can be concluded that VL in the northwest of Iran is expanding to new areas along the border with the Republic of Azerbaijan, and the northeastern section of this region is a high-risk area. Leishmania parasites belonging to the Kinetoplastida class and the family Trypanosomatidae Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: No**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: No**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Dear AuthorsPlease consider the following comments:1- Please یouble-check the spelling of the words in all manuscript. For example in title please change leishmaniosis to leishmaniasis.2- The article needs to be edited in English by a native.3- Please define in methodology how did you calculated the incidence of VL?4- Please explain more about the method of analysis for Moran's Index and General G analysis. Did you used the points of disease or considered the counties as the analysis units.5- Please cite a relevant reference for MaxEnt model.6- The reference 24 cited in line 104 seems to be irrelevant to the analysis.7- Fig 5 for the result of spatial auto-correlation should be changed with a map resulted from this analysis.8- In all maps please use \"Urmia Lake\"9- Table 1 was not included in the reviewed file.10- Please discuss more about the role of weather and environmental variables on the habitat suitability for life cycle of VL.Reviewer #2: This study analyzes the spatial patterns of visceral leishmaniasis in three provinces  in northwestern Iran from 2009 to 2017, as well as its relationship with environmental and geographical factors. The manuscript has a relevant theme, but needs an important review/organization in its content and writing.-I suggest a comprehensive review of the English written in the manuscript ;-Please describe clearly and comprehensively the main problem/object addressed in the manuscript;-The introduction does not provide an entirely relevant description of the proposal and significance of the study. The problem is not significantly and concisely stated in the text;-The methods needs to include some additional methodological details in the text , as well as a comprehensive and detailed description of all analyzes presented in the results;-Overall, the results are not clearly presented and the interpretations and conclusions are not supported and justified by the results, but rather general statements. The limitations of this study are not presented in the text ;-The titles of figures and tables are not provided in the manuscript, and the tables were not included in the main file.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  13 May 2020Dear editor of the Plos One1) Thank you for your reply. I noted that our ethics committee waived the need for informed consent from the patients and the patients provided consent for the records to be used in this research. 2.) Yes, the Supporting Information files named “GIS Data of VL.xlsx” and “VL Data.xls,” along with the data located in our manuscript, constitute the minimal data set required to replicate the conclusions in our study.3.) Yes, “All relevant data are within the manuscript and its Supporting Information files.”My dear editor, Thank you very much for that patiently and carefully to your comments and we will guide you. I hope I have answered your questions correctly. We worked hard on this study and took the time. This study was conducted in a very important area of endemic disease and with this study, high-risk areas are easily identified for VL disease control and prevention programs.Best regards 1) Thank you once again for your attention to our queries. You have stated that \"This data not including the patients characters (name or address).\" but it is unclear whether while reviewing patients’ hospital and health records any of the authors had accessed to potentially identifiable information or whether the authors have only accessed fully anonymized records. Could you please clarify this? Please also state in your ethics statement whether the ethics committee waived the need for consent from the patients or whether you obtained informed consent from the patients for their medical records to be used in research/this study. We after receiving the ethics code of the ethics committee of Ardabil University of Medical Sciences We used the database of health centers. This data includes patients who have been previously treated. We used demographic characteristics of patients and place of residence to study. The decision of the ethics committee will also be sent to the appendix.2) Thank you for your additional data sharing information. If you would like to provide an Excel file containing your data, please upload your minimal anonymized data set to your PLOS ONE submission as a Supporting Information file.Ok, we're sending two Excel files that include:A. File the number of patients in the study area.B. The incidence of VL files to different locations for GIS software is used.My dear Anita Estes ,PONE-D-19-35837R1Spatial Autocorrelation and Epidemiological Survey of Visceral Leishmaniasis in an endemic area of Azerbaijan Region, Northwest of IranThank you for better comments. 1) Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate \"Supporting Information\" files. 2) Thank you for removing the funding-related text from the Acknowledgments Section of your manuscript. Please let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:\"Ardabil University of Medical Sciences\"3) Please amend your authorship list in your manuscript file to include all authors. (The names of the authors were checked and there was no problem).4) We note that with regard to the copyright of Figures 1, 6, and 7, you wrote: \"I dragged figures 1, 6 and 7 with the Arcmap GIS 10.4.1 software and uploaded the original file of GIS.\" .https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). This may include: a.) The values behind the means, standard deviations and other measures reported; b.) The values used to build graphs; c.) The points extracted from images for analysis. .5) Please confirm whether the data uploaded as Supporting Information files constitutes the minimal data set, defined as the data set used to reach the conclusions drawn in the manuscript with related metadata and methods, and any additional data required to replicate the reported study findings in their entirety .6) If there are ethical or legal restrictions to sharing third-party data used in your study, and the data included as Supporting Information files in your submission does not meet the requirements in the minimal data set definition above, please provide the following: a.) A description of the data set and the third-party source; b.) If applicable, verification of permission to use the data set; and c.) All necessary contact information others would need to apply to gain access to the data. Please note that it is not acceptable for an author to be the sole named individual responsible for ensuring data access. You can find more information on PLOS ONE’s policies regarding acceptable restrictions and third-party data via the following link: Thank you Dr.Eslam Moradi-Asl My dear editor and reviewers ,I'm so sorry. We're in control of corona virus that's why it's late.We tried to respond to all comments individually.We answered every question or comment in front of you.The English were Native.All shapes and maps were edited.We created all the maps ourselves with the Arcmap GIS 10.4.1 software.We wish you all health and good time.PONE-D-19-35837Spatial Autocorrelation and Epidemiological Survey of Visceral Leishmaniosis in an endemic area of Azerbaijan Region, Northwest of IranPLOS ONEAdditional Editor Comments (if provided):-Improve titles in Results section to be more descriptive.  - Include legends to figures. - Check figures axes titles. For example, Y axis in figure 4 is labeled ‘Vl’ instead of ‘VL’. Ok , Edited )-. Please ensure you have thoroughly discussed any potential limitations of this study within the Discussion section. Ok, the limitations of the study were discussed at the end.Journal Requirements:When submitting your revision, we need you to address these additional requirements:http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 2. In the ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records used in your retrospective study. Specifically, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. 3. Please ensure you have thoroughly discussed any potential limitations of this study within the Discussion section. 4. During your revisions, please note that a simple title correction is required to ensure correct spelling. The title should read: \"Spatial Autocorrelation and Epidemiological Survey of Visceral Leishmaniasis in an endemic area of Azerbaijan Region, Northwest of Iran\". Please ensure this is updated in the manuscript file and the online submission information. 5. Thank you for stating the following in the Acknowledgments Section of your manuscript:\"This work was supported by the Ardabil University of Medical Sciences (Project numbers 92218).\"We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:\"Ardabil University of Medical Sciences \" http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.6. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see The Excel and GIS format of the data and maps is sent to the attachment. In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see We will update your Data Availability statement on your behalf to reflect the information you provide.http://journals.plos.org/plosone/s/licenses-and-copyright.7. We note that Figures #1, 6 and 7 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software . For more information, see our copyright guidelines: We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:1. You may seek permission from the original copyright holder of Figures #1, 6 and 7 to publish the content specifically under the CC BY 4.0 license. *** I dragged figures 1, 6 and 7 with the Arcmap GIS 10.4.1 software and uploaded the original file of GIS.*****http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:We recommend that you contact the original copyright holder with the Content Permission Form (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0  remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. 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Please include a copy of Table 2 which you refer to in your text on page 6 and Table 1 and 3 which you refer to in your text on page 7.Ok , was done Reviewer #1: Dear AuthorsPlease consider the following comments:1- Please double-check the spelling of the words in all manuscript. For example in title please change leishmaniosis to leishmaniasis. 2- The article needs to be edited in English by a native.( It was native).3- Please define in methodology how did you calculated the incidence of VL?R:  of the disease was evaluated using SPSS version 234- Please explain more about the method of analysis for Moran's Index and General G analysis. Did you used the points of disease or considered the counties as the analysis units.R:  were calculated and used to evaluate the significance of the index (27). Moran’s I is a commonly used indicator of spatial autocorrelation. In this study, global Moran’s I was used as the first measure of spatial autocorrelation. Its values range from−1 to 1. The value “1” means perfect positive spatial autocorrelation , while “−1” suggests perfect negative spatial auto-correlation (a checkerboard pattern), and “0” implies perfect spatial randomness  . The High/Low Clustering  tool measures how concentrated the high or low values are for a given study area. This tool calculates the High/Low General G value (observed & expected) & the associated Z score & p-value for a given input feature class(30).)5- Please cite a relevant reference for MaxEnt model.. Fort Collins, Colorado. 2011.)6- The reference 24 cited in line 104 seems to be irrelevant to the analysis.( Reference edited ) 7- Fig 5 for the result of spatial auto-correlation should be changed with a map resulted from this analysis. .8- In all maps please use \"Urmia Lake\" , . 9- Table 1 was not included in the reviewed file. ( This is for Editor) 10- Please discuss more about the role of weather and environmental variables on the habitat suitability for life cycle of VL.Reviewer #2: This study analyzes the spatial patterns of visceral leishmaniasis in three provinces  in northwestern Iran from 2009 to 2017, as well as its relationship with environmental and geographical factors. The manuscript has a relevant theme, but needs an important review/organization in its content and writing. -I suggest a comprehensive review of the English written in the manuscript ; Ok , The manuscript was edited native-Please describe clearly and comprehensively the main problem/object addressed in the manuscript; The introduction was edited -The introduction does not provide an entirely relevant description of the proposal and significance of the study. The problem is not significantly and concisely stated in the text;  and determine the high-risk areas of the disease in the provinces and its relation with environmental and geographical factors. Another objective of the study was to determine the environmental suitability for VL in northwestern Iran for prevention and control of diseases.)-The methods needs to include some additional methodological details in the text , as well as a comprehensive and detailed description of all analyzes presented in the results; Ok, was edited  positive serologic tests was more than 1:3200 cases with medical records, diagnosed by physicians and treated in different parts of the province. By referring to health centers in different counties and reviewing patients’ hospital and health records, demographic characteristics, including age, sex, place of residence, month and year of the disease as well as the results of the DAT test were collected. Then, the data collected were compared with the general statistics in the offices of the vice-chancellor for the health of the provinces of East Azerbaijan, West Azerbaijan, and Ardabil, and the results of the comparison were recorded. The collected data were stored in an ArcMap environment based on the patients’ addresses.))-Overall, the results are not clearly presented and the interpretations and conclusions are not supported and justified by the results, but rather general statements. The limitations of this study are not presented in the text ; Once again the whole article was edited according to your comments and it was native.-The titles of figures and tables are not provided in the manuscript, and the tables were not included in the main file.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 24 Jun 2020PONE-D-19-35837R1Spatial Autocorrelation and Epidemiological Survey of Visceral Leishmaniasis in an endemic area of Azerbaijan Region, Northwest of IranPLOS ONEDear Dr. Moradi-Asl,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.More specifically, please fully incorporate the two remaining comments made by reviewer 1 in the revised version of the manuscript (i.e. R1). Also, address the few editorial comments made in the ´Aditional Editor Comments´ space right after the signature field at the bottom of this message.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Aug 08 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). 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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Albert Schriefer, M.D., Ph.D.Academic EditorPLOS ONEAdditional Editor Comments (if provided):Introduction, line 43. I suggest adjusting ´there is a high correlation between the life cycle of the VL disease and the environmental factors involved in it ...´ to  ´there is a high correlation between the occurrence of the VL disease and the environmental factors involved in it ...´.www.worldclime.com) with a resolution of 30 sec (�1 sq.km) and were used 112 along with an elevation variable at the same resolution to evaluate and determine the appropriate 113 ecological niches (Table 1)´.Methods, lines 111-114. Please, refer to table 1 here: ´A total of 19 climate variables were downloaded from the 111 Worldclime website  and mean diurnal range (21.60%)´. As currently written it is confusing.Results, line 176. Please, correct ´(Table 1and 3)´ to (Table 1)´.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your \"Accept\" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: No**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: No**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: No**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Dear AuthorsThank you for addressing most of the comments. Still I think two comments are not addressed:1- Did you used the points of VL disease for Moran's analysis or considered the counties as the analysis units?2- Fig 5 for the result of spatial auto-correlation should be replaced with a map resulted from this analysis. You can ask Arcmap to plot the map i this analysis.Good luckReviewer #2: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  1 Jul 2020My dear editor,Thanks for you and Reviewers and good time.Additional Editor Comments (if provided):1. Introduction, line 43. I suggest adjusting ´there is a high correlation between the life cycle of the VL disease and the environmental factors involved in it ...´ to ´there is a high correlation between the occurrence of the VL disease and the environmental factors involved in it ...´. Ok, was done. www.worldclime.com) with a resolution of 30 sec (�1 sq.km) and were used 112 along with an elevation variable at the same resolution to evaluate and determine the appropriate 113 ecological niches (Table 1)´. Ok, was done.2. Methods, lines 111-114. Please, refer to table 1 here: ´A total of 19 climate variables were downloaded from the 111 Worldclime website  and mean diurnal range (21.60%)´. As currently written it is confusing. Ok, was done and edited . In addition, the impact of all variables together on the model were precipitated again. The model showed that two factors; seasonality (35.40%) and mean diurnal range (21.60%) had the greatest impact on the occurrence of the disease)6. Results, line 176. Please, correct ´(Table 1and 3)´ to (Table 1)´. Ok, was done.Reviewer #1: Dear AuthorsThank you for addressing most of the comments. Still I think two comments are not addressed:1- Did you used the points of VL disease for Moran's analysis or considered the counties as the analysis units? We used the points of VL disease for Moran's analysis.2- Fig 5 for the result of spatial auto-correlation should be replaced with a map resulted from this analysis. You can ask Arcmap to plot the map i this analysis. To determine the autocorrelation, the Moran and G index have been used, and the Arcmap software output is in the form of a graph, not a map.Best regards Dr.Eslam Moradi-AslAttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 8 Jul 2020Spatial Autocorrelation and Epidemiological Survey of Visceral Leishmaniasis in an endemic area of Azerbaijan Region, Northwest of IranPONE-D-19-35837R2Dear Dr. Moradi-Asl,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Albert Schriefer, M.D., Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 5 Aug 2020PONE-D-19-35837R2 Spatial autocorrelation and epidemiological survey of visceral leishmaniasis in an endemic area of Azerbaijan region, the northwest of Iran Dear Dr. Moradi-Asl:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Albert Schriefer Academic EditorPLOS ONE"}
{"text": "Escherichia coli from river-water and river-sediment in pre-, during- and post-mass bathing events in river Kshipra, Central India.Antibiotic resistance is one of the major global health emergencies. One potential source of dissemination of resistant bacteria is mass gatherings, e.g. mass bathing events. We evaluated the physicochemical parameters of water quality and the antibiotic resistance pattern in commensal E. coli to 17 antibiotics were tested.Water and sediment samples were collected from three selected points during eight mass bathing events during 2014–2016. Water quality parameters  were analyzed using standard methods. In river water and sediment samples, antibiotic susceptibility patterns of isolated E.coli with multi drug resistance (MDR) or extended spectrum beta-lactamase (ESBL) production were between 9–44% and 6–24%, respectively in river-water as well as river-sediment. Total coliform count/ml and E. coli count were higher during-and post-bathing in river water than in pre-bathing period. Thus, the percentage of resistance was significantly higher during and post-bathing period (p<.05) than in pre-bathing. Colony forming unit (CFU)/ml in river-sediment was much higher than in river-water. Percentage of resistance was significantly higher in river-water (p<.05) than in river-sediment.pH, turbidity and dissolved oxygen were significantly lower and total dissolved solid, free carbon dioxide were higher during mass bathing, whilst TSS, BOD and COD were lowest in pre-bathing and highest in post-bathing period. E.coli isolated from the Kshipra River showed significant variation during mass bathing events. Guidelines and regulatory standards are needed to control environmental dissemination of resistant bacteria.Antibiotic resistance in  Clean, pure and safe water exists briefly in nature, as it is immediately polluted by prevailing environmental factors and anthropogenic activities. Industrialization and urbanization has polluted rivers mostly via agricultural runoffs and industrial effluents containing many used or unused antibiotics . AntibioEscherichia coli (E. coli), usually a commensal coliform of humans and animals, enters into the river mainly from livestock operations, their waste products and human septate  images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software . For more information, see our copyright guidelines: We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:1. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:We recommend that you contact the original copyright holder with the Content Permission Form (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0  remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.The following resources for replacing copyrighted map figures may be helpful:http://viewer.nationalmap.gov/viewer/USGS National Map Viewer (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/The Gateway to Astronaut Photography of Earth (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.htmlMaps at the CIA (public domain): http://earthobservatory.nasa.gov/NASA Earth Observatory (public domain): http://landsat.visibleearth.nasa.gov/Landsat: http://eros.usgs.gov/#USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://www.naturalearthdata.com/Natural Earth (public domain): Additional Editor Comments:Expert reviewers have pointed out the need to revise the manuscript updating information on antimicrobial susceptibility testing methodology and controls. Do you have data on colistin susceptibility using MIC method in agar or broth dilution? Please respond to reviewer comments point by point.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: 1. CLSI Performance Standards for Antimicrobial Susceptibility Testing are updated every year. Consequently, testing the antimicrobial susceptibility for Colistin requires MIC with agar or broth microdilution, and can no longer be reported using disk diffusion.2. Kindly justify why this study carried out in 2014-16 is submitted for publication in 2019.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  23 Jan 2020PONE-D-19-25307Mass-bathing events in River Kshipra, Central India- influence on the water quality and the antibiotic susceptibility pattern of commensal E.coliWe are thankful to the Editor and reviewer for the comments and suggestions required for the improvement of the manuscript. Following are the reply to the various queries and concerns.Editors Comments1. When submitting your revision, we need you to address these additional requirements.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttp://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfAuthor's Reply: Formatting done as suggested.2. Funding Statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Author's Reply: Added the statement.http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see We will update your Data Availability statement on your behalf to reflect the information you provide.Author's Reply: Supporting files are added.4. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software . https://gadm.org/) and display only three sites of sampling. The figure 1 legend now shows all details. Author's Reply: We have now replaced the previous figures and upload the map that is freely available in GADM database :The reviewers comments have been addressed satisfactorily.Reviewers' comments: 20 Feb 2020PONE-D-19-25307R1 E.coli Mass bathing events in River Kshipra, Central India- influence on the water quality and the antibiotic susceptibility pattern of commensal Dear Dr. Purohit:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Iddya Karunasagar Academic EditorPLOS ONE"}
{"text": "The Loran-C system is an internationally standardized positioning, navigation, and timing service system. It is the most important backup and supplement for the global navigation satellite system (GNSS). However, the existing Loran-C signal acquisition methods are easily affected by noise and cross-rate interference (CRI). Therefore, this article proposes an envelope delay correlation acquisition method that, when combined with linear digital averaging (LDA) technology, can effectively suppress noise and CRI. The selection of key parameters and the performance of the acquisition method are analyzed through a simulation. When the signal-to-noise ratio (SNR) is −16 dB, the acquisition probability is more than 90% and the acquisition error is less than 1 μs. When the signal-to-interference ratio (SIR) of the CRI is −5 dB, the CRI can also be suppressed and the acquisition error is less than 5 μs. These results show that our acquisition method is accurate. The performance of the method is also verified by actual signals emitted by a Loran-C system. These test results show that our method can reliably detect Loran-C pulse group signals over distances up to 1500 km, even at low SNR. This will enable the modern Loran-C system to be a more reliable backup for the GNSS system. The Loran-C system is an internationally standardized radio positioning, navigation, and timing (PNT) service system. It has the advantages of high transmission power, long propagation distance, and good phase stability ,2,3. It The Loran-C signal acquisition involves finding the first pulse position of the master and secondary stations without manual intervention. This is a key step in signal processing performed by navigation and timing receivers . With thTherefore, this article proposes an envelope delay correlation (EDC) acquisition method, combined with linear digital averaging (LDA) and adaptive threshold techniques, which can effectively reduce the effect of noise and CRI on the signal acquisition process. Furthermore, the correlation peaks can also be repaired by this method, so that the acquisition results have higher accuracy. The method in this article can accurately and simultaneously detect the signals of the master and secondary stations, thus improving the sensitivity and effective range of Loran-C receivers.The Loran-C signal is formally defined by the United States Coast Guard (USCG) as a sequence of pulses of radio frequency (RF) energy at a central frequency of 100 kHz . A singlThe Loran-C navigation chain is composed of one master station and two to five secondary stations. The pulse groups signal of the master and secondary stations are shown in The interval between the Loran-C pulse groups is called the group repetition interval (GRI) . The GRIThe inherent interference in Loran-C signals is mainly from noise and cross-rate interference ,22. TheyNoise is an integral part of any radio system, and it can originate from sources external to the receiving system or from within the receiving system itself. External sources include lightning discharges, precipitation, electronics and power transmission. The main internal noise source is thermal noise generated in the receiver antenna and front end ,23. The Although each Loran-C station has a different GRI, all Loran-C transmitters transmit their signals with the same frequency and format. Therefore, the signal is often interfered with by signals from other stations in the navigation chain. This is called cross-rate interference (CRI) ,24. A scThe first step in the signal detection process is the acquisition of Loran-C signals, which enables the starting position of the master and secondary pulse group signals to be found. In this article, Loran-C pulse group signals are detected using the envelope delay correlation (EDC) method. This acquisition method is shown in Step 1: The received signal passes through a band-pass filter (BPF) to remove interference and noise outside the band. Its bandwidth is 30 kHz. Step 2: The signal envelope is obtained through quadrature demodulation. The signal envelope is delayed by a GRI, and the correlation peak is obtained using the EDC method, which can reduce the influence of CRI.Step 3: The correlation peak is accumulated and averaged according to the GRI period by using linear digital averaging (LDA) technology, which can restore the Loran-C correlation peak and reduce the influence of interference on the delay correlation peak amplitude.Step 4: The detection threshold is calculated according to the statistical characteristics of the noise in the correlation results. Based on the characteristics of the Loran-C pulse group signal, the correlation peak amplitude is compared with the detection threshold. With this, the acquisition of the master and secondary pulse group signal is achieved.L values of 32, 64, 96, 128, 160, 192, and 224 We assume that the digital Loran-C signal, after passing through the band-pass filter, isrding to ,28:(3)SI signals ,30:(5)I defined as (17)SIR=The waveform of the simulated signal a shows tNoise is the most basic source of interference in the received signal, and it is also inevitable. Generally, the receiving system works normally when the SNR is greater than −10 dB . TherefoCRI is caused by overlapping signals from different Loran-C chains. This will become more severe as the number of stations increases. When the interference signal overlaps with the expected signal, the waveform of the expected signal is affected. The receiving system is required to work normally when the intensity of the CRI signal is lower than the intensity of the expected signal . This seWe test the acquisition method with signals actually transmitted by a real Loran-C system. The test scheme is shown in The actual collected signal is the signal emitted by the BPL timing system in China. The coordinates of the BPL timing system are In order to compare the method in this article with the existing method, we use the existing method to process the collected data. The Loran-C system is a remote land-based radio system that can provide high-precision PNT services. However, the traditional Loran-C acquisition method is unable to effectively suppress noise and CRI. Therefore, with the development of modern Loran-C systems, more advanced Loran-C signal processing capacity is required. In this article, we proposed a Loran-C acquisition method based on the EDC method and analyzed the selection of key parameters in the method. In addition, LDA technology was used to restore the correlation peaks and improve the SNR. This article also described the method of calculating the adaptive detection threshold. We demonstrated that our acquisition method can detect the pulse group signals of the master and secondary Loran-C stations at the same time.In addition, a simulation was used to verify the effectiveness of the acquisition method and analyze its anti-noise and anti-interference performance. The simulation results show that this method can quickly acquire the master and secondary station signals. When the SNR > −16 dB, the acquisition probability reaches more than 90% and the acquisition error is less than 1 μs. When the SIR of the CRI is −5 dB, the method can still detect the pulse group signal effectively because of its ability to suppress interference. Finally, we used our acquisition method to process actual received signals. Those results proved that our method can reliably detect Loran-C pulse group signals over distances up to 1500 km, even at low SNR. Thus, the method has high engineering application value and it is optimized for the design of the new Loran-C navigation and timing receivers. This will improve the performance of the modern Loran-C system, making it a more reliable backup for the GNSS system."}
{"text": "Loran-C is the most important backup and supplement system for the global navigation satellite system (GNSS). However, existing Loran-C demodulation methods are easily affected by noise and skywave interference (SWI). Therefore, this article proposes a demodulation method based on Loran-C pulse envelope correlation–phase detection (EC–PD), in which EC has two implementation schemes, namely moving average-cross correlation and matched correlation, to reduce the effects of noise and SWI. The mathematical models of the EC, calculation of the signal-to-noise ratio (SNR) gain, and selection of the EC schemes are given. The simulation results show that compared with an existing method, the proposed method has clear advantages: (1) The demodulation SNR threshold under Gaussian channel is only −2 dB, a reduction of 12.5 dB; (2) The probability of the demodulated SNR threshold, being less than zero under the SWI environment, can reach 0.78, a 26-fold increase. The test results show that the average data availability of the proposed method is 3.3 times higher than that of the existing method. Thus, our demodulation method has higher engineering application value. This will improve the performance of the modern Loran-C system, making it a more reliable backup for the GNSS. The positioning, navigation, and timing (PNT) system is the key infrastructure in any country considering national economy and security. It provides PNT services for military, commercial, and civil users worldwide ,2. One oThe signal-to-noise ratio (SNR) required for Loran-C data demodulation is higher than that for signal acquisition and detection. Therefore, the demodulation performance determines the timing capability of the Loran-C receiver. In recent years, studies on Loran-C signal receiving methods have mainly focused on ways of enhancing the accuracy of TOA measurements, such as by signal acquisition and detection ,17,18, sTherefore, this article proposes an envelope correlation–phase detection (EC–PD) method to demodulate the Loran-C signal at low SNRs. In this method, two EC schemes, namely moving average-cross correlation (MA–CC) and matched correlation (MC), are used to reduce the effects of noise and SWI, thus significantly improving the timing capability and sensitivity of the Loran-C receiver.The Loran-C signal has been formally defined by the United States Coast Guard (USCG) as a sequence of pulses in the radio frequency (RF) energy range with a central frequency of 100 kHz . The defA(n) denote the normalized envelope of the reference pulse, as shown in λ, and the delay of the SWI is represented by τ. Let m-th Loran-C pulse envelopes in the Loran-C pulse group; thus, the mathematical model of the Loran-C pulse envelope can be expressed as:Let nd noise ,20,21. TN is equal to 200. According to [τ ≥ 35 μs. Therefore, the range of τ discussed in this article is 35–200 μs. In addition, to distinguish the data pulse from the reference pulse, the subscript k is used to express the data pulse, where To reduce the complexity of demodulation, and considering that the energy of a Loran-C pulse signal is mainly concentrated near the envelope peak, we take the duration of the Loran-C pulse signal as 200 μs, i.e., In this study, the EC–PD method is used for a low SNR demodulation of the PPM, in which EC has two implementation schemes: MA–CC and MC. Before the demodulation, the receiver needs to complete the acquisition of the Loran-C pulse signal, which is used to determine the starting positions of the reference and data pulses. Moreover, it needs to identify the skywave to obtain the estimated values of N. The stored ESD of the reference and data pulses are represented by column vectors The first step in the demodulation process is to store the envelope sampling data (ESD) of the reference and data pulses; the storage depth is 1)if 2)if 3)if The phase detection can be carried out using an inverse tangent function with return values in the interval Let This section presents the mathematical models of the EC schemes, laying a foundation for the calculation and analysis of the SNR gain.R zeros before and after the stored ESD, and then replacing the middle value of First, we define a In scheme 2, R. In this simulation, we set From Equation (6), we can easily prove that the noise terms in According to Equation (7), and from the above analysis process, we can easily obtain the equation for calculating the SNR gain of scheme 2, as follows:The magnitude of the SNR gain is an important basis for selecting the EC schemes. Evidently, when there is no SWI, There are two strategies, namely strategy A and strategy B, for selecting the EC schemes. In strategy A, the receiver needs to obtain We present a method to determine We verified the effectiveness of the demodulation method from three aspects. In −3, and is recorded as In addition, we take the SNR threshold as one of the quality parameters to compare the demodulation performances of the above three methods. The SNR threshold mentioned in this article refers to the minimum In this section, the Furthermore, based on the above simulation data, we determined the statistical characteristics of the SNR threshold, represented by the probability distribution, as shown in We test the data demodulation method with signals transmitted by a real Loran-C system. The actual received signal is the Loran-C signal (the GRI is 74.30 ms) emitted by the main station (station ID is 09) of Shandong Rongcheng. This station belongs to China’s Changhe 2 navigation system. The test platform is placed in national time service center of China, 1227.1 km away from the station. (1).In RF signal processing, the input Loran-C signal is sampled by analogue-to-digital and filtered by an adaptive notch and finite impulse response band-pass, thus obtaining the digital signal.(2).The complex envelope of the Loran-C pulse is obtained through orthogonal down conversion.(3).The baseband signal processing includes signal acquisition , carrier(4).The EC–PD and EPD–MD methods are alternately selected for signal demodulation every half an hour.(5).The experimental data are composed of message frames, as shown in The following is the description of the test platform:In the next step, we define two variables, namely the maximum data availability The realization and application of the Loran-C system data link technology can make it possible to build a relatively perfect PNT system by combining with the satellite-based PNT system. However, the existing demodulation method used in the Loran-C system cannot effectively suppress noise and SWI. Therefore, with the development of modern Loran-C systems, a more advanced Loran-C signal processing capability is required. In this study, we developed a Loran-C demodulation method at low SNRs based on the EC–PD, where EC includes two schemes: MA–CC and MC. The mathematical models of the MA–CC and MC, calculation of the SNR gain, and selection of the EC schemes based on the skywave identification results were described in detail. The theoretical analysis results showed that the MA–CC is more suitable for scenarios with SWI, whereas the MC is more suitable for scenarios with only noise. Therefore, the combination of the MA–CC and MC could effectively reduce the effects of noise and SWI on the demodulation process.In addition, a simulation was conducted to verify the effectiveness of the demodulation method and analyze its anti-noise and anti-SWI performances. The simulation results showed that compared with the existing method in , the proThe EC technology proposed in this article has a very low implementation complexity compared with some techniques, such as singular value decomposition ,33 and w"}
{"text": "The study aimed to examine the changing incidence of geriatric trauma and evaluate the predictive ability of different scoring tools for in-hospital mortality in geriatric trauma patients.Annual reports released by the National Trauma Database (NTDB) in the USA from 2005 to 2015 and the Trauma Register DGU® in Germany from 1994 to 2012 were analyzed to examine the changing incidence of geriatric trauma. Secondary analysis of a single-center cohort study conducted among 311 severely injured geriatric trauma patients in a level I trauma center in Switzerland was completed. According to the in-hospital survival status, patients were divided into the survival and non-survival group. The differences of the ISS (injury severity score), NISS (new injury severity score), TRISS (Trauma and Injury Severity Score), APACHE II , and SPAS II (simplified acute physiology score II) between two groups were evaluated. Then, the areas under the receiver-operating characteristic curve (AUC-ROC) of different scoring tools for the prediction of in-hospital mortality in geriatric trauma patients were calculated.The analysis of the NTDB showed that the increase in the number of geriatric trauma ranged from 18 to 30% between 2005 and 2015. The analysis of the DGU® showed that the mean age of trauma patients rose from 39.11 in 1993 to 51.10 in 2013, and the proportion of patients aged ≥ 60 years rose from 16.5 to 37.5%. The findings from the secondary analysis showed that 164 (52.73%) patients died in the hospital. The ISS, NISS, APACHE II, and SAPS II in the death group were significantly higher than those in the survival group, and the TRISS in the death group was significantly lower than those in the survival group. The AUCs of the ISS, NISS, TRISS, APACHE II, and SAPS II for the prediction of in-hospital mortality in geriatric trauma patients were 0.807, 0.850, 0.828, 0.715, and 0.725, respectively.The total number of geriatric trauma is increasing as the population ages. The accuracy of ISS, NISS and TRISS was higher than the APACHE II and SAPS II for the prediction of in-hospital mortality in geriatric trauma patients. Trauma is the fourth leading cause of death overall for all ages and the leading cause of death among young people aged less than 45 years . Introduhttps://www.facs.org/quality-programs/trauma/tqp/center-programs/ntdb/docpub) and the Trauma Register DGU® in Germany from 1994 to 2012 (http://www.traumaregister-dgu.de/) were searched to examine the changing incidence of geriatric trauma. Through analysis of the NTDB, the proportion of geriatric trauma and corresponding death rates in the USA were calculated. Based on the data in the DGU® obtained by contacting the Prof. Dr. Rolf Lefering , we calculated the mean age of trauma patients and the proportion of trauma patients aged ≥ 60 years and their trends over time. There is a lack of specific trauma database in China. Thus, we searched the annual reports of the National Bureau of Statistics of the People’s Republic of China (http://www.stats.gov.cn/), and the proportion of population aged ≥ 65 was calculated.The annual reports of the National Trauma Database (NTDB) in the USA from 2005 to 2015 . All statistical process was performed using IBM SPSS 20.0 (Zhejiang University) and P < 0.05 was regarded as statistical significance.Secondary analysis of a single-center cohort study conducted among 311 severely injured geriatric trauma patients in a level I trauma center in Switzerland was completed , 17. Thehttp://www.stats.gov.cn/), the proportion of the population who aged 65 or above increased from 7.7% to 10.1% between 2005 and 2014 , NISS , APACHE II , and SAPS II  in the death group were significantly higher than that in the survival group. The median TRISS was significantly lower in the death group than that in the survival group  .The AUCs were calculated to assess the performance of different scoring tools for the prediction of in-hospital mortality in geriatric trauma patients. Table With the increasingly aging of the population, the incidence of geriatric trauma tends to increase. The study aimed to examine the changing incidence of geriatric trauma and evaluate the predictive ability of different scoring tools for the prediction of in-hospital mortality in geriatric trauma patients. Firstly, through analysis of the annual reports of two large trauma databases, we found the mean age of trauma patients and the proportion of geriatric trauma patients are increasing. Then, secondary analysis of a cohort study showed that the ISS, NISS, and TRISS were better than the SAPS II and APACHE II for the prediction of in-hospital mortality in geriatric trauma patients.Increased life expectancy and independent and active lifestyle expose a great number of elderly people to serious accidents , 20. AddAccurate prognostic expectations are important for decision-making in geriatric trauma patients. Several scoring systems have been developed in an attempt to accurately predict outcomes for trauma patients . The ISSThe APACHE II and SAPS II are generally used to measure the severity and predict outcome for critically ill patients. There was no significant difference between the APACHE II versus the TRISS for the prediction of mortality in severe trauma patients –36. The Some limitations of this study should be acknowledged. Firstly, only the annual reports of two large trauma databases were analyzed to describe the age profile of trauma patients, which may limit the generalization of the results. Then, the geriatric specific scoring system, known as the Geriatric Trauma Outcome Score (GTOS), was not evaluated in the present study , 40. HowThe number and proportion of geriatric trauma patients are increasing rapidly. The ISS, NISS, and TRISS have better performance for the prediction of in-hospital mortality in geriatric trauma patients in comparison with the APACHE II and SAPS II.Additional file 1. List of different scoring tool."}
{"text": "Circular RNAs (circRNAs) are endogenous, single-stranded, most frequently non-coding RNA (ncRNA) molecules that play a significant role in gene expression regulation. Circular RNAs can affect microRNA functionality, interact with RNA-binding proteins (RBPs), translate proteins by themselves, and directly or indirectly modulate gene expression during different cellular processes. The affected expression of circRNAs, as well as their targets, can trigger a cascade of events in the genetic regulatory network causing pathological conditions. Recent studies have shown that altered circular RNA expression patterns could be used as biomarkers in psychiatric diseases, including schizophrenia (SZ); moreover, circular RNAs together with other cell molecules could provide new insight into mechanisms of this disorder. In this review, we focus on the role of circular RNAs in the pathogenesis of SZ and analyze their biomarker and therapeutic potential in this disorder.  The rapid development of novel DNA and RNA sequencing technologies has shed light on the mechanisms of multifactorial disorders, including their probability, origin, development, and pathogenesis ,2,3.Schizophrenia (SZ) is a serious psychiatric disorder that is caused by both environmental and molecular factors . This diIn 2020, the number of published studies aimed at the molecular portrait of SZ has exceeded tens of thousands. However, a significant number of genes, molecular mechanisms, and pathways that are involved in SZ origin and pathogenesis still remain unknown or unclear . UnderstGenome-wide association studies (GWASs) have described only approximately 200 genome SNPs’ susceptibility loci for SZ . Recent It has long been considered that non-coding RNAs (ncRNAs), initially found in eukaryotes, are nothing more than non-functional “junk” RNAs accumulated during the long evolutionary process. Nowadays, many studies have shown that a significant part of this genomic “dark matter” could affect RNA silencing and post-transcriptional regulation of gene expression. As a result of recent advances in high throughput sequencing technologies, thousands of transcribed non-coding RNAs have been described in the vast majority of eukaryotic organisms . These mCircular RNAs (circRNAs) are most frequently non-coding, endogenous, single-stranded RNA molecules that play an important role in cell life. CircRNAs are covalently closed RNA molecules and often originate from protein-coding genes. Previously, it was thought that circular RNAs result from non-functional splicing errors that sometimes occur during the post-transcriptional processes in cells . CurrentRecent studies have shown a significant impact of circRNAs on the origin and development of SZ, as well as an important role of circRNAs in other non-coding and protein-coding gene expression regulation . RecentlMolecular genetic data for circRNAs’ activity in SZ remain limited, despite significant progress made in functional and prediction analysis for this type of RNA ,24,25. TOne analysis of dorsolateral prefrontal cortex (DLPFC) of postmortem healthy and schizophrenic human brains showed significant changes in circRNA expression. A comparison of schizophrenic individuals and healthy controls resulted in the identification of 574 differentially expressed circRNAs, and more than two-thirds of them  were downregulated in SZ. Most importantly, the vast majority of these differentially expressed circular RNAs had miRNA targets and were considered to be potential sponges for microRNAs regulating their activity in the brain . HoweverIt has been also shown that neuronal-enriched circHomer1a  apparently played an important role in cognition processes, as its expression was downregulated in SZ postmortem prefrontal cortex (PFC) area of the human brain, while an intronic circCUL4A  was significantly upregulated .PLEKHA2) was significantly upregulated in peripheral blood mononuclear cells transcriptome ..126].The importance of research and development advances in the molecular therapy for SZ cannot be overestimated, since SZ is the most common variant of psychiatric disorders with cognitive and sensory symptoms and a prominent hereditary component. We suppose that future studies should focus on a deep search for prognostic biomarkers based on genomic, transcriptomic, and epigenetic data and focus on the development of molecular tools and delivery systems to target tissues and organs."}
{"text": "Background and Objectives: Raw electroencephalographic (EEG) signals are rarely used to monitor the depth of volatile induction of general anaesthesia (VIGA) with sevoflurane, even though EEG-based indices may show aberrant values. We aimed to identify whether response (RE) and state entropy (SE) variations reliably reflect the actual depth of general anaesthesia in the presence of different types of epileptiform patterns (EPs) in EEGs during induction of general anaesthesia. Materials and Methods: A randomized, prospective clinical study was performed with 60 patients receiving VIGA using sevoflurane with the increasing concentrations (group VIMA) or the vital capacity (group VCRII) technique or an intravenous single dose of propofol (group PROP). Facial electromyography (fEMG), fraction of inspired sevoflurane (FiAA), fraction of expired sevoflurane (FeAA), minimal alveolar concentration (MAC) of sevoflurane, RE and SE, and standard electroencephalographic evaluations were performed in these patients. Results: In contrast to periodic epileptiform discharges, erroneous SE and RE values in the patients’ EEGs were associated with the presence of polyspikes (PS) and rhythmic polyspikes (PSR), which were more likely to indicate toxic depth rather than false emergence from anaesthesia with no changes in the FiAA, FeAA, and MAC of sevoflurane. Conclusion: Calculated RE and SE values may be misleading during VIGA when EPs are present in patients’ EEGs. During VIGA with sevoflurane, we recommend monitoring raw EEG data in scientific studies to correlate it with potentially erroneous RE and SE values and the end-tidal concentration of sevoflurane in everyday clinical practice, when monitoring raw EEG is not available, because they can mislead anaesthesiologists to reduce sevoflurane levels in the ventilation gas and result in unintentional true emergence from anaesthesia. Further studies are required to investigate the behaviour of EEG-based indices during rapid changes in sevoflurane concentrations at different stages of VIGA and the influence of polyspikes and rhythmic polyspikes on the transformation of EEG signals into a digital form.  General anaesthesia is sometimes associated with unwelcome events. Both volatile induction of general anaesthesia (VIGA) with sevoflurane and intravenous induction of general anaesthesia with propofol may induce seizure-like movements or seizures accompanied by haemodynamic instability ,2, whichIntraoperative awareness during general anaesthesia is a distressing complication with the potential for long-term psychological consequences , and theElectroencephalographic (EEG) signals are derived from a frontal sensor, which is quickly and easily applied to the patient’s forehead and does not entail complex preoperational preparations. The EEG signal is then transformed to a digital score between 0 and 100 visible on the anaesthetic monitor to control the hypnotic component of general anaesthesia: values over 80 indicate an awake state; between 60 and 80, sedation; between 40 and 60, a level of unconsciousness suitable for surgery; between 30 and 40, too deep anaesthesia; and under 30, overdose of anaesthetic ,20. ThisBIS values during the presence of EPs on the EEG  under GAThe abovementioned results suggest that EEG-based indices do not always provide a reliable measure of anaesthetic depth; hence, anaesthesiologists should not rely exclusively on the BIS and SE readings when assessing depth of anaesthesia. We had previously conducted a comparative, prospective study to assess the influence of VIGA with sevoflurane using two different techniques and intravenous induction of general anaesthesia using propofol on the possible presence of EPs . This stPatients who were scheduled to undergo elective orthopaedic knee surgery under combined general and regional anaesthesia in the Clinic of Orthopaedics at St. Barbara’s Memorial Hospital no. 5 in Sosnowiec, Poland, and met the inclusion criteria were asked to participate in the study. Sixty adult patients under 70 years of age with American Society of Anaesthesiologists (ASA) scores of I-II were enrolled after obtaining written informed consent. Ethical approval for this study (NN-6501-196/06) was provided by the Ethical Committee of Medical University of Silesia on December 19, 2006; the study was registered in the Clinical Trial Registry .The exclusion criteria were as follows: a history of epilepsy, medical treatment that might interfere with the EEG assessments , or a neurological disease or a neurosurgical operation that would impair EEG or entropy EEG monitoring; pregnancy; drug or alcohol abuse; a history of pulmonary disease; or signs predicting difficult mask ventilation or intubation. To exclude any pre-existing epileptic EEG patterns, standard 30 min initial EEG recordings were performed for all patients participating in the study. The initial EEG was taken in a dark quiet room for 5 min as a baseline, followed by three eye-opening and closing sequences of 10 s each and photostimulation lasting 10 min . Then, another baseline reading was obtained, and the patients were asked to achieve a state of hyperventilation by taking 20 forceful breathes per minute for five minutes. Finally, another baseline reading was obtained. Only patients without EPs in their EEGs were included into final analysis (Prof. Ville Jäntti). The fifty-five patients included in the study were randomly assigned to one of the three groups  using blind random allocation via sealed envelopes. 2 was maintained at 35–45 mmHg since hyperventilation may trigger epileptiform activity before and during the induction of general anaesthesia. After LMA placement, before the operation started, sevoflurane concentration was maintained at the level of 1 MAC. Before the induction of anaesthesia, none of the patients received any medication on the day of surgery. Directly before surgery, the patients were preoxygenated for 5 min with 100% oxygen and intravenously administered 10 mL/kg per body weight of Ringer solution. The patients in the VIMA group were anaesthetised with sevoflurane using the increasing concentrations technique. The patient was breathing spontaneously via a face mask and the sevoflurane concentration in the inhaled gas was doubled every 10 breaths, starting from 0.3 vol% and proceeding in the sequence 0.3–0.6–1.2–2.4–4.8–8 vol.% until a minimal alveolar concentration (MAC) of 2 was obtained in the exhalation gas. The patients in the VCRII group were anaesthetised with 8% sevoflurane and 92% oxygen using the vital capacity technique. The anaesthetic circuit was prefilled with 8% sevoflurane, and a face mask was applied to the patient’s face to allow them to breathe spontaneously. In group C, the patients were preoxygenated with 100% oxygen and received propofol intravenously at a single dose of 2.5 mg/kg of body weight with subsequent propofol infusion at an infusion speed of 4 mg/kg body weight/hour. Assisted ventilation was initiated in all groups after loss of consciousness (LOC) when hypoventilation appeared and the ciliary reflex disappeared. When the minimum MAC of 2% was reached for sevoflurane in patients from the VIMA and VCRII groups or 5 min after the bolus of propofol with subsequent propofol infusion in patients in the PROP group, the patients in all groups were paralysed with a standard intravenous dose of 0.08–0.1 mg/kg cisatracurium , and a laryngeal mask (LMA) was applied after 45 s. CO2), fraction of inspired oxygen in the gas mixture (FiO2), facial electromyography (fEMG), fraction of inspired sevoflurane (FiAA), fraction of expired sevoflurane (FeAA), exhaled carbon dioxide concentration (EtCO2), minimal alveolar concentration of sevoflurane (MAC), and depth of anaesthesia using response entropy (RE) and state entropy (SE)  [Throughout anaesthesia induction and surgery, standard monitoring procedures were utilised and close attention was paid to vital parameters such as non-invasive arterial pressure (BP), heart rate (HR), standard electrocardiography (ECG) II, arterial blood saturation (SaOFinland) .2 cup electrodes (Spes Medica) attached to the scalp with EC2 Electrode Cream (Grass Technologies). The electrodes  were positioned on both temporal bones lateral to the eyes, on both mastoid bones, on Fp1, Fp2  and Fpz, and the ground electrode was positioned in the centre of the forehead between the eyebrows. Four-channel EEG was recorded using the following electrode pairs: Fp1—left mastoid, Fp2—right mastoid, Fpz—left temporal, and Fpz—right temporal [The data were collected and digitised at 10 s intervals using the S5/Collect (Datex-Ohmeda Division) and Bispectrum Analyzer for BIS (BSA for BIS version 3.22B2 for A-2000—S. Hagihira) installed on a notebook computer (Fujitsu-Siemens Amilo Pi1515). Four EEG channels were recorded using Ag/AgCltemporal  . Since NMBDs are reported to decrease the BIS value , the momp < 0.05 .Statistical analyses were performed using MS Excel, STATISTICA 12, Stat Soft , and R 3.3.2, GNU General Public License. The measured data were characterized using the mean and standard deviation (X ± SD) as well as medians with interquartile range (M IQR). Normality of distribution was checked with the Shapiro–Wilk W test. The significance of differences between means was tested using ANOVA for multiple groups, and for skewed distributions, their compatibility in groups was examined using the Kruskal–Wallis test by ranks. Comparison of average values of tested parameters at different stages was performed via one-way analysis of variance (ANOVA) for repeated measurements and the Friedman Rank Sum test. Two-way analysis of variance (ANOVA) for repeated measures and the nonparametric aligned rank test for repeated measures were conducted in two-factor analysis, with the group and the stage of the study serving as factors. For nominal data, we used percentages. Relationships between nominal variables were verified by the Fisher’s exact test for n by m tables. Evaluation of the relationship between two continuous variables was performed using the Spearman correlation coefficient R’ with an appropriate test of significance. Statistical significance was set at the level p = 0.664), body weight , height , and female-to-male participant ratios . In the VCRII group, EPs were registered in 7 (21.9%) patients, while in the VIMA group, EPs were registered in 10 (31.3%) patients. Out of the total of 60 patients allocated in the current study, 20 patients from the VIMA group, 20 from the VCRII group, and 20 from the PROP group were included in further analyses. Four patients each from the VIMA and VCRII groups were excluded due to possible artefacts in their EEG signals . Patients in the VIMA, VCRII, and PROP groups showed no significant differences in terms of age . In the VCRII group, EPs were registered in 7 (21.9%) patients, while in the VIMA group, EPs were registered in 10 (31.3%) patients. The patients’ parameters were analysed during the following six stages of VIGA: stage 1, onset of VIGA; stage 2, after LOC defined as absence of ciliary reflex; stage 3, 10 s before appearance of the very first EP; stage 4, the first 10 s of the very first EP; stage 5, when the highest RE and SE values appeared in the presence of EPs; and stage 6, when the highest RE and SE values appeared at least 10 s after disappearance of EPs. The time intervals from the start of VIGA (stage 1) to the very first EP in the EEG (stage 4) were significantly different between the VIMA and VCRII groups .The patients’ parameters were analysed during the following six stages of VIGA: stage 1, onset of VIGA; stage 2, after LOC defined as absence of ciliary reflex; stage 3, 10 s before appearance of the very first EP; stage 4, the first 10 s of the very first EP; stage 5, when the highest RE and SE values appeared in the presence of EPs; and stage 6, when the highest RE and SE values appeared at least 10 s after disappearance of EPs. The time intervals from the start of VIGA (stage 1) to the very first EP in the EEG (stage 4) were significantly different between the VIMA and VCRII groups  before the induction of anaesthesia (stage 1); (2) after loss of consciousness (LOC) (stage 2); (3) before the presence of the first EP in the EEGs in the VIMA-EP and VCRII-EP groups, and the mean time to the potential presence of the first EP in the EEGs in the VIMA-nEP and VCRII-nEP groups (stage 3); (4) during the presence of the first EP in the EEGs in the VIMA-EP and VCRII-EP groups and during the mean time interval to the potential presence of the first EP in the EEGs in the VIMA-nEP and VCRII-nEP groups (stage 4); (5) during the presence of the highest RE and SE values associated with the first EP in the EEGs in the VIMA-EP and VCRII-EP groups and the highest RE and SE values in the EEGs in the VIMA-nEP and VCRII-nEP groups; and (6) during the presence of the highest RE and SE values associated with the disappearance of EPs in the EEGs in the VIMA-EP and VCRII-EP groups and the highest RE and SE values in the EEGs in the VIMA-nEP and VCRII-nEP groups . The RE The influence of induction on the levels of these parameters at individual stages in the study is presented in There was a significant decline in the SAP, MAP, fEMG, SE, and RE values in patients induced using the VCRII technique with no EPs (VCRII-nEP group) in the final stages of the study . The lower fEMG values at the fourth and fifth stages were noted in patients induced using the VIMA technique with EPs in their EEGs, a significant increase in the value at the final stage in the case of SE and RE was observed . We observed normative values of SE and RE (40–60) in less than 10% of anaesthetised patients regardless of the analysed stage of VIGA. A significantly higher percentage of patients who possibly recovered from anaesthesia  among the patients induced using the VCRII technique was observed at the third stage, while most of those in deep anaesthesia at this stage belonged to the VIMA group. For all patients induced with the VCRII technique, we noted deep anaesthesia (based on the parameter RE) at the fifth stage of the study . Among patients induced using the VCRII technique and showing EPs (VCRII-EP group), significantly higher values of SE and RE were associated with higher values of HR. Significant negative correlations between SE and SAP, DAP and MAP were observed for the patients who were induced with the VCRII technique and showed no EPs (VCRII-nEP). In the case of RE, correlation parameters were related to DAP and MAP. Negative correlation of RE with fiAA values was noted in patents induced with VCRII technique and showing the development of EPs (VCRII-EP group) . During the presence of EPs, PS and PSR that may falsely indicate intraoperative awareness were present regardless of the anaesthetic regimen used, whereas PEDs appeared mainly during SE values that may falsely indicate deep anaesthesia . Interestingly, in one male patient aged 41 years  induced with sevoflurane using the VIMA technique, before the appearance of EPs (stage 3), the values of SE and RE were 60 and 79, respectively . When PS appeared in his EEG recordings, during the first 10 s, the SE and RE values decreased to 45 and 59, respectively  and subsequently increased to 80 and 94, respectively, 90 s later , indicating false recovery from anaesthesia, whereas the depth of anaesthesia measured by clinical parameters did not change during the transition from stages 3 to 4 and 5. Epileptiform activity during the abovementioned increase in RE and SE is presented in Surprisingly, in another male patient aged 23 years, 140 s after induction was started using VIGA with sevoflurane (stage 1) and 50 s after LOC (stage 2), we observed convulsions following tonic-clonic generalized seizures. Further EEG pattern analysis revealed the development of spiky activity. In this case, evident epileptiform activity in the patient’s EEG recordings accompanied by clinically manifested tonic-clonic seizures that did not result in a rapid increase of RE and SE values, because the observed values of RE and SE were 7–18 and 7–15, respectively, indicating toxic concentrations of sevoflurane in the ventilation gas, whereas at the time of the lowest RE and SE values , the FeAA, FiAA, and MAC values were 4.52, 7.62, and 2.20, respectively .In our study, induction of VIGA with sevoflurane resulted in the presence of EPs in patients’ EEGs at a similar rate to previous studies concerning the presence of EPs during GA with sevoflurane ,7,26,27.To our knowledge, the problems associated with aberrant values of EEG-based indices in the presence of EPs have been quite well recognized in terms of BIS ,26,27, bVisual inspection of the EEG signals for detection of normal, interictal, and ictal activities is a challenging task due to the huge volumes of EEG segments that have to be studied offline or the speed of EEG pattern changes when monitoring online during GA. Therefore, attempts have been made to utilise non-linear methods to study EEG signals for automatic monitoring of epileptic activities. The ultimate goal of clinical anaesthesiologists is to develop a computerized technique that can classify all the three classes of EEG segments , and the need for such software directs current research to investigate the possibility of application of automatic seizure monitoring in the near future, which could provide a real-time EEG-based brain monitoring system for epileptic seizure prediction .The exemplary studies in this field investigating either the utility of observance of distribution entropy values  sample aIn our study on the fifth stage after induction using the VIMA technique, where EPs were present, RE and SE values were significantly higher than those in patients who showed no EPs in their EEGs at the same stage, similar to the findings obtained by Li P et al. . At the third stage of the study analysis (before the appearance of EPs in patients’ EEGs), higher values of SE and RE were observed in the group of patients with EPs in their EEGs after induction using the VCRII technique (VCRII-nEP group), in comparison to stages 4 (first 10 s of the presence of EPs) and 5, when RE and SE values where the highest in the presence of EPs, similar to findings of Lu WY et al. . Rapid decrease in RE and SE in the VCRII-EP group during the transition from stage 3 to 4 in the current study analysis, and contrary to our observation in the VIMA-EP group, where during the transition from stage 3 to 4, such reductions were not present were also observed by Weng WC et al. . Globally, at the third stage of VIGA with the potential presence of EPs, 13 patients presented with SE values indicating recovery from GA, 5 patients showed SE values indicating potential recovery from GA with recall, whereas no patient showed values indicating a surgical level of anaesthesia, and 13 patients showed values indicating almost toxic concentrations of sevoflurane in the ventilation gas. However, no differences in FiAA, FeAA, and MAC were observed at this time point. At the fourth stage in 3 patients from the VCRII group, after appearance of EPs, both RE and SE values indicated potential recovery from GA, but in patients with no EPs in their EEGs, such values were not observed. Therefore, we conclude that the appearance of EPs may elevate RE and SE values falsely, falsely indicating insufficient concentration of sevoflurane in ventilation gas. Interestingly, in 24 patients, the SE values represented almost toxic concentrations of sevoflurane that almost doubled during the transition from stage 3 to 4 with no corresponding changes in FiAA, FeAA and MAC. At the fifth stage, in one patient from the VIMA group who showed EPs, both RE and SE values elevated to a level corresponding to recovery from GA. For almost 30 patients, these values stayed < 40, indicating too deep anaesthesia, with no statistically significant changes in FiAA, FeAA and MAC during the transition from stage 4 to 5; this was identical to our observations for a patient presenting with generalised tonic-clonic seizures whose RE and SE values were <20 during spiky activity in his EEG recordings despite moderate FiAA, FeAA, and MAC at this time. Monitors showing EEG-based indices, which evaluate the hypnotic component of anaesthesia by analysing patients’ EEGs, proved helpful in decreasing the number of incidences of intraoperative awareness with recall . On the We observed that a significantly higher percentage of patients falsely recovered from anaesthesia  among the patients induced using VCRII technique at the third stage (before EPs appeared), while among those in deep anaesthesia, most patients were induced using the VIMA technique. We suppose that abovementioned difference might have been caused by a shift in transformation of the EEG signal detected by a frontal sensor into RE and SE value in patients who underwent VIGA with the VCRII technique, which is very fast. Conversely, such correlations were not observed in the VIMA group since induction of VIGA in these patients was relatively slow, so the shift in transformation of EEG signal into RE and SE value might have been of no clinical relevance.In our study, EPs appeared on average after 209 ± 195 s after the onset of VIGA in patients anesthetized using VCRII. In three patients from the VCRII group, RE and SE values reflected potential false recovery from anaesthesia during the presence of EPs in their EEGs, whereas in patients anesthetized using the same technique who showed no EPs in their EEG at the fourth stage (VCRII-nEP group), all patients had RE values < 40 and SE values < 30, possibly falsely indicating toxic concentrations of sevoflurane in the ventilation gas. At the fifth stage, the SE values decreased below 40, mainly falsely representing a toxic level of anaesthesia (SE < 30 in 26 patients) in the VCRII-EP group despite the continuous presence of PS-type EPs. Surprisingly, in one patient anesthetized using the VIMA technique, at fifth stage of VIGA, SE and RE increased to >80 during the presence of PS-type EPs, possibly falsely indicating recovery from anaesthesia despite the steadily high concentration of volatile anaesthetic in ventilation gas at stages 3, 4 and 5. In this case, a potential shift in transformation of the EEG signal into a digital form is rather not likely to explain the sudden increase in RE and SE values, as at the fourth stage their indicated surgical level of anaesthesia  and concentration of sevoflurane in ventilation gas was not higher than that in the previous stages. Since the RE and SE values in a huge majority of patients at stage 5 reflect toxic concentrations of sevoflurane, regardless of technique used and despite the absence of changes in FeAA, FiAA, and MAC of volatile anaesthetics, further studies are required to investigate if RE and SE values may not falsely show too deep anaesthesia than it really is. Similar observations were obtained Laitio RM et al.  who obseIf SE values unreliably show too deep a level of anaesthesia as a result of the contribution of high-amplitude delta activity, it may possibly be hazardous for the patient because the anaesthesiologist could decrease the concentration of sevoflurane in ventilation gas and unintentionally awake the patient, if depth of anaesthesia is blindly guided based on observance of RE and SE values only. On the other hand, the time needed for transformation of EEG signal derived from frontal sensor might be the explanation for the fact that the concentration of end-expiratory sevoflurane increases faster during VIGA than the values of RE and SE decrease, and there may be a time delay in the reflection of the actual depth of anaesthesia in a huge majority of cases regardless of the appearance of EPs in patients EEGs, especially when the VCRII technique is utilised. Therefore, we suggest that further studies are needed to investigate if RE and SE reliably reflect the actual depth of anaesthesia during rapid changes of sevoflurane concentration in the end-expiratory gas by comparing values of different EEG-based indices that are popularly used to monitor depth of anaesthesia, and comparing them with EEG patterns, especially during the presence of PS-type EPs in patients’ EEGs. On the margin, in all patients who had EPs in their EEGs, we observed patterns of the PED type, but they tended to appear after NMBD administration, so we did not perform analysis afterwards. NMBD are known to influence values displayed by depth of anaesthesia monitors, which is well-recognised in the case of BIS scores ,50,51,52In the end, since a lag in time is probably needed for transformation of the EEG signal recorded from a frontal sensor into a digital form displayed on the monitor, this factor could constitute the main limitation of the present study’s assessments, especially concerning VCRII group. Therefore, from a more practical point of view, we recommend on-line observation of variations in RE and SE values as well as FeAA, FiAA, and MAC of volatile anaesthetics, especially during the VCRII technique, because their verification against raw online EEG patterns in everyday practise is not available. Relying only on RE and SE values to guide the depth of anaesthesia may possibly lead to making a wrong decision resulting with either too deep anaesthesia or intraoperative awareness with recall. During different stages of VIGA with sevoflurane, RE and SE values may vary, showing a wide variety of values from >80, which may falsely represent recovery from anaesthesia during presence of EPs in patients’ EEGs, to <30, which may falsely indicate a toxic concentration of sevoflurane, regardless of the steady concentration of sevoflurane in the ventilation gas. The values of SE and RE during the presence of EPs in patient’s EEGs indicating false recovery from anaesthesia were associated with PS, whereas the presence of PEDs did not result in the presence of aberrant RE and SE values. RE and SE values during the presence of EPs are more likely to indicate toxic depth of anaesthesia rather than false recovery from anaesthesia with no change in FiAA, FeAA of sevoflurane, and MAC values between stages. Abovementioned aberrant RE and SE values could mislead anaesthesiologists into changing sevoflurane levels in the ventilation gas and either possibly lead to unintentional either recovery from anaesthesia or administering toxic concentration of sevoflurane, if depth of VIGA with sevoflurane is blindly guided based on observance of RE and SE values only. Further studies are required to investigate the behaviour of EEG-based indices during rapid changes in sevoflurane concentration at different stages of general anaesthesia and the influence of EPs on the transformation of EEG signals into a digital form. Therefore, during VIGA with sevoflurane, we recommend monitoring raw EEG in scientific studies to correlate it with the values of RE and SE and the end-tidal concentration of sevoflurane in everyday clinical practice, when monitoring raw EEG is not available."}
{"text": "When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures  RNA structure is a jack-of-all-trades. Besides being crucial to the function of structural non-coding RNAs (ncRNAs), RNA structural elements are now widely recognized as key players in almost any essential biological process, ranging from transcriptional and post-transcriptional control of gene expression, to catalysis and sensing of environmental stimuli such as metabolite and temperature changes .n nucleotides-long RNA can theoretically fold in up to 1.8n distinct conformations  (A widely used approach to define the putative structure of an RNA based on its sequence relies on predicting its minimum free energy (MFE) structure, the theoretically most stable structure, based on a set of experimentally-determined thermodynamic parameters known as r rules) . Unfortur rules) . This isr rules) . The accr rules) .in vivo, while the majority of them are only suitable for in vitro and ex vivo RNA structure probing experiments. Two classes of compounds are mainly used to date. On the one hand, nucleobase-specific probes, such as dimethyl sulfate (DMS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), can be respectively used to probe adenine (A) and cytosine (C), or guanine (G) and uracil (U) bases  bases ,14. On tU) bases , as theyU) bases ,17, withU) bases ,19.Typical readout of RNA probing experiments is the detection of reverse transcription (RT) drop-off events induced by the probing reagent-modified RNA bases . More rein vivo RNA probing by synthesizing six new SHAPE reagents and by assaying their ability to probe in vivo Gram-positives, Gram-negatives and mammalian cells. One of these compounds, 2-aminopyridine-3-carboxylic acid imidazolide (2A3), showed both increased reactivity with RNA and higher permeability to biological membranes, resulting in a significantly higher signal-to-noise ratio when compared to NAI. Importantly, when used to perform experimentally-informed prediction of in vivo RNA structures, 2A3 produced markedly more accurate predictions than NAI.While the use of SHAPE compounds over nucleobase-specific probes has in theory a considerable advantage, as they enable the unbiased probing of all four RNA bases, reagents such as DMS still exhibit a higher signal-to-noise ratio in MaP experiments. To this end, we here sought to optimize Ka close to (or slightly above) 7. The pKa values in water were obtained (where possible) using the DataWarrior tool  and added to D2O to achieve quantifiable concentrations. Samples were incubated at 25°C or 37°C and kinetic 1H NMR scanning was performed. The disappearance of product resonances and reappearance of starting materials (correlating to hydrolysis) was monitored as previously described . 2 ml aliquots were collected by centrifugation at 1000 × g (4°C) for 5 min. Cell pellets were resuspended in 1 ml of Resuspension Buffer , and lysozyme was added to a final concentration of 100 μg/ml. After incubation at 22°C for 5 min and on ice for 10 min, protoplasts were collected by centrifugation at 5000 × g (4°C) for 5 min. Pellets were resuspended in 120 μl Protoplast Lysis Buffer , supplemented with 0.2 μg/μl Proteinase K. Samples were incubated for 5 min at 22°C and for 5 min on ice. Sample was then extracted 2 times with phenol:chloroform:isoamyl alcohol , and once with chloroform. 20 U SUPERase•In™ RNase Inhibitor  were then added and RNA was equilibrated at 37°C for 20 min prior to probing.Deproteinized escribed , with miE. coli rRNAs in RNA folding buffer, pre-equilibrated at 37°C for 20 min, were mixed with 20 μl of each SHAPE compound . RNA was then allowed to react at 37°C for 15 min, with moderate shaking, after which 200 μl of 1 M DTT were added, to quench the reaction. Samples were then vortexed briefly and 1 ml of ice-cold QIAzol Lysis Reagent  was added to each sample, followed by extensive vortexing.180 μl of deproteinized E. coli K-12 DH10B was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ∼ 0.5). 2 ml aliquots were collected by centrifugation at 1000 × g for 5 min. Cell pellets were resuspended in 450 μl 1× PBS (pH 7.4) and 50 μl of each SHAPE compound were added, followed by brief vortexing. Cells were allowed to react at 37°C for 20 min, with moderate shaking, after which 500 μl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 17 000 × g for 2 min (4°C) and supernatant discarded. Cell pellets were then resuspended in 100 μl of Buffer A , supplemented with 5 U SUPERase•In™ RNase Inhibitor, by vigorously vortexing for 30 s. 25 μl of Buffer B , supplemented with 100 μg/ml final lysozyme, were then added, followed by 30 s vortexing. Samples were incubated 1 min at room temperature, after which 125 μl of Buffer C  were added to lyse protoplasts. Samples were then incubated at room temperature for 5 min, after which 100 μl of lysate were transferred to 1 ml ice-cold QIAzol and thoroughly mixed by vortexing.A single colony of Bacillus subtilis 168 was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ∼ 0.5). 2 ml aliquots were collected by centrifugation at 1000× g for 5 min. Cell pellets were resuspended in 450 μl 1× PBS (pH 7.4) and 50 μl of each SHAPE compound were added, followed by brief vortexing. Cells were allowed to react at 37°C for 20 min, with moderate shaking, after which 500 μl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 17 000 × g for 2 min (4°C) and supernatant discarded. Cell pellets were then resuspended in 200 μl TE Buffer , supplemented with 1.1% final SDS. 0.25 g glass beads were then added and samples were beated in a Mini-Beadbeater-24 (Glen Mills) for 1 min. Samples were then incubated on ice for 1 min, after which beating was repeated once. 1 ml ice-cold QIAzol was then added, followed by vigorous vortexing. Samples were then incubated at 70°C for 20 min, to allow complete cell lysis.A single colony of d-glucose), supplemented with 10% heat-inactivated FBS, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 U/ml penicillin and 25 μg/ml streptomycin, at 37°C (5% CO2), to ∼75% confluence. Cells were then covered with a thin layer of trypsin–EDTA (0.25%) solution, incubated at room temperature for 1 min, then dissociated by pipetting up and down with complete medium. Cells were centrifuged at 180 × g for 1 min and medium discarded. Cells were then resuspended in 450 μl 1× PBS (pH 7.4) and 50 μl of each SHAPE compound were added, followed by gentle mixing by tapping the tube. Cells were allowed to react at 37°C for 15 min, with moderate shaking, after which 500 μl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 10 000 × g for 1 min (4°C) and supernatant discarded. Cells pellets were lysed by direct addition of 1 ml ice-cold QIAzol, followed by a brief incubation at 56°C for 5 min to completely dissolve cell aggregates.HEK293 cells were grown in DMEM (4.5 g/l g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 ml tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column  and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Besides samples treated with I6, all other samples appeared to be perfectly intact. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase  at 37°C for 20 min.After samples have been collected in 1 ml QIAzol, 200 μl of chloroform was added, followed by vigorous vortexing for 15 s. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12 500 × 2], then purified with NucleoMag NGS Clean-up and Size Select beads , supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 μl (for SSII and HIV RT) or 2.5 μl (for TGIRT-III) NF H2O. Eluted RNA was supplemented with 0.5 μl 20 μM random hexamers and either 0.25 μl (for SSII) or 0.5 μl (for HIV RT and TGIRT-III) dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 μl. For HIV RT, reaction was supplemented with 1 μl 5× RT Buffer , 0.5 μl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 5 U HIV RT . For TGIRT-III, reaction was supplemented with 1 μl 5× RT Buffer , 0.25 μl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 50 U TGIRT-III RT . For SSII, reaction was supplemented with 1 μl 5× RT Buffer , 0.5 μl DTT 0.1 M, 0.25 μl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT . Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 37°C (for HIV RT), 2 h at 57°C (for TGIRT-III) or 3 h at 42°C (for SSII). SSII and HIV RT were heat-inactivated by incubating at 75°C for 20 min. As TGIRT-III tightly binds the cDNA-RNA complex, 0.5 μg of Proteinase K were added and reaction was incubated at 37°C for 20 min, after which 0.5 μl of a 1:2 dilution of protease inhibitor cocktail  in water was added to stop the reaction. For SSII, the buffer exchange step was omitted. Instead, 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module . Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® , following manufacturer instructions.Total RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer {3})’. For each hairpin-loop, we then calculated the median reactivity on the first and last two bases of the loop and the median reactivity on the three bases of the stem preceding and following the loop. The signal-to-noise value was then calculated as the ratio between the median reactivity across all the loops and the median reactivity across all the stems.E. coli cells, under conditions previously shown to preserve the native RNA folding  and HIV RT (N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) adducts (HIV RT). Analysis of the distribution of per-base mutation frequencies across 16S and 23S rRNAs showed that, while TGIRT-III has lower background mutation frequencies compared to SSII (median DMSO control: 0.75 × 10−3 for TGIRT-III versus 2.19 × 10−3 for SSII) as previously reported , we sought to find the best reverse transcription conditions. To this end, we extracted deproteinized total RNA from  folding ,23,33 and HIV RT ,24,34. Treported , SSII shreported . Accordie models  and inspe models . Altogetin vivo probing experiments); (ii) high signal over background ; (iii) easy and safe to synthesize. The two best-characterized in vivo SHAPE reagents to date are NAI and FAI. These compounds can be easily synthesized via the carbonyldiimidazole (CDI)-mediated activation of carboxylic acids, resulting in the corresponding carboxylic acid imidazolide, an activated carbonyl, that can acylate nucleophiles present in the system, especially 2′ hydroxyl groups of RNA. We decided to adopt the same strategy, trying to optimize the acylating group. To this end, we selected seven commercially-available carboxylic acids, preferring those containing nitrogen groups or for which the putative adduct-forming group had a pKa in water close to (or slightly above) 7  permeable to biological membranes  as it was too reactive, hence resulting in very fast RNA degradation (data not shown). When looking at overall distributions of mutation rates, indoline-5-carboxylic acid imidazolide (I5) and 1-methylimidazole-4-carboxylic acid imidazolide (1M4) showed a lower reactivity towards RNA compared to NAI . Oppositely, the other four compounds, nicotinic acid imidazolide (NIC), benzotriazole-5-carboxylic acid imidazolide (B5), 6-aminopyridine-3-carboxylic acid imidazolide (6A3) and 2-aminopyridine-3-carboxylic acid imidazolide (2A3), all showed increased reactivity compared to NAI , with 2A3 having ∼2.4X higher mutation frequencies than NAI , independently of DMSO background signal subtraction , independently of the probed cell type, partially followed by B5 in B. subtilis and NIC in HEK293 cells.At first, we looked again into the distribution of mutation frequencies for the different reagents. Unexpectedly, while the observed distributions for HEK293 cells approximately mirrored those observed on ied RNAs , we obsee Figure . On the ex vivo-probed RNAs, ROC curves built with respect to unpaired versus paired residues from E. coli, B. subtilis and H. sapiens rRNAs  revealed a marked difference in sensitivity/specificity of NAI compared to 2A3, with 2A3 showing up to 17–20% increased discrimination of unpaired versus paired residues, independently of DMSO background signal subtraction  with respect to NAI , independently of the examined cell type  and the intercept (b). As these parameters depend on the experimental setup and on the employed reagent, we first used the ViennaRNA Package 2.0  and positive predictive value  for both 16S and 23S rRNAs. Notably, grid search already revealed that the accuracy of 2A3-driven predictions was largely independent of the employed slope/intercept pair and that, even the most accurate NAI-driven prediction  was ∼10% less accurate than most 2A3-driven predictions, independently of the used m/b pair and ∼18% less accurate that the top-scoring 2A3-driven prediction . As an example, although incorporation of NAI data slightly increased the accuracy with respect to the unconstrained prediction , the NAI-guided prediction resulted in the misfolding of the 3′ domain of the 16S rRNA, while the 2A3-guided prediction correctly recapitulated most of the known base-pairs from the accepted reference structure . This unwanted side product increased over time, eventually reaching a maximum of ∼90% as compared to 2A3 in solution during an overnight reaction in a sealed vessel. The formation of 2A3 proceeds rapidly as CDI is added to the 2-aminonicotinic acid. The conversion of 2A3 to 3AIA then proceeds at room temperature in the crude reaction mixture. The unwanted side reaction proceeds slowly and can be minimized with vigorous stirring under a constant flow of inert gas.in vivo SHAPE probe, we obtained commercially-available pure 3AIA  and used it to probe living E. coli cells. Notably, 3AIA showed modification frequencies indistinguishable from those of the DMSO control in vivo  showed partial improvements with respect to NAI. Surprisingly, when tested in vivo, 2A3 significantly outperformed NAI and all the other tested reagents under all the analyzed conditions, with the most striking differences being observed in bacterial cells. Indeed, for both E. coli and B. subtilis, several regions of the ribosome resulted to be blind to probing by NAI and most of the other tested reagents, while they were readily probed by 2A3. In this context, 2A3 better captured local nucleotide dynamics, as confirmed by in depth analysis of available ribosome 3D structures. This reveals that, besides showing a higher ability to react with RNA, 2A3 has also a greater capacity to permeate biological membranes. It is conceivable that the peptidoglycan of the bacterial cell wall might represent the main limitation when it comes to in vivo SHAPE probing. Indeed, although 2A3 is able to probe both gram-positive and gram-negative bacteria, we observed higher efficiencies in gram-negatives, suggesting that the thicker peptidoglycan of gram-positives limits the permeability to the SHAPE compound. The presence of an amine rather than a methyl group at position C2, that differentiates 2A3 from NAI, might explain this increased permeability, in agreement with a recent study showing that small molecules containing amine groups are more likely to permeate and accumulate into bacteria  even if providing information on roughly only a half of RNA bases.Furthermore, we showed that the higher signal-to-noise ratio achieved by 2A3 results in a significantly higher RNA structure prediction accuracy when performing 2A3-informed RNA folding. It is worth noticing that 2A3, as opposed to NAI, outperforms probing with dimethyl sulfate (DMS) as well. Indeed, probing with DMS usually shows a higher signal-to-noise ratio with respect to NAI, hence resulting in more accurate experimentally-informed RNA structure predictions . It may be that 2A3 falls in a ‘sweet’ spot for reactivity compared to the other tested electrophiles both in vivo RNA structurome.In summary, 2A3 represents a concrete and significant advance in our ability to interrogate RNA structures in living cells. We can anticipate that, being able to readily probe even regions of ribonucleoprotein complexes that are normally hidden to other compounds, 2A3 will rapidly overtake conventional SHAPE reagents, revealing new and previously unobserved features of the ex vivo probing of E. coli RNA with DMSO and NAI has been previously deposited under the accession GSE151327, as part of another study. Additional processed files are available at: http://www.incarnatolab.com/datasets/2A3_Marinus_2020.php.SHAPE-MaP data has been deposited to the Gene Expression Omnibus (GEO) database, under the accession GSE154563. Data for gkaa1255_Supplemental_FileClick here for additional data file."}
{"text": "Experimental data from in vivo tests showed that test compounds 3a–3c, 3f, and 3g (50 mg/kg) significantly prolonged reaction times of animals in tail-clip and hot-plate tests compared to the controls, indicating that these compounds possess centrally mediated antinociceptive activities. Furthermore, these compounds reduced the number of writhing behaviors in the acetic acid-induced writhing tests, showing that the compounds also possess peripheral antinociceptive activity. In the mechanistic studies, naloxone pre-treatments abolished the antinociceptive activities of compounds 3a–3c, 3f, and 3g, indicating that opioidergic mechanisms were involved in their antinociceptive effects. Molecular docking studies demonstrating significant interactions between the active compounds and µ- and δ-opioid receptor proteins supported the pharmacological findings. This study is the first showing that molecules designed to bear thiazole and piperazine moieties together on their structure exert centrally and peripherally mediated antinociceptive effects by activating the opioid system.In this study, we aimed to design and synthesize novel molecules carrying both the thiazole and piperazine rings in their structures and to investigate their antinociceptive activity. Targeted compounds were obtained by reacting thiosemicarbazide derivative and appropriate 2-bromoacetophenone in ethanol. The structures of the obtained compounds were determined using data from various spectroscopic methods (IR,  Pain is a health problem affecting the quality of life of patients due to its prevalence and accompanying disabilities. The pharmacological agents used in the treatment of pain include nonsteroidal anti-inflammatory drugs (NSAID), opioid analgesics, and analgesic adjuvants  . AlthougNitrogen- and sulfur-containing heterocycles are frequently used in drug synthesis. Thiazole, a five-membered ring system carrying three carbons, one nitrogen, and one sulfur atom, is one such structure. It has been reported that thiazole-bearing compounds have some central nervous system (CNS)-related effects, such as anti-schizophrenic , anti-pa2 adrenergic, adenosinergic, and D2/3 dopaminergic receptors +: 442.09.Yield: 85%, Rf = 0.57, M.P. = 210–212 °C, FTIR : 3294 (-NH), 2856 (C-H), 817. 1H-NMR : δ = 2.32 , 2.93 , 3.23–3.26 , 3.33–3.36 , 7.03 , 7.19–7.22 , 7.53 , 7.74 , 7.94 , 11.93 . 13C-NMR : δ = 21.27, 34.32, 45.59, 47.77, 102.74, 115.93, 125.67, 125.91, 127.89, 128.75, 129.62, 132.56, 137.19, 141.98, 151.45, 168.69. ESI-MS (m/z): [M + H]+: 456.11.Yield: 82%, Rf = 0.68, M.P. = 226–230 °C, FTIR : 3294 (N-H), 2845 (C-H), 819. 1H-NMR : δ = 2.93 , 3.23–3.25 , 3.33–3.36 , 3.78 , 6.96 , 7.02 , 7.10 , 7.52 , 7.78 , 7.93 , 11.93 . 13C-NMR : δ = 34.31, 45.59, 47.77, 55.55, 101.51, 114.03, 114.40, 115.93, 125.69, 127.28, 127.88, 128.07, 141.96, 151.44, 159.20, 168.68. ESI-MS (m/z): [M + H]+: 472.10.Yield: 87%, Rf = 0.77, M.P. = 175–178 °C, FTIR : 3292 (N-H), 2845 (C-H), 2222 (C≡N), 817. 1H-NMR : δ = 2.92 , 3.23–3.26 , 3.33–3.37 , 7.02 , 7.53 , 7.59 , 7.86 , 7.96 , 8.03 , 12.01 . 13C-NMR : δ = 34.42, 45.58, 47.76, 107.58, 109.98, 115.90, 119.47, 125.51, 126.56, 127.99, 133.13, 139.32, 142.57, 149.27, 151.55, 169.13. ESI-MS (m/z): [M + H]+: 467.09.Yield: 89%, Rf = 0.34, M.P. = 237–239 °C, FTIR : 3305 (N-H), 2845 (C-H), 1504, 1334 (NO2), 819. 1H-NMR : δ = 2.92 , 3.23–3.26 , 3.34–3.37 , 7.02 , 7.53 , 7.67 , 7.96 , 8.10 , 8.27 , 12.05 . 13C-NMR : δ = 34.42, 45.58, 47.75, 108.60, 115.89, 124.55, 125.47, 126.77, 128.01, 141.22, 142.66, 146.63, 148.97, 151.57, 169.22. ESI-MS (m/z): [M + H]+: 487.08.Yield: 88%, Rf = 0.79, M.P. = 220–223 °C, FTIR : 3344 (N-H), 2841 (C-H), 817. 1H-NMR : δ = 2.92 , 3.23–3.25 , 3.33–3.35 , 6.98 , 7.22–7.27 , 7.49–7.52 , 7.64 , 7.83–7.86 , 7.96 , 11.24 . 13C-NMR : δ =34.42, 45.58, 47.64, 47.79, 103.41, 115.75 (J = 26.08 Hz), 125.20, 125.64, 127.92, 128.99, 142.19, 143.07, 151.49, 151.85, 162.04 (J = 244.24 Hz), 168.90, 177.84. ESI-MS (m/z): [M + H]+: 460.08.Yield: 79%, Rf = 0.31, M.P. = 190–193 °C, FTIR : 3300 (N-H), 2835 (C-H), 817. 1H-NMR : δ = 2.93 , 3.23–3.26 , 3.34–3.37 , 7.03 , 7.36 , 7.46 , 7.53 , 7.87 , 7.95 , 11.98 . 13C-NMR : δ = 34.34, 45.57, 47.78, 106.55, 115.95, 125.57, 126.04, 126.08, 126.50, 127.99, 138.82, 142.50, 149.30, 151.48, 169.07. ESI-MS (m/z): [M + H]+: 476.05.Yield: 81%, Rf = 0.71, M.P. = 204–208 °C, FTIR : 3304 (N-H), 2827 (C-H), 844. 1H-NMR : δ = 2.93 , 3.23–3.26 , 3.34–3.36 , 7.04 , 7.52–7.55 , 7.76 , 7.96 , 8.06 , 12.06 . 13C-NMR : δ = 34.42, 45.58, 47.64, 104.56, 115.58, 120.89, 125.20, 127.98, 128.99, 129.97, 131.96, 142.30, 143.07, 151.85, 177.84. ESI-MS (m/z): [M + H]+: 510.08.Yield: 77%, Rf = 0.78, M.P. = 142–145 °C, FTIR , obtained from the Anadolu University Research Unit for Experimental Animals, Eskişehir, Turkey, were used in the study. The animals were housed in well-ventilated rooms with a 12/12 h dark/light cycle at a temperature of 24 ± 1 °C. The food in the cages was withdrawn 12 h before the experiments to avoid a possible food interference with the absorption of the test compounds. The experimental protocol of this research has been approved by the Local Ethical Committee on Animal Experimentation of Anadolu University, Eskişehir, Turkey. The mice were divided into 10 groups of seven animals each. The test compounds were dissolved in sunflower oil and administered (p.o.) to animals at doses of 50 mg/kg in a volume of 0.1 mL . SunflowMeasurements were taken 60 min after the administration of the test compounds or sunflower oil, and 30 min after the administration of morphine .A tail-clip test was used to assess the response of animals to mechanically induced noxious stimuli. A metal artery clamp that applies standardized pressure was placed 2–2.5 cm from the base of the tail and latency for turning and biting the clamp was recorded by a stopwatch . A sensiA hot-plate test was used to assess the reaction of animals against thermal noxious stimuli. The hot-plate device consists of a heated surface kept at a constant temperature of 55 ± 1.0 °C . The animals were placed on the surface of the aluminum plate and pain thresholds were determined before and after the test compounds’ administrations. Paw licking or jumping latencies of each animal were recorded in seconds. In sensitivity tests, animals that failed to show a nociceptive response within 15 s were discarded from the experiments. The cut-off time for this test was accepted as 30 s to avoid an injury to the paws ,55.In both the tail-clip and hot-plate tests, analgesic efficacies of the test compounds were expressed as a percentage of the maximum possible effect (MPE %) using the following equation: v/v acetic acid in a volume of 0.1 mL/10 g. The mice were then placed in transparent boxes and the number of writhing behaviors was recorded for 10 minutes, 5 minutes after acetic acid injections [The acetic acid-induced writhing test was used to evaluate the responses of animals to chemically induced noxious stimuli elicited by i.p. injection of 0.6% jections ,56. ReduThe possible involvement of opioid receptors in the antinociceptive effects of test compounds was examined by mechanistic studies using a non-selective opioid receptor antagonist, naloxone. For antagonism studies, mice were pre-treated with naloxone at a dose of 5.48 mg/kg 15 min before the administrations of test compounds ,41. ThenThe Rota-Rod test device  was used to evaluate the possible effects of test compounds on the motor coordination of animals. Before experiments, mice were trained on the rotating rod of the apparatus set at 16 rpm for 3 consecutive days. Animals that could stay on the rotating mill for more than 180 s were used for the tests. The falling time of the mice was considered as a parameter for motor coordination. The cut-off time for this test was accepted as 10 min ,41,57.p < 0.05.Statistical evaluation of the experimental data was carried out using GraphPad Prism ver. 8.4.3 software . The data used in the statistical analyses were acquired from seven animals in each group. The differences between experimental groups were determined by one-way analysis of variance (ANOVA) followed by a post-hoc Tukey’s test. The results were presented as mean ± standard error of the mean (SEM) and considered statistically significant when GraphPad Prism ver. 8.4.3 software were used for creating the figures.www.pdb.org, accessed date 28 April 2021). Active conformations of these receptors were used. Molecular docking studies were performed using an in silico procedure to define the binding modes of obtained compounds in active regions of opioid receptors. X-ray crystal structures of µ-opioid receptor (PDB ID: 5C1M) , δ-opioiThe structures of proteins were built using the Schrödinger Maestro  interfacTo the best of our knowledge, this is the first study showing that molecules designed to carry thiazole and piperazine moieties together on their structures have convenient pharmacokinetic profiles and show notable antinociceptive efficacies mediated by the opioid receptors at the spinal, supraspinal, and peripheral sites."}
{"text": "Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fatal interstitial lung disease characterized by abnormal transition and proliferation of fibroblasts. The uncontrolled transition of fibroblasts, commonly known as myofibroblasts, are the principal source of the enormous extracellular matrix (ECM) depositing in lung parenchyma, leading to gradual failure of gas exchange and mortality of the patients. However, up to now, rare effective therapeutic strategies have been developed to blockade fibroblast-to-myofibroblast transition (FMT) in IPF.Method: We illustrated that the lungs originated from IPF patients and mice with pulmonary fibrosis are characterized by the overexpression of sushi-repeat-containing protein, X-linked 2 (SRPX2). Further functionality studies identified the pivotal role of SRPX2 in FMT. Mechanistically, SRPX2 was involved in a TGFβR1/SMAD3/SRPX2/AP1/SMAD7 positive feedback loop. Specifically, SRPX2 was upregulated by TGF-β1 in a TGFβR1/SMAD3-dependent manner, after which SRPX2 in turn repressed the expression of AP1, subsequently minimized SMAD7 expression, through which it reduced the formation of inhibitory complex with TGFβR1 and enhanced SMAD signaling pathway to promote FMT and exacerbate pulmonary fibrosis. Notably, intratracheal administration of siRNA-loaded liposomes could effectively suppress the expression of Srpx2 in the lung and remarkably protect mice against BLM-induced pulmonary fibrosis, concomitant with a significant reduction of FMT.Results: Accordingly, these data indicate that Srpx2 plays an essential role in the pathogenesis of pulmonary fibrosis and suggests the strategy aiming at silencing Srpx2 could be a promising therapeutic approach against pulmonary fibrosis in clinical settings. Idiopathic pulmonary fibrosis (IPF), a fatal lung interstitial disease, is characterized by progressive scarring of the pulmonary parenchyma with a median survival of only 2-3 years after diagnosis Current pathogenic theories demonstrate that fibroblast-to-myofibroblast transition (FMT) plays a central role in the pathogenesis of IPF, featured by uncontrolled production of extracellular matrix (ECM) depositing in lung parenchyma To identify more promising targets for the blockade of FMT induced by TGF-β1, herein, we employed deep RNA-Sequence to delineate the transcriptome changes in myofibroblasts. Notably, sushi repeat-containing protein X-linked 2 (SRPX2), a chondroitin sulfate proteoglycan, was strikingly overexpressed in TGF-β1-induced fibroblasts. Although recent studies have revealed the critical role of SRPX2 in the occurrence and progression of various cancers Srpx2 siRNA-loaded liposomes to suppress FMT for the treatment of pulmonary fibrosis. Indeed, both of in vitro and in vivo studies provided compelling evidence that suppression of Srpx2 by siRNA-loaded liposomes significantly protected mice against BLM-induced lung injury and fibrosis, coupled with the pronounced reduction of fibroblast transition in the lung. In a mechanistic study, for the first time, we demonstrated that silencing SRPX2 expression could repress fibroblast differentiation by blocking the AP1/SMAD7/SMAD2/3 signaling axis. Collectively, our data support the notion that Srpx2 plays a critical role in the progression of pulmonary fibrosis and the liposomes-based strategy aiming at silencing Srpx2 could be a viable therapeutic approach against pulmonary fibrosis in clinical settings.Previous studies Antibodies against Collagen type I, α-SMA, SMAD2, SMAD3 were purchased from Cell Signaling Technology . Antibodies against p-SMAD2, p-SMAD3 and p-SMAD2/3 were obtained from Boster Biological Technology Co., ltd . Antibodies against SRPX2, β-ACTIN, SMAD7 and FIBRONECTIN were acquired from Proteintech Group, Inc . Cholesterol and DSPC were acquired from Sigma-Aldrich, Inc.  and mPEG2000-DMG (MW2660) was purchased from NOF Co., Ltd. (Kawasaki Japan). The cationic lipidoid C12-200 was generated through ring opening of epoxides by amine substrates with a previously reported method Lung tissues were collected from patients with non-small cell lung cancer  and patients with IPF (n = 6) in Tongji Hospital after receiving informed consent. IPF was diagnosed according to the American Thoracic Society (ATS)/European Respiratory Society (ERS) consensus diagnostic criteria SRPX2  and a nontargeting control siRNA (named scramble siRNA) were purchased from RiboBio . The specific SRPX2-targeted siRNA sequences were as follows: si-SRPX2_001: 5'-CAG ATG AAA GCT ACA ATG A-3', si-SRPX2_002: 5'-CAG ATG AAA GCT ACA ATG A-3', and si-SRPX2_3: 5'-GAG GAA ATC TTC ACA TTC A-3'. For transfection, Lipofectamine 3000  was used according to previously reported Three siRNAs targeting Liposomes were raised as carriers to encapsulate siRNA. The siRNA was dissolved in citrate buffer  and rapidly mixed with a lipid mixture by vortexing. The lipid mixture was composed of C12-200, cholesterol, DSPC and mPEG-DMG dissolved in ethanol at a molar ratio of 50:38.5:10:1.5. The unentrapped siRNA was removed by ultrafiltration centrifugation. The entrapment efficiency was measured by RiboGreen assay. SiRNA-loaded liposomes were used after being diluted with PBS. The characteristics of liposomes  were listed in Srpx2 siRNA-loaded liposomes, scramble siRNA-loaded liposomes or unloaded liposomes  were injected via intratracheal to the corresponding group of the mice. Eventually, all mice were euthanized 21 days after BLM injection for analysis of pulmonary fibrosis.All male C57BL/6 mice (8-10 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . The mice were all housed in a specific pathogen-free (SPF) animal facility at Tongji Hospital in a 12:12 h light/dark photocycle with sterile acidified water and irradiated food. All animal experimental procedures were allowed by the Animal Care and Use Committee at Tongji Hospital. The mice were anesthetized with 1% pentobarbital sodium (60 mg/kg) by intraperitoneal injection, and the 2.5 mg/kg BLM diluted in PBS or PBS alone was administered via intratracheal injection as previously reportedThe left lungs were collected and fixed in 4% neutral buffered paraformaldehyde for 24h at room temperature, followed by embedded in paraffin. Subsequently, the lungs were sliced into 5 µm sections and stained with hematoxylin and eosin (H&E), Sirius red and Masson's trichrome as previously reported A hydroxyproline assay kit  was used to measure the hydroxyproline content of the lungs to assess the lung collagen deposition according to previously reported 2 humidified incubator. The cell culture medium was refreshed every 3 days, and the cells were passaged at a 1:2 ratio every 3-4 days.Human lung tissues from IPF patients and control subjects were minced and allowed to adhere to the bottom of the culture flask. Samples were cultured for 24h after attachment to the culture flask bottom, in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum  at 37 °C in a 5% COin vivo imaging system   as previous reported ex vivo fluorescence imaging and biochemical detection, respectively.DiR-labelled liposomes were constructed as the above method. And then, the prepared liposomes were intratracheal injected to the C57/B6 mice. The mice were then anesthetized and imaged at different time points  by an RNAs from mouse lung fibroblasts after TGF-β1 stimulated for 24 h were extracted using an RNA isolation kit . RNA quality and integrity were determined using the Nanodrop 2000 Spectrophotometer  and Agilent Bioanalyser . RNA-seq libraries were multiplexed and loaded per lane into the Illumina HiSeq flow cell v3. All sequencing protocols were carried out as the manufacturer's instructions using the Illumina HiSeq 1000 and HiSeq control software.SRPX2 forward, 5′- TGG CTG GTT GAT TTT GTA GAG AAA-3′ and reverse, 5′- TAG AAA AGA GTT AGG TGT CAC ATT GAA TAA-3′; human FIBRONECTIN forward, 5′- GAT GTC CGA ACA GCT ATT TAC CA-3′ and reverse, 5′- CGA CCA CAT AGG AAG TCC CAG-3′; human COL1A1 forward, 5′- GAG GGC CAA GAC GAA GAC ATC-3′ and reverse, 5′- CAG ATC ACG TCA TCG CAC AAC-3′; human ACTA2 forward, 5′-GAC GCT GAA GTA TCC GAT AGA ACA CG-3′ and reverse, 5′- CAC CAT CTC CAG AGT CCA GCA CAA T-3′; human ACTB forward, 5′-AGC GAG CAT CCC CCA AAG TT-3′ and reverse, 5′-GGG CAC GAA GGC TCA TCA TT-3′; human AP-1 forward, 5′- CAA ACC TCA GCA ACT TCA ACC-3′ and reverse, 5′- CTG GGA CTC CAT GTC GAT G-3′; human SMAD7 forward, 5′- ATG TTC AGG ACC AAA CGA TCT-3′ and reverse, 5′- GGA TGG TGG TGA CCT TTG G-3′.mouse Srpx2 forward, 5′-ATG AAG TGT TCC AGC GAT GGT GA-3′ and reverse, 5′-TGG CAT ACT CGG GCA GGA CTA C-3′; mouse Fibronectin forward, 5′- GAT GTC CGA ACA GCT ATT TAC CA-3′ and reverse, 5′-CCT TGC GAC TTC AGC CAC T-3′; mouse Col1a1 forward, 5′-TAA GGG TCC CCA ATG GTG AGA-3′ and reverse, 5′-GGG TCC CTC GAC TCC TAC AT-3′; mouse Acta2 forward, 5′-GGA CGT ACA ACT GGT ATT GTG C-3′ and reverse, 5′- TCG GCA GTA GTC ACG AAG GA-3′; mouse Actb forward, 5′-GCC ACA GCA CTC CAT CGA C-3′ and reverse, 5′-GTC TCC GAT CTG GAA AAC GC-3′, mouse AP-1 forward, 5′-AAG ATG GAA ACG ACC TTC TAC G-3′ and reverse, 5′-CTT AGG GTT ACT GTA GCC GTA G-3′; mouse Smad7 forward, 5′-CTG TGT TGC TGT GAA TCT TAC G-3′ and reverse, 5′- GAG ACT CTA GTT CAC AGA GTC G-3′.Total RNA was extracted from fibroblasts and the mice lungs with TRlzol reagent  as previously reported Fibroblasts and lung tissues were homogenized in RIPA lysis buffer. Proteins were subjected to Western blot with the indicated primary antibodies using established techniques For the immunofluorescence experiment, the slides were treated with antibody at 4 °C overnight. The primary antibodies used for staining were as follows: mouse anti-SRPX2 (1:100), rabbit anti-FIBRONECTIN (1:100), rabbit anti-COLLAGEN I (1:100), rabbit anti-α-SMA (1:100), and rabbit anti-p-SMAD2/3 (1:100). All slides were incubated with an Alexa 488-conjugated anti-mouse and Alexa 594-labeled anti-rabbit antibody  after washed with PBS. And then, the nuclei were counterstained with DAPI (4'-6-diamidino-2-phenylindole) for 5 min. Finally, the slides were analyzed under a fluorescence microscope .3 cells/well. Cell proliferation was then measured using the EdU proliferation assay  as previously reported Human pulmonary fibroblasts (HPFs) were purchased from ScienCell Research Laboratories, Inc  and cultured in 96-well plates at a density of 2×104 cells/well. The lower chambers were filled with culture medium containing 10% FBS to function as a chemoattractant. After incubation at 37 °C for 24 h, the cells migrated through the membrane filter and were stained with 0.1% crystal violet .Cell migration assays were performed using Transwell inserts with a membrane with a pore size of 8.0 mm  according to previously reported methods p<0.05 was used to indicate statistical significance.Experimental data are expressed as the Mean ± SEM. All statistical analyses were performed using GraphPad Prism . Two experimental groups were compared using the Student's t test or the Student's t test with Welch's correction for unpaired data. More than two groups were compared using one-way ANOVA with Bonferroni's correction. in vitro results, SRPX2 was indeed up-regulated in IPF patients' lung compared to control subjects  myofibroblasts  were quantified by RT-PCR. Interestingly, the expression level of FMT related genes illustrated a positive correlation with the expression of SRPX2  on day 14 and day 18 (Figure Srpx2 siRNA-loaded liposomes group, as evidenced by the H&E, Masson's trichrome and Sirius red staining (Figure Srpx2 siRNA-loaded liposomes group (Figure  Srpx2 siRNA-loaded liposomes treated mice than that in scrambled siRNA group (Figure The therapeutic effects of Srpx2 siRNA-loaded liposomes displayed attenuated expressions of fibronectin, collagen I and α-SMA (Figure in vitro studies (Figure in vivo studies also indicated that suppression of Srpx2 in lung could effectively repress the phosphorylation of Smad2/3, coupled with a dramatical overexpression of Smad7 and AP-1 (Figure Srpx2 siRNA-loaded liposomes could be a potent therapeutic approach against pulmonary fibrosis via blocking attenuated FMT.To further evaluate the therapeutic effect of the liposomes, we next conducted Western blot, RT-PCR and immunostaining to examine the fibrotic markers in the lung. Consistent to above results, mice administered with A Figure . In lines Figure , the in Srpx2 siRNA reversed BLM-induced lung injury and fibrosis (Figure IPF is a chronic and progressive interstitial lung disease of unknown origin with an average life expectancy of 2-3 years after diagnosis s Figure . Collectvia different signaling pathways Previous studies have demonstrated that SRPX2 could efficaciously regulate proliferation, migration, differentiation and apoptosis of tumor cells Myofibroblasts are critical in the process of pulmonary fibrosis by their secretion of ECM proteins, leading to tissue stiffness and respiratory failure Another critical issue is the underlying mechanisms by which SRPX2 regulated the transition, proliferation and migration of fibroblasts. It is compelling noted that TGF-β1 is one of the most potent and well-studied inducers for FMT via binding with activated TGFβR1 via AP-1 depend manner, as evidenced by inhibiting AP-1 reversed the overexpression of SMAD7 induced by SRPX2 siRNA and activated TGF-β/SMADs signaling pathway. Additionally, similar data were also obtained in the following in vivo studies. Collectively, SRPX2 was upregulated by TGF-β1 in a TGFβR1/SMAD3-dependent manner. After that, the elevated SRPX2 would inhibit the expression of AP1, and subsequently down-regulated the expression of SMAD7, leading to an enhanced TGF-β/SMADs signaling pathway. This positive feedback loop centered on SRPX2 would further promote fibroblasts differentiating into myofibroblasts, exacerbating pulmonary fibrosis.To address the notion that SRPX2 regulated the TGF-β/SMADs signaling pathway, we next examined the expression of SMAD7, which interferes with the phosphorylation of SMAD2 and SMAD3 Srpx2 siRNA could be efficiently phagocytized by the fibroblasts in fibrotic lesions of the lung following intratracheal injection and suppress the expression of Srpx2 for at least 6 days. More importantly, Srpx2 siRNA-loaded liposomes was intratracheally injected into mice during the “fibrotic” phase of the model, which is more applicable and reflective to the clinical management of IPF patients. Since our previous work Srpx2 siRNA-loaded liposomes on macrophages. No obvious difference was detected on the polarization of alternative activated macrophages, which were one of major sources of TGF-β Srpx2 siRNA-loaded liposomes on pulmonary fibrosis is duo to suppression of fibroblasts behavior, not macrophages activation. Overall, intratracheal injection of Srpx2 siRNA loaded liposomes could block TGF-β signaling pathway and subsequently decelerate the process of FMT, contributing to the protection of mice from BLM-induced lung injury and fibrosis, which provide an available, safe and effective therapeutic approach for IPF.Although numerous clinical trials have been carried out to characterize viable drugs for IPF, unfortunately, no effective therapeutic approach was currently available to cure or even halt the progression of IPF Srpx2 siRNA significantly reduced the expression of Srpx2 and then suppressed FMT, contributing to the improvement of the pulmonary fibrosis induced by BLM. Briefly, our data supported that intratracheal administration of Srpx2 siRNA loaded liposomes could be a promising therapeutic approach against pulmonary fibrosis in clinical settings.In conclusion, we firstly demonstrated that SRPX2 was overexpressed in the fibroblasts originated from IFP patients and pulmonary fibrosis mice. Mechanistically, SRPX2 was upregulated by TGF-β1 canonical signaling pathway during the process of FMT. In addition, the elevated SRPX2 would inhibit the expression of AP1, and then attenuated SMAD7 expression, forming a positive feedback loop to enhance TGF-β/SMADs signaling, which could finally promote FMT and exacerbate pulmonary fibrosis. Inspiringly, administration of liposomes carrying Supplementary figures.Click here for additional data file."}
{"text": "Moreover, the PQS ELISA here reported has been foundto be robust and reliable, providing accurate results in culture media.The technique allowed us to follow up the PQS profile of the releaseof bacterial clinical isolates obtained from patients of differentdisease status. A clear correlation was found between the PQS immunoreactivityequivalents and the chronic or acute infection conditions, which supportsthe reported differences on virulence and behavior of these bacterialstrains due to their adaptation capability to the host environment.The results obtained point to the potential of the PQS as a biomarkerof infection and to the value of the antibodies and the technologydeveloped for improving diagnosis and management of P. aeruginosa infections based on the precise identificationof the pathogen, appropriate stratification of the patients accordingto their disease status, and knowledge of the disease progression.Quorumsensing (QS) is a bacterial cell density-based communicationsystem using low molecular weight signals called autoinducers (AIs).Identification and quantification of these molecules could providevaluable information related to the stage of colonization or infectionas well as the stage of the disease. With this scenario, we reporthere for the first time the development of antibodies against thePQS , the main signaling molecule fromthe  Accordingto the U.S. Centers for Disease Control and Prevention (CDC), about51,000 P. aeruginosa HAI cases occureach year in U.S. hospitals. This pathogen has developed a vast adaptiveresponse and significant resistance to innate immune effectors andto antibiotics, particularly during chronic infections. Such is thecase in patients suffering from cystic fibrosis (CF), a disease causedby a genetic disorder that predominantly affects the mucociliary clearanceof the respiratory tract,2 creating anideal scenario for opportunistic pathogens such as P. aeruginosa. This pathogen colonizes the lungsof these patients at the age of 2–3 years old and ends up persistingin 60–80% of CF patients as a chronic infection, compromisingtheir life and frequently ending in respiratory failure and prematuredeath.3 On the other hand, antibiotic therapyrarely leads to elimination of a pathogen from chronically infectedCF respiratory airways. In 2017, multidrug-resistant P. aeruginosa caused an estimated 32,600 infectionsamong hospitalized patients and 2700 estimated deaths in the UnitedStates.4Healthcare-associatedand community-acquiredinfections  have become a major burdenfor public health worldwide,P. aeruginosa.5 The lackof reliable tools for rapid diagnostic of bacterial infections hascontributed to the overprescription and misuse of broad spectrum antibiotics,strengthening the generation of multidrug-resistant bacteria.6 Current diagnostic methods based on a cultureplate require several days to obtain conclusive results, which oftenis an unaffordable time for effectively treating the infection.7 Many diagnostic alternatives have emerged aspotential complements of the standard techniques, providing more completeand accurate results. For instance, matrix-assisted laser desorptionionization–time of flight–mass spectrometry (MALDI-TOF-MS)analysis has been successfully used to characterize the protein footprintfrom bacterial isolates of CF.8 Also, polymerasechain reaction (PCR) methods have appeared as interesting alternativesto identify pathogens.9 However, thesetechniques require complex selection of the targets, tedious and time-consumingextractions, highly trained personnel, and/or expensive equipment,10 which prevent their wide implementation on clinicalsettings. Point of care (PoC) devices may be a promising option infuture routine clinical diagnostics, providing rapid reliable responsesand being low cost and user-friendly to use. PoC performance wouldallow us to change the management of the patient and would providefaster diagnosis than conventional methods, ideally in almost everyplace and situation. Most of the time, these devices rely on the useof a biorecognition element that specifically binds the correspondingbiomarker target,11 such is the case ofantibodies.Recently, the World HealthOrganization (WHO) has published a listdeclaring the urgent need of finding new antibiotics for the treatmentof infections caused by resistant bacteria, such as carbapenem-resistant 13 Because of their key role in the development of pathogenesis, thesignaling molecules of the quorum sensing (QS) system could be a potentialbiomarker of infection. QS is a bacterial communication mechanismbased on the release of low molecular weight signals that regulatethe coordinated genetic expression of a wide array of physiologicalactivities.14 These signals, also calledautoinducers (AIs), promote their own biosynthesis and their concentrationincrease as a function of population density. At a particular AI concentrationthreshold, the genetic expression of decisive factors such as thesecretion of virulence factors and biofilm formation is triggered. P. aeruginosa QS is a well-studied communicationnetwork composed of four systems  performing in acomplex hierarchical manner. Particularly, the quinolone-based pqs system, responsible for the regulation of virulencefactors including pyocyanin, elastase, lectin, and rhamnolipids,15 has particularly attracted the attention ofthe scientific community due to its QS-dependent and independent functions.16 Its main signaling molecule, 2-heptyl-3-hydroxy-4(1H)-quinolone or the PQS ,specific for P. aeruginosa, is responsiblefor important functions besides its signaling activity. For instance,the PQS mediates cytotoxicity and has been indicated to balance thegrowth and death in P. aeruginosa populations.17 The PQS also mediates iron acquisition due toits strong chelating character and its activity to prompt the expressionof genes related to the biosynthesis of the siderophores pyoverdineand pyochelin.19 Furthermore, the PQS promotesthe formation of outer membrane vesicles (OMVs) due to its remarkedlipophilic character. The molecule induces the curvature of the bacterialmembrane and resides as an integral component, maintaining its biologicalactivity and achieving the communication between bacteria throughthe aqueous media.20 Eventually, the PQSwas also found to modulate the host immune response by dysregulatingseveral defense mechanisms and cytokine expression.21Host–pathogen interaction involves the releaseof a largeamount of molecules, which have potential as biomarkers for diagnosis,patient stratification, disease monitoring, or for guiding antibiotictreatment as being the subject of investigation.23 such as, for example, in sputum,approximately at the micrometerrange by thin-layer chromatography (TLC) analysis,24 although other more selective techniques found it at concentrationsin the low nanometer range.25 While massspectrometry has been the most commonly used technology to quantifythese molecules,27 electrochemical28 or bioreporter methods29 havealso been reported. These measurements require complex sample treatmentmethods such as extraction using organic solvents and tedious purificationor enrichment steps to accomplish the necessary detectability. Onthe other hand, only in very few cases, these alkylquinolones havebeen measured in clinical samples, partially due to the increasedsample complexity and low detectability attained by some of thesetechniques. With this scenario, we have focus our attention on thedevelopment of specific antibodies against the main signaling moleculeof the pqs QS system, with the final aim of developingimmunochemical diagnostic technologies suitable to assess the potentialvalue of these specific QS molecules as biomarkers of infection. Inthis paper, we report, for the first time, the development of a highlysensitive microtiter-based enzyme-linked immunosorbent assay (ELISA)to quantify the PQS and its use to analyze the concentration levelsof this molecule in culture media where clinical isolates obtainedfrom infected patients have been grown. Preliminary data point tothe potential value of this QS molecule as the biomarker to diagnose P. aeruginosa infection and to stratify patientsin respect to the stage of infection.The PQS has been found in several body fluids ofinfected patients,Supporting Information.See the (13) was synthesized following a similar approach to thatdescribed by Reen et al.30 for the synthesisof the PQS through a five-step synthetic pathway from 3-(4-aminophenyl)propanoicacid and methyl 3-oxodecanoate (3), obtained by a nucleophilicreaction of 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum’s acid)with octanoyl chloride and subsequent methanolysis. All the intermediatesand the final product were purified and characterized by nuclear magneticresonance (NMR) spectroscopy and ultraperformance liquid chromatography(UPLC)-MS/MS.31The hapten3-propanoic acid (13) in anhydrous DMF (400 μL) was cooled to 4 °C. Afterward,tri-n-butylamine  andisobutyl chloroformiate  were addedto the hapten solution and the mixture was left stirring for 15 minat 4 °C and 30 min at room temperature. Then, 200 μL ofthe reaction mixture was slowly added over the protein solution  or keyhole limpet hemocyanin (KLH), 2.5 mg/mL,2 mL in 10 mM phosphate buffered saline (PBS)). Then, the mixturewas left stirring for 2 h at RT and overnight at 4 °C withoutagitation. The bioconjugates were purified by dialysis against 0.5mM PBS (5 × 5 L) and Milli-Q water (1 × 5 L) and storedfreeze-dried at −80 °C. A small fraction (20 μL)of PQS-BSA was kept for MALDI-TOF-MS analysis, rendering a haptendensity of 17 haptens per molecule of BSA (see Table S1). Hapten densities of PQS-BSA and PQS-KLH were alsoassessed by fluorescence, measuring the intensity of the emissionat 445 nm, of the native protein and the bioconjugates, and interpolatingthe value on a calibration curve build with the PQS hapten, renderingthe same value of previous MALDI-TOF-MS analysis .A solution of the PQS hapten Supporting Information.See the 2SO4 solution(50 μL/well), and the absorbance was read at 450 nm.Microtiter plates were coated withthe HHQ-BSA conjugate in coating buffer overnight at 4 °C and covered with an adhesive plate sealer.The day after, plates were washed with PBST (4 × 300 μL/well)and solutions of PQS standards  followed by the As385  addition and the microplates were left without agitationfor 30 min at room temperature. After another washing step, a solutionof diluted goat anti-rabbit IgG-HRP (1/6000 in PBST) was added (100μL/well) and incubated for 30 min at room temperature. The plateswere washed again, and the substrate solution (100 μL) was addedand left for 30 min at room temperature in the dark. The enzymaticreaction was stopped by adding 4 N HPerformance of the assays wasevaluated through the modification of different physicochemical parameters, solubilitywith addition of organic solvents, competence time, incubation time,or cation complexation by EDTA) in the competitive step.50 value was used to calculate the cross-reactivityaccording to the following equation CR (%) = IC50(crossreactant)/IC50 × 100.HHQ, PQS, and HQNO calibrationcurves (10 μM to 1 pM) were built in PBST and measured on theAs385/HHQ-BSA ELISA following the procedure described above. The standardcurves obtained were fitted to the four-parameter equation mentionedabove, and the ICP. aeruginosa isolates were selected and stored frozen at −20 °C.Prior to the analyses, P. aeruginosa isolates were cultured overnight at 37 °C in blood agar plates.The day after, a portion of the grown bacteria was diluted in Mueller–Hinton(MH) culture broth (3 mL) and shaken at 37 °C. When the opticaldensity at 600 nm (OD600) reached a value of 0.2–0.3,a dilution (1/1000 in 20 mL of MH) was performed and the resultingsolution was shaken at 37 °C. Aliquots were taken at the selectedtimes for measurement of OD600, for colony forming unit(CFU) calculation, and measurement of the PQS by the ELISA. The restof the isolates were grown using the same experimental procedure,yet the aliquots were extracted just at a selected time of 8 h. PQSconcentrations measured by the ELISA were expressed as PQS immunoreactivityequivalents (IRequiv.) due to the potential specific interferencescaused by other alkylquinolones of different chain lengths potentiallypresent in the culture media.Clinicalisolates were obtained from lowerrespiratory tract samples, mainly sputum specimens, of patients diagnosedof acute or chronic respiratory infections. Clinical samples werecultured in MacConkey agar, blood agar, and chocolate agar and incubatedfor up to 5 days at 37 °C. Culturebroth samples were analyzed following the same procedure as describedabove except for coating the plates with HHQ-BSA at 0.04 μg/mL and diluting As385 64,000 timeswith PBST-EDTA. The standards used to build the standard curves wereprepared in MH broth diluted 10 times with PBST-EDTA, and the sampleswere diluted 10 times with PBST-EDTA before measuring them with theELISA. To assess the accuracy of the method, blind spiked samplesusing diluted MH culture broth (1/10) were prepared and measured usingthe above-described ELISA for three different days and three replicateswithin the same day. The results are expressed as the mean of allreplicates.Understandingquorum sensing (QS) is key to apprehend about pathogenesis.QS depends on specific signaling molecules or autoinducers, whichare responsible for triggering the expression of a series of genes,biosynthesizing virulence factors, and favoring biofilm progression.Rapid detection of these signaling molecules could give cliniciansan early indication of infection or knowledge about the stage of thedisease.Over the last few years, biosensors and PoC deviceshave emergedas promising alternatives for a more rapid and efficient detectionof biomarkers of the disease. Among them, antibody-based technologieshave demonstrated their capability of providing accurate and reliablediagnostic results thanks to their great affinity, which allows usto develop highly sensitive technologies and their specificity. However,accomplishing high quality antibodies with the necessary affinityand specificity is a crucial factor, which is mainly dependent onthe immunogen and of the strategy used to produce such antibodies.P. aeruginosa to regulate virulence .34The pseudomonasquinolone signal (PQS) is an autoinducer used specifically by ence see 1 and for35 basedon two sequential modifications of the C-3 position in the PQS biologicalprecursor, 2-heptyl-4-quinolone or HHQ. Hapten synthesis (see (9). The aniline derivative was subsequentlyreacted by amination reaction with β-ketoester (3) in the presence of a catalytic amount of p-toluenesulfonic acid to obtain the intermediate (10). Afterward,the quinolone derivative (11) was prepared through Conrad–Umpachthermic cyclization in diphenyl ether at 270 °C. Formylationof the C-3 position through the Duff reaction36 allowed us to obtain the quinolone derivative (12) asthe precursor of the desired hapten. Concomitant hydrolysis of themethyl ester group occurred; however, this fact no longer affectedthe synthetic procedure. Finally, oxidation of the C-3 position throughDakin reaction allowed us to obtain the PQS hapten (13). This reaction relies on a characteristic hydrogen peroxide nucleophilicaddition that promotes the -aryl migration and elimination, subsequentlyobtaining an hydroxyl group directly bonded to the previously formylatedposition.37 Overall, the pure proposedPQS proposed hapten (13) was obtained in five syntheticsteps with an overall yield of 16%.The synthesis of the PQS hapten was inspired from a strategy previouslydescribed by Pesci and co-workersesis see 1 started38 Hapten bioconjugateswere obtained with a high density ratio , as demonstrated by MALDI-TOF-MS and fluorescenceanalysis .The PQS hapten was usedto prepare KLH and BSA protein bioconjugatesfollowing the anhydride mixed method. This method relies on the carboxylicacid activation using a chloroformate and a sterically hindered base,forming an anhydride that afterward reacts with the lysine residuespresent in the protein.Table S2), from which the combination of As385/HHQ-BSA was selected for furtherstudies due to the higher detectability in further experiments.Antisera  were increased against PQS-KLH in female New Zealand whiterabbits. The avidity of obtained As for the BSA conjugates (HHQ-BSAand PQS-BSA) and the competition of the PQS for the binding to theantibodies were assessed by combined experiments, comparing the absorbanceof the assay in the presence and absence of the analyte. After a preliminaryselection, the most promising combinations were analyzed by two-dimensionaltitration experiments. The first set of experiments allowed us toselect the best As/bioconjugate combinations for competitive assaysbased on the largest reduction of the signal when the PQS was presentin different combinations, while the second one allowed us to selectthe concentrations of the immunoreagents providing the best ratio/noisesignal. As a result, four different As/bioconjugate combinations wereevaluated in a competitive format . A significant variationof the assay features by introducing a preincubation step was notobserved, and the optimum competition time was found to be 30 min.The performance of the assay under different physicochemical conditionssuch as the effect of time, pH, concentration of a non-ionic surfactant,ionic strength, or the presence of an organic solvent was evaluated, while the IC50 value wassignificantly lower (better detectability) than under the standardconditions using a buffer with 0.05% of this detergent. However, sinceit is known that the presence of a small amount of Tween 20 helpsin minimizing nonspecific interactions and solubilizing the analyte,it was decided to keep a concentration of just 0.01%. The detectabilitywas slightly better at conductivity values greater than 15 mS/cm;however, the Amax decreased substantially,and for this reason, it was decided to keep the conductivity at 15mS/cm for further experiments. Furthermore, it was added to the assaybuffer (0.1 mM) of EDTA as a result of the accuracy experiments (explainedin the section below).The assay was able to workat pH values between 4.5 and 9.5, althougha slightly better performance was observed at pH 7.5 based on itsdetectability and maximum absorbance. The addition of an organic solventsuch as DMSO did not produce a significant enhancement; although the P. aeruginosa bacterial cultures (μM) and in the same range of concentrations(low nanometer range) compared to the ones found in sputum samplesby Abdalla and co-workers.39 Furthermore, the detectability achieved with the As385/HHQ-BSA ELISAis greater than those obtained with other techniques such as thosebased on bioreporters40 or electrochemical methods.28 Regarding LC-MS/MS methods, the ELISA reportedhere allows us to obtain higher or similar detectability without includingadditional preconcentration steps. These methods require the use ofsolid phase extraction and/or organic solvent extraction steps, endingup in a 20 times concentration factor of the sample for obtainingthe reported LOQs.39Under these conditions,the assay showed an LOD of 0.17 ± 0.01 nM and a dynamic rangecompressed between 0.53 ± 0.04 and 24.37 ± 3.18 nM see 2, which P. aeruginosapqs QS system. However, other quinolones produced by this pathogen havealso an important role in communication or interspecies interaction.The other two molecules are HHQ, the precursor of the PQS in the biosyntheticpathway and involved in the signaling process, and HQNO, an N-oxidequinolone able to inhibit the electron transport chain in other bacteriashowing remarkable antistaphylococcal properties.41 The only structural difference between HHQ, HQNO, and thePQS is one oxidized position (C-3 or N-1), the reason why the recognitionpattern by the As385/HHQ-BSA ELISA had to be assessed. Despite thesimilarity between the different molecules, the percentage of cross-reactivitywas less than 2% for HQNO (IC50 = 236.2 nM) and about 13%in the case of HHQ (IC50 = 27.2 nM) (see Table S5). Moreover, additional specificity experiments wereperformed in which calibration curves of the PQS were built underthe presence of constant concentrations of either HHQ or HQNO . It allowed us to evaluate the potentialcross-reactivity for each point of the calibration curve at differentconcentrations of cross reactants. The experiments showed that, indeed,the percentages of cross-reactivity were not constant and diminishedwhen the concentrations of the PQS increased. Hence, the ELISA developedrecognized a much better PQS despite the great structural similaritiesof the other two autoinducer molecules of the pqs system, which pointed to the possibility to quantify specificallythe PQS from samples where all three quinolones will be present. Specificitystudies using other molecules structurally related  and non-structurally related-thiazole-4-carbaldehyde,the main signaling molecule of the iqs QS system),potentially present in clinical or bacterial culture samples, werealso performed. In all cases, the As385/HHQ-BSA ELISA did not respondto concentration values up to 10 μM of all mentioned molecules,and thus, the CR was assumed to be less than 0.01% for all of them.The PQS is the most important and active signalingmolecule inthe Table S3, where the percentage of the coefficient of variation (%CV)at three different concentrations is shown . The reproducibility is only slightlylower when measuring concentrations close to the limit of quantificationduring different days, but even at those very low concentration values,the %CV is below near 10% on intra-plate and inter-plate studies.As385/HHQ-BSA ELISA intra-plate, inter-plate, and inter-day precisionwere assessed, as it can be observed in P.aeruginosa was grown. The total amount of exo-productsand production profile of QS molecules such as the PQS can vary amongstrains coming from different infection types or infection status3 and might have clinical relevance. However, suchtypes of samples may contain a variety of components from the mediaor from the bacteria, which may interfere with the assay. For thisreason, on the first instance, the matrix effect caused by measurementin Mueller–Hinton (MH) culture broth, frequently used to grow P. aeruginosa strains, was evaluated. These mediacontain nitrogenous compounds, vitamins, starch, and amino acids,which added to the products released by the bacteria itself such asvirulence factors, proteins, and other exo-products, which could makethe sample quite complex. To estimate the extension of this effect,calibration curves were built in MH culture media, at different dilutionfactors, and measured with the As385/HHQ-BSA ELISA. The resultingcalibration curves and the effect caused by MH in the assay performancecan be seen in 50 value was 6.05 ± 0.16 nM, while the obtainedLOD was 0.36 ± 0.14 nM  in a non-deliverable manner19 causes an iron starvation response and stimulates a concentration-dependentincrease production of iron scavenging siderophores such as pyoverdineand pyochelin. PQS-Fe3+ has been found to accumulate inthe membrane of the cell, acting as a storage molecule. Consideringsuch behavior, the PQS might be forming complexes with the cationspresent in MH culture broth. To probe this hypothesis, the calibrationcurve was run in the presence of different concentrations of ethylenediaminetetraaceticacid (EDTA), a powerful ion chelator able to displace the divalentcation complexation by the PQS . The addition of EDTA caused a substantial increase of the maximumabsorbance; nevertheless, it produced an increase in detectability.In light of these results, we assessed the effect of EDTA in the buffer(PBST-EDTA) on the assay accuracy. As it is shown in Accuracy of the assay was evaluated by preparing blind spiked samplesin MH culture broth and measuring them with the developed ELISA. Interestingly,the results showed a critical underestimation of the spiked PQS concentrations3. We hyp the PQS S4. The aP. aeruginosa airway infection, respectively, were initially measured.With these results, we addressed the investigation of thePQS productionprofile of clinical isolate growth on MH broth media. Since previouslyperformed experiments pointed at a different production profile ofHHQ, the PQS biosynthetic precursor, depending on whether the clinicalisolates belonged to patients with a chronic or an acute infection,and the PQS profiles of the release of isolates from patients withan acute (PAAI6) and a chronic (PACI6) 600 andthe calculated CFUs. The decrease in the OD600 at 48 hof growth observed in PAAI6 might be explained by the prolonged exposureto high concentrations of self exo-products with lytic activity, suchas pyocyanin and the PQS.44Interestingly,similarly to HHQ, PQS quantifiable levels were reachedafter 5 and 12 h of growth for the clinical isolates PAAI6 and PACI6,respectively, while after 48 h, the PQS IRequiv. value of PAI6 washigher than that of PAAI6 see 4, as alrP. aeruginosa infectionwith distinct severity degrees. For this purpose, clinical isolatesfrom 12 patients with acute or chronic infections were grown in MHat 37 °C. After 8 h (time chosen as a compromise based on theabove-described behavior), sample aliquots were taken and measuredwith the PQS ELISA developed. As it can be observed in The next set of experiments was seeking to demonstrate that thiswas a general behavior for clinical isolates coming from patientssuffering from P. aeruginosa adapts to the host environment by evolving toward a state of reducedbacterial invasiveness that favors bacterial persistence without causingoverwhelming host injury. In that respect, the results found withthe here reported PQS ELISA are in agreement with this situation.The low levels of the PQS released by the isolates from chronic infectedpatients would justify a potential lower production of virulence factors.From a diagnostic perspective, in addition to identifying the microorganismscausing the disease, being able to distinguish between a chronic andan acute infection is very relevant to apply appropriate therapeuticstrategies and improve disease management.In light of this outcome, it seems clear that the concentrationof QS molecules released to the media can be correlated with the typeof infection or the disease status of the patient. It has been oftenreported that the behavior of strains inducing acute or chronic infectionsis substantially different in terms of virulence and exo-product releasecaused by an adaptation to the hostile host environment. Thus, onchronic lung infections, pqs QSsystem of P. aeruginosa have been reported.The produced antibodies were used to develop the indirect competitiveAs385/HHQ-BSA ELISA for the quantification of the PQS, achieving adetectability that is within the range of concentrations found inbiological and clinical matrices. The results obtained when measuringthe PQS IRequiv. released by clinical isolate growth on MH culturebroth demonstrated the ability of the As385/HHQ-BSA ELISA to stratifypatients depending on the state of the disease. If this immunochemicalstrategy is applied for a higher number of QS molecules and/or infectionbiomarkers, then it might provide interesting information for diagnosticor prognostic purposes as well as about the pathogenic strain, thestatus of the patient, and/or the progression of the disease. In sucha case, the technique developed in this work could be envisaged asa future complement of the standard clinical analysis and help clinicianson taking decisions about the treatment and the management of infectedpatients. On the other hand, the obtained limits of detection anddynamic ranges led us to consider applying the method for the directquantification of the PQS in clinical respiratory samples from infectedpatients and its evaluation as the biomarker of infections. Eventually,the versatility and specificity of antibodies would also allow usto implement the developed technique in a wide variety of sensorsand/or PoC devices, supporting the rapid and straightforward diagnosticof infections caused by P. aeruginosa and subsequently improving the management of the patients.Inthis work, for the first time, antibodies against the most relevantand biologically active quinolone from the"}
{"text": "Addressing physical activity (PA) barriers is an essential component of increasing PA among the 56-73% of community-dwelling adults 50 years and older who are not performing the recommended 150 minutes of moderate-to-vigorous PA. As there is no feasible, multi-factorial tool to assess PA barriers among this population, we developed and validated a PA barrier assessment tool called the Inventory of Physical Activity Barriers (IPAB). We collected cross-sectional data on 503 adults (mean age 70.1), with 79 participants completing the scale twice for test-retest reliability and 64 completing a cross-over design examining the ability to use two administration formats interchangeably. Our analyses consisted of exploratory and confirmatory factor analysis, Cronbach alpha, intraclass correlation coefficient, Bland-Altman Plot, and t-tests. Using factor analysis, we identified and confirmed an eight-factor solution consisting of 27 items. The 27-item IPAB is internally consistent , has a high test-retest reliability (intraclass correlation coefficient=0.99), and can differentiate between individuals who meet the recommended levels of PA and those who do not (p < 0.001). The IPAB scores ranged between 1.00-3.11 for the paper format (mean=1.78) and 1.07-3.48 for the electronic format (mean=1.78), with no statistical difference between the paper and electronic administration formats (p=0.94), resulting in the conclusion that the two administration formats can be used interchangeably. Participant feedback illustrates that the IPAB is easy to use, has clear instruction, and is an appropriate length. The newly validated IPAB scale can be used to develop individualized PA interventions that address PA barriers among patients 50 years and older."}
{"text": "Human and veterinary pharmaceuticals are being increasingly used for disease treatment; hence, their distribution and factors influencing them in the aquatic environment need to be investigated. This study observed the effect of human and animal populations, usage, purchasing criteria (prescription vs. non-prescription), and land use to identify the spatio-temporal distribution of eight pharmaceuticals at twenty-four sites of the tributaries of the Han River watershed. In rural areas, the mean concentration (detection frequency) of non-prescription pharmaceuticals (NPPs) was higher (lower) compared to that of prescription pharmaceuticals (PPs); in urban areas, a reverse trend was observed. Pharmaceutical concentrations in urban and rural areas were mainly affected by wastewater treatment plants (WWTPs) and non-point sources, respectively; concentrations were higher downstream (4.9 times) than upstream of the WWTPs. The concentration distribution (according to the target) was as follows: human–veterinary > human > veterinary. Correlation between total concentration and total usage of the pharmaceuticals was high, except for NPPs. Most livestock and land use (except cropland) were significantly positively correlated with pharmaceutical concentrations. Concentrations were mainly higher (1.5 times) during cold seasons than during warm seasons. The results of this study can assist policymakers in managing pharmaceutical pollutants while prioritizing emerging pollutants. Pharmaceuticals, including non-steroidal anti-inflammatory drugs (NSAIDs) and antibiotics, are used for treating human and animal diseases but are considered emerging pollutants, categorized into two classes ,2. PrescVarious factors influence the distribution patterns of pharmaceuticals in urban and rural surface water, including intake and usage patterns ,16,17, pUnfortunately, large quantities of pharmaceuticals in surface water can endanger aquatic organisms such as bacteria, algae, invertebrate, and fish. Biotoxicity and susceptibility of organisms to the pharmaceuticals vary depending on the compounds and species ,34,35,36While pharmaceuticals can affect water environments in various ways ,51, theiStudies have identified that WWTPs affect the distribution of pharmaceutical concentrations in surface water of the Han River watershed, yet, other factors affecting the distribution have not been identified ,54,55,56In the present study, we discuss the results from a field study conducted from October 2015 to September 2016. The main objectives of this study were to: (1) investigate the overall distribution trend of eight pharmaceuticals; (2) compare PPs and NPPs at upstream and downstream areas of WWTPs; (3) identify the effects of human and animal populations, drug use (prescription and non-prescription), and land use on the distribution of the pharmaceuticals and the respective correlations; (4) describe the spatio-temporal variations in pharmaceutical concentrations and their possible pollutant sources in the tributaries of the Han River watershed, South Korea.4 (SMZ-d4), and ibuprofen-d3 (IBP-d3), were purchased from Toronto Research Chemicals . HPLC grade solvents such as methanol and acetonitrile, and Na2-EDTA  were obtained from Sigma-Aldrich. Reagent water was prepared by Milli-Q water purification system . Individual stock solutions of each compound were prepared in methanol; standard mixtures were prepared by diluting the stock solution and stored in the dark at –20 °C. All glassware were baked at 250 °C overnight in the oven, and rinsed with methanol prior to use.Eight pharmaceutical compounds (purity > 98%): naproxen (NPX), acetylsalicylic acid (ASA), carbamazepine (CBZ), trimethoprim (TMP), clarithromycin (CTM), sulfamethazine (SMZ), sulfamethoxazole (SMX), and sulfathiazole (STZ) were purchased from Sigma-Aldrich . The details of these pharmaceutical compounds are given in The Han River watershed is the largest in South Korea, consisting of four major rivers  and includes Seoul  and Gyeonggi Province . Several tributaries flowing into the Han River are impacted by human activities in Seoul. In this study, human and livestock population and land use data were retrieved from the Korean Statistical Information Service (KOSIS) . For thi3/day), were identified; details are presented in Twenty-five WWTPs, except for those with a small treatment capacity (<1.0 m2-EDTA in water. Oasis HLB (500 mg–6 mL) solid phase extraction (SPE) cartridges were used to extract the compounds from the water samples. Preconditioning of cartridges was carried out in the following order: 4 mL water → 4 mL methanol → 4 mL water → 5 mL water . The sample was loaded at a rate of about 10 mL/min using SPE cartridges after conditioning. Cartridges were washed with 6 mL acidified water to extract any residual Na2-EDTA prior to drying under vacuum, for approximately 1 h. Analytes were eluted with 6 mL methanol and 12 mL of methanol/acetone . The extracts were evaporated to 0.5–1 mL with a gentle stream of high-purity N2.Water samples (2 × 500 mL), filtered using 0.22 µm cellulose acetate membrane , were spiked with 25 µL isotopically labeled standard mix and 1000 µL of NaTM, Milford, MA, USA) equipped with an Agilent Eclipse plus C18 column . The eluent flow rate was 0.25 mL/min with a gradient of eluent A (acetonitrile) and B (5 mM ammonium acetate). The gradient program was as follows: 15% A to 2 min, linear increase of B, A to 90% in 3 min and held for 2 min. The total run time was 15 min. A returned to 15% in 3 min and was stabilized for 5 min to finish the cycle. Injection volume of 10 µL was used for analysis and the column temperature was maintained at 40 °C. For MS detection, the instrument was operated in the positive electrospray ionization and multiple reactions monitoring (MRM) mode. The MS/MS parameters were optimized as follows: nebulizer pressure, 35 psi; gas flow, 8 L min−1; mass range, 100–400 amu; scan time, 300 ms; temperature, 350 °C. Other parameters of MS/MS and retention time are summarized in The eight pharmaceuticals and ISs were quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled to Quattro Premier MS . Detailed information is given in The pharmaceuticals’ method detection limit (MDL) and limit of quantitation (LOQ), classified as the concentrations corresponding to the signal-to-noise (S/N) ratios of 3 and 10, respectively, were 0.0008–0.013 µg/L and 0.0026–0.0413 µg/L, with spiked concentration between 0.005 µg/L and 0.03 µg/L. The pharmaceutical recovery and precision rate for the filtered surface water samples ranged from 91.2% to 113.7% and 1.8% to 15.2% for all analytes. The calibration curve was generated over a broad concentration range (0.2–1000 µg/L) to demonstrate high linearity  was used to generate graphics, and ArcGIS 9.2  was used to produce a digital map of the Han River watershed.Minitab 15  and Excel 2016  were used to conduct the statistical analysis. Pearson’s correlation analysis was conducted to identify sources using pharmaceutical concentrations, and one-way analysis of variance (ANOVA) and two-sample paired Concentrations and detection frequencies of the target pharmaceuticals in the tributaries of Han River watershed are shown in ow influences the sorption of different chemicals in WWTPs, pH may also influence sorption behavior; hence, Dow’s pH-dependent n-octanol water distribution ratio was estimated using the median pH 7 observed in this study [ow > 4; Log Dow > 2.5) and have a large molecular weight (MW > 500 g/mol) [ow < 4; Log Dow < 2.5), removal by sorption would be negligible. Although pharmaceuticals are not recalcitrant pollutants in surface water (T ½ = 151.2–1440 h), the importance of continuous discharge should not be overlooked.The distribution of concentration of the pharmaceutical compounds is related to their properties . Althougis study  (Table S0 g/mol) ,72. HoweWhile most pharmaceuticals generally require a prescription, some are readily available at convenience stores without a prescription . TherefoThe high detection frequency of the PPs in both urban and rural areas can be attributed to the continuous discharge from WWTPs (point source), whereas the relatively high mean concentration of NPPs in the rural areas can be attributed to the untreated discharge from public and private facilities such as parks, museums, camping sites, and arboretums, as well as to a floating population ,56,74. AThe concentration of pharmaceuticals upstream and downstream of the WWTPs was examined. Of the twenty-four sites, nine and fifteen sites were present upstream and downstream, respectively. The total concentration and detection frequency downstream of the WWTPs was 4.9- and 2.4-times higher than those upstream, as shown in Pharmaceutical compounds selected in this study are divided into human, human and veterinary, and veterinary according to their intended use and detection concentration . PharmacBy contrast, the relatively high proportion of rural areas and natural tourist attractions can cause the pharmaceuticals to be generated in the form of non-point pollutants, and their high concentrations were detected owing to the low removal efficiency of relatively old and small WWTPs . SMZ, whSun et al.  reportedConcentration of pharmaceuticals in surface water have been reported to be a function of the population, livestock species and number, and land use types, as well as the population not connected to WWTPs as the potential pollutant sources; studies on correlation among these variables have been conducted in different countries ,20,81. Hp < 0.05). In cropland, herbicides and chemicals necessary for farming are mainly used rather than pharmaceuticals; hence, the proportion of pharmaceuticals utilized in the human and veterinary category is relatively very little [y little . Livestoy little . In addiy little  concludeThe concentration distribution of pharmaceutical compounds in independent rivers in the Han River watershed was analyzed . In the HR-4, where the highest pharmaceutical concentration was detected in the HR area, is located directly under the largest WWTP-19 in Seoul; the mean concentration was 10.1 times higher than that at HR-3 which is located upstream on the same river and is not affected by WWTPs. The pharmaceutical concentration in the HR area was found to be in the following order: HR-4 > -5 > -2 > -7 > -1 > -6 > -3 ranging from 0.0042 µg/L to 0.8661 µg/L with 12.5–75.0% detection frequency.The second highest pharmaceutical concentration and detection frequency were in found in the IHR area. IHR-1 was detected having the second highest concentration among the 24 sites. However, unlike HR-4, this site does not have a WWTP located upstream, but a large recreational park is located nearby. This park is considered a non-point pollution source, unlike the WWTPs, which is a point source of pollution. Poor park management and careless waste discharge (such as that from toilet facilities) can lead to pharmaceutical compounds being present in high concentrations in the nearby areas. Other sites, IHR-2, -3, and -4, similarly detected low concentrations ranging from 0.0052 µg/L to 0.0859 µg/L.The third highest concentration was detected in the SR area. SR-6 and -7 are not characterized by a WWTP upstream; they showed the lowest concentrations of pharmaceuticals while relatively high concentrations were detected at SR-4. Due to the mixed impact of non-point pollution sources such as livestock and farming, the Bok River, where SR-4 is located, receives pollutants from non-point sources such as livestock and farming . Two WWTThe NR area was characterized by the least amounts of pharmaceutical compounds among the areas studied; each site had very low levels of pollutants. The concentration of pharmaceutical compounds was different at each site in each river, and this is attributed to the capacity of WWTPs, the removal rate of pollutants by the WWTPs, and the content of organic matter in the WWTP effluent .The water temperature was divided into cold  and warm , corresponding to the seasons, and the concentration of pharmaceutical compounds in each season is shown in This study has several strengths and limitations. First, to the best of our knowledge, this is the first study to identify the distribution characteristics of prescription and non-prescription pharmaceuticals in tributaries of the Han River. Second, we found that pharmaceuticals originated from human and animal populations and drug and land use. Third, we demonstrated high-quality analytical performance  due to proper equipment maintenance during the analysis of pharmaceuticals in surface water samples. These results provide insight into the distribution of pharmaceutical substances in surface water based on environmental factors.However, some limitations should be considered when interpreting our findings. First, a total of four water samples may not reflect the concentration of general pharmaceutical compounds representing the Han River watershed. Although sampling was performed at the same place and time, no data were collected for unusual environmental events, such as heavy rain and water structure construction. Second, confounding factors, such as regional pharmaceutical usage, an aging population, and major diseases may affect the results of this study. These factors could be the focus of future research.Therefore, continued research and data collection will inform policymakers, securing the health of the aquatic ecosystem in surface water. Moreover, policymakers can use the comprehensive results of this study to determine the concentration and detection frequency of pharmaceuticals in the Han River watershed and consider the possibility of pharmaceutical discharge due to environmental factors.p < 0.05). Higher concentrations were mainly observed during the cold season, with the mean concentration being 1.3 times higher than that during the warm season. Spatial analysis revealed that the pharmaceutical concentrations in different areas were impacted by different sources. This study provides scientific information to policymakers for regulating the concentrations of pharmaceutical pollutants in natural aquatic environments, as well as identifying priority emerging pollutants.In this study, eight pharmaceutical compounds of different therapeutic classes were investigated at twenty-four sites of the tributaries in the Han River watershed. The effects of various factors on pharmaceuticals distribution were investigated. Each of the studied pharmaceuticals were detected at least once in all of the samples. The mean concentrations of the pharmaceuticals showed the following order: CTM > NPX > CBZ > ASA > TMP > SMX > SMN > STZ with detection frequency ranging from 6.3% to 78.1%. In urban areas, the mean concentration and detection frequency of the PPs were higher than those of the NPPs, and in urban and rural areas, the pharmaceutical concentrations were significantly higher downstream of the WWTPs than upstream; this is mainly attributed to the WWTPs. Overall, human and human–veterinary pharmaceuticals were more abundant and were measured in higher concentrations compared to the veterinary pharmaceuticals. All livestock and land use (except cropland) were found to have a significant positive correlation with the concentration of pharmaceuticals ("}
{"text": "Consumer choice models have been introduced in eldercare services in several Western welfare societies. Choice models in eldercare emphasise the importance of individuals’ abilities to make informed choices and therefore entail a risk for increased inequalities among older adults with care needs. In the Nordic countries, such inequality risks are in stark contrast to universal policy ambitions of equal access to care services. Care managers, who are responsible for needs assessment for eldercare services, have a central role in implementing policies and, thus, have first-hand experience of their impact on older adults’ access to care. The aim of this study was to explore care managers’ experiences of how user choice affects older adults’ access to care services in three Nordic cities: Copenhagen, Tampere, and Stockholm. These cities were purposely selected as forerunners in marketisation, with different ways of implementing choice models. Semi-structured interviews with care managers were conducted in Copenhagen, Tampere, and Stockholm and analysed thematically. The findings indicate there are difficulties related to older adults’ ability to access information needed to make informed choices, as well as limitations in choice related to available services and personal finances. Further, care managers find that older adults’ abilities to overcome these difficulties are shaped by their health, education, language skills, and assistance from relatives. In order to reduce the risk of choice models increasing the gap between older adults with different resources and capabilities, there is a need to develop accessible information, as well as models for professional guidance. Choice models have reorganised the provision of long-term care (LTC) across all Western societies  providers in both home care and residential care. Distinctive for Stockholm was the large number of service providers. For example, a home care user could choose between more than 60 providers in each city district.According to the law regulating choice models, the Act of System of Choice, local authorities have to appoint a provider, which could be public or private, for those who do not choose for themselves . Users could also choose a ‘self-appointed carer’, who was employed by the municipality after an assessment of the carer’s qualifications. In Denmark, the Law on Free Choice of Provider of Practical Assistance and Personal Care has obliged local authorities to adopt choice models in home care since 2003, whereas it was optional in residential care  to make informed choices  or personal (help with the body). As a consequence, there was a general lack of services addressing social needs, as one care manager in Copenhagen illustrated: ‘There are those who have needs related to loneliness or anxiety, and it’s really hard to support them with that. We have no services aimed at these needs.’The standardisation of services entails difficulties in meeting complex needs, such as mental health problems and substance abuse in combination with physical problems or dementia. In all three cities, care managers stated that it is hard to meet needs that do not match the available services. A care manager in Tampere concluded: ‘I am quite helpless because I have no services to offer them.’When it comes to having services granted, relatives can be of importance. Care managers reported that older adults who have relatives who know what services there are and what arguments to use in order to get these services are in a better position to receive services since these relatives tend to act as advocates for their next-of-kin and argue their case for them. This is especially so if the older adults are on the verge of being eligible for a service, as illustrated by a care manager in Copenhagen: ‘Sometimes, you might give in a little, and it might be a little easier to grant the service than if nobody is pushing for it.’ In line with this, care managers expressed concerns that older adults who do not have relatives to inform them might miss out on services because they are unaware of them.Another aspect of limitations in choice regarded personal finances. This subject was most commonly raised by care managers in Tampere, where older adults with enough financial resources could access choices either through the private market for home care services or through the voucher system for residential care. One care manager explained the importance of personal finances: ‘What gives you options is money. If you receive a low pension and you only have a little bit of money at your disposal, then you don’t have that freedom of choice.’However, the importance of personal finances extends beyond the choice model in Tampere, where care managers experienced that older adults cut back on services or even refrained from services due to high customer fees, even for public services. One care manager in Tampere illustrated: ‘Yesterday, I received a call from a customer who had just started receiving a service. The customer told me their monthly income, and I calculated what the monthly fee would be. The customer was terrified and had to cut back on the services.’Care managers in Stockholm had also experienced users being reluctant to accept services due to user fees, although they did not report this to the same extent as the care managers in Tampere. In Stockholm, this was perceived as most common among users with smaller care needs, who felt that the costs for services were disproportionally high. One care manager in Stockholm said: ‘There are some who think they cannot afford it, that their financial situation would become too strained. And they sometimes decline services.’ In Copenhagen, where there were no fees for home care services, the care managers did not report that financial resources affected access to care services.Care managers rarely mentioned any positive aspects of choice models, and when they did, there was no consistency between the cities. In Stockholm, care managers declared the emergence of providers with different language profiles to be an improvement for older migrants since the opportunity to receive care in one’s own language makes services more accessible. One care manager in Stockholm said: ‘Then they can understand the staff because they speak the same language. So, it is very beneficial that there are different providers of home care services who know different languages.’ However, other care managers problematised that this opportunity was only present for some of the largest language groups, and the providers usually made no guarantees that all staff could speak a specific language.In Tampere, the city where older adults’ personal finances mattered the most, some of the care managers emphasised that the choice model benefited older adults with a good personal economy. However, they approached this fact from different perspectives. As shown in the section on personal finances, some expressed concern for those least well off. Others argued that it is only fair that people with more money can access more or better services. When discussing the voucher for residential care services, which is only available for the more well off, one care manager in Tampere stated: ‘Why should everybody have the same ‘shit’, so to speak? Why shouldn’t everyone who has money use it for their own well-being? I think it is a great addition.’This paper has explored care managers’ experiences of how choice models affect older adults’ access to LTC in three Nordic cities. The findings suggest that choice models entail more difficulties than benefits. Across all cities, the findings revealed difficulties related to accessing information needed to make informed choices. These difficulties are related to the fact that the information older adults receive in relation to making choices is general, while older adults are expected to find more specific information needed to make informed choices by themselves. In line with earlier studies, the care managers in our study stressed that difficulties in accessing information are exacerbated when the older adult is in a poor state of health and has a lack of energy and/or reduced cognitive capacity  or personal (help with the body), which means that people with complex needs and social needs, such as loneliness, are disadvantaged. However, these problems are more related to cutbacks and standardisation of services (Kröger and Leinonen Taken together, these findings have implications for two different aspects of inequality in access to care: unequal opportunities to make informed choices and unequal opportunities to having ones needs met. This shows that sameness in treatment is not enough to achieve equality in outcomes Fineman , 2017. EThe differences between the cities regarding the benefits of choice models and the importance of financial resources show that choice models may have different implications in different contexts. The large number of providers in Stockholm can be an obstacle in choice but also entails the benefit of providers with language profiles. Most variation between the cities is related to the importance of personal finances. In Tampere, the impact of personal income seems much more prominent than in the other cities. This can be attributed to high user fees in publicly funded Finnish LTC, in combination with a voucher system for residential care that is only accessible for those with a solid personal economy. Also, in Stockholm, the care managers experienced that older adults with fewer resources struggle with the user fees. In Denmark, there are no fees for home care services, and care managers in Copenhagen did not perceive personal finances as a barrier to care. The fact that care mangers experience that user fees restrict access to LTC in two of the three cities is worrying from an equality perspective, and deserves more attention in the future research.Altogether, the findings indicate that the operations and organisations of choice models reproduce existing inequalities (cf. Fineman In addition, the findings of this study point to the conclusion that general knowledge of the welfare system is an advantage, whereas newcomers to the system, such as older migrants, are disadvantaged. Although non-native speakers are generally identified as a disadvantaged group in research on choice models (cf. Baxter et al. One limitation of this study is that we only present the perspectives of care managers and, thus, have no empirical data of older adults’ experiences. By interviewing older adults about their experiences, we could have gained insight into individual experiences of choice models. However, interviewing care managers, who meet many older adults in their work, provided an overview of how choice models affect access to services. Another limitation is that we rely solely on interview data reflecting the care managers’ experiences, which means we cannot determine actual differences in older adults’ access to LTC within, or between, the three cities. Despite these limitations, the study points to significant design flaws and contributes to the knowledge on inequality implications of the interplay between how choice models are implemented and general features of LTC.In line with other studies, our findings suggest that older adults have different resources and abilities to overcome barriers raised by design flaws. Like Fineman , we percThe present study has identified general system design features that could potentially ameliorate inequalities among older adults: low user fees, services that match actual needs and not only the most basic ones, and more transparent choice models with tailored information. There is also a need for more targeted interventions, such as information in different formats, including easy-to-read formats; information in foreign languages; and professional guidance for those who do not have a network of relatives to rely on, as well as other vulnerable groups, such as non-native speakers and people with dementia (cf. Baxter et al. The findings revealed difficulties related to design features of choice models but also difficulties that are not directly related to choice models. According to care managers, older adults with poor health, low education, poor language skills, and no close relatives are the most negatively affected by these difficulties. To prevent the gap from increasing between older adults with greater resources and those with fewer resources, measures are needed to make LTC more transparent and accessible for everyone."}
{"text": "Patients successfully resuscitated from cardiac arrest often develop organ dysfunction caused by systemic inflammation and increased coagulation, leading to disseminated intravascular coagulation (DIC). The involvement of histones in DIC and organ dysfunction in patients with sepsis and trauma has been previously reported, raising the probability that histones may also be associated with pathophysiology in patients after cardiac arrest and resuscitation. This study evaluated the relationship between histones and organ dysfunction related to coagulofibrinolytic changes in patients with post-cardiac arrest syndrome (PCAS).This prospective single-center observational study assessed 35 adult patients with PCAS who were divided into two groups, i.e., 15 patients with multiple organ dysfunction syndrome (MODS) and 20 patients without MODS. MODS was defined as a sequential organ failure assessment score of ≥12. The plasma levels of histones and coagulofibrinolytic markers, including soluble fibrin, tissue-type plasminogen activator, plasminogen activator inhibitor-1, plasmin-alpha 2-plasmin inhibitor complex (PIC), and soluble thrombomodulin, were measured in patients with PCAS immediately after admission to the emergency department, and 3 and 24 h after arriving at the hospital.p = 0.012] and higher mortality rates  than those without MODS. Moreover, patients with MODS exhibited higher histone levels than those without MODS during the early phase of the post-resuscitation period. Severe endothelial injury and higher thrombin and plasmin generation were observed in the MODS group. Plasma levels of histones were positively correlated with those of soluble fibrin immediately after resuscitation  and PIC 3 h after arriving at the hospital . This correlation was prominent in the patient population with MODS .PCAS patients with MODS had higher DIC scores [4 (3.0–5.0) vs. 1 (0.0–3.0), This study demonstrated that elevated histone levels were associated with increased levels of thrombin, and subsequent plasmin generation in PCAS patients, especially those with MODS. Further studies are required to elucidate the causal relationship between histones and organ dysfunction related to DIC in PCAS. Over the past half-century, the rate of return of spontaneous circulation (ROSC) has considerably improved owing to the widespread use of cardiopulmonary resuscitation (CPR) among the public and advances in treatment methods for patients after cardiac arrest. However, the long-term survival of resuscitated patients remains poor. The high mortality rate in patients who are successfully resuscitated after cardiac arrest may be ascribed to the development of post-cardiac arrest syndrome (PCAS) . The IntRecent studies have shown the importance of bidirectional interactions between inflammation and coagulation that are triggered by damage-associated molecular patterns (DAMPs), such as histones— in response to cellular injury and neutrophil extracellular traps (NETs) released by activated neutrophils—in the pathophysiology of DIC , 8. AlthWe conducted a single-center prospective observational study. A total of 47 patients who experienced out-of-hospital cardiac arrest were enrolled between September 2018 and June 2020. Patients were excluded if they did not achieve ROSC or were under 20 years of age. Written informed consent was obtained from the patients' next of kin to draw blood samples and record essential data necessary for scoring DIC, acute physiology and acute chronic health evaluation (APACHE) II, and sequential organ failure assessment (SOFA) scores. If the next of kin did not accompany any of the patients, informed consent was retrospectively obtained after collecting blood samples at 0 h. In addition, blood samples from 15 healthy volunteers , who were medical students and medical staff in our hospital), were collected to measure plasma levels of histones and coagulofibrinolytic markers. The study was approved by the Institutional Review Board of the Ethics Committee of Hokkaido University School of Medicine (017-0518).DIC was diagnosed according to the Japanese Association for Acute Medicine (JAAM) DIC score  14). Th. Th14). Blood samples were collected from an arterial catheter in patients using a syringe at three-time points: immediately after ROSC (0 h), 3 h after ROSC (3 h), and 24 h after ROSC (24 h). Blood samples from healthy volunteers were collected once per person. Blood samples were immediately placed into tubes with sodium citrate and centrifuged at 1,500 × g for 15 min at 4°C. The plasma was stored at −80°C until further examination.We measured the plasma levels of histones   and the Data necessary for scoring such as DIC score, SOFA score, APACHE II score, and other general coagulation markers, including antithrombin, fibrinogen/fibrin degradation products (FDPs), and D-dimer were retrieved from the medical records.U-test. Categorical data were compared among the groups using Fisher's exact test or Pearson's chi-squared test, depending on the size of the data. Correlations were evaluated using Spearman's rank test. Receiver operating characteristic (ROC) curve analyses with the area under the curve (AUC) calculation were used to quantify the predictive performance of the plasma levels of histones at each time point. Statistical significance was set at P < 0.05. All statistical analyses and calculations were performed using GraphPad Prism for Windows version 9.3.1  and JMP Pro 16.0 .Numerical variables were presented as medians and quartiles, and categorical variables were summarized as numbers and percentages. Numerical data were compared among groups using the two-sided non-parametric Mann–Whitney n = 15) and non-MODS (n = 20) groups. The characteristics of the patients in both groups are shown in Out of the 47 patients, 12 were excluded from the study. Among the excluded patients, 11 did not achieve ROSC. We also excluded one patient because the samples were not treated appropriately. In the present study, we analyzed the final cohort of 35 patients. The patients were divided into MODS . There were no significant differences in PAI-1 levels at any time point between the MODS and non-MODS groups, whereas PAI-1 levels at 24 h in the MODS group tended to be higher than those in the non-MODS group.Serial changes in coagulofibrinolytic molecular markers  are shown in p = 0.030). This correlation was prominent in a population with MODS . There was also a positive correlation between histone levels and PIC measured at 3 h, when the fibrinolysis is considered to peak . This correlation became stronger in the MODS group.We evaluated the correlation between plasma levels of histones and coagulofibrinolytic markers . There w to peak , 22,  curves. The area under the curve (AUC) for plasma levels of histones measured at 0 was 0.710  . In ordeIn the current study, we found that PCAS patients with MODS had higher DIC scores and higher mortality rates than those without MODS. In addition, patients with MODS exhibited massive thrombin and plasmin generation with higher levels of histones than those without MODS during the acute phase of PCAS. The levels of histones were positively correlated with SF, a thrombin generation marker, and PIC, a plasmin generation marker. According to our analysis, the plasma level of histones immediately after ROSC was a good predictor of MODS development in patients with PCAS.Histones are commonly known as DNA-binding proteins that play an important role in compacting DNA to be stored in the nucleus –25. HistA previous study demonstrated that the levels of circulating histones increase in mouse models of sepsis in a neutrophil-dependent manner, suggesting that histones are mainly derived from NETs . HoweverAlthough this study cannot demonstrate a causal relationship between histones and the pathophysiology of PCAS-associated DIC, we believe that extracellular histones may be a promising therapeutic target for PCAS-associated coagulopathy and consequent organ dysfunction, as previous studies showed that the administration of neutralizing antibodies to histones can prevent death from severe sepsis in a murine model , 28. ThiThis study has several limitations. First, we excluded 11 patients without ROSC from initial cohort because this study aimed to elucidate the pathophysiology of PCAS. This may be a study limitation. Second, the sample size was small. This limitation may have resulted in wide variations in the measured parameters as shown in To the best of our knowledge, this is the first study to investigate the relationship between histones and the coagulofibrinolytic changes associated with PCAS and to suggest the potential of histones for predicting the MODS development in patients with PCAS. Although the causal relationship between histones and organ dysfunction related to DIC in PCAS requires investigation, we expect that the neutralization of histones may represent a new therapeutic strategy for treating PCAS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of the Ethics Committee of Hokkaido University School of Medicine (017-0518). The patients/participants provided their written informed consent to participate in this study.AM analyzed the study results, interpreted the data, and drafted the manuscript. TW oversaw the entire research and contributed to the research concept, the analysis, and interpretation of the data, and was responsible for drafting, editing, and submitting the manuscript. TT contributed to the data acquisition, interpreted the data, and revised the manuscript for important intellectual content. SG had a significant influence on data interpretation and critical appraisal of the manuscript. All authors read and approved the final version of the manuscript before submission.This work was supported in part by JSPS KAKENHI .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
{"text": "All patients received combined immunotherapies (anakinra ± corticosteroids ± intravenous immunoglobulin ± tocilizumab) according to our local treatment algorithm. The endpoint was the composite event of intubation due to severe respiratory failure (SRF) or mortality. Median (interquartile range) sIL-2R levels were significantly higher in patients with severe disease, compared with those with moderate disease (6 (6.2) vs. 5.2 (3.4) ng/mL, p = 0.017). sIL-2R was the strongest laboratory predictive factor for intubation/death  after adjustment for other known risk factors. Youden’s index revealed optimal sIL-2R cut-off for predicting intubation/death at 9 ng/mL . Delta sIL-2R between the day of event or discharge minus admission date was higher in patients that intubated/died than in those who did not experience an event (2.91 (10.42) vs. 0.44 (2.88) ng/mL; p = 0.08)). sIL-2R on admission and its dynamic changes during follow-up may reflect disease severity and predict the development of SRF and mortality.Risk stratification of coronavirus disease-19 (COVID-19) patients by simple markers is critical to guide treatment. We studied the predictive value of soluble interleukin-2 receptor (sIL-2R) for the early identification of patients at risk of developing severe clinical outcomes. sIL-2R levels were measured in 197 patients (60.9% males; median age 61 years; moderate disease,  Coronavirus disease 2019 (COVID-19) has a wide clinical spectrum, ranging from asymptomatic to severe respiratory failure (SRF), multiorgan failure, and death ,4. IndeeEvidence suggests that the pathophysiology of COVID-19 deterioration is based on immune dysregulation and hyperinflammation resembling cytokine release syndrome, driven by several pro-inflammatory cytokines ,9. ExcesHowever, to date, few studies have investigated the correlation between COVID-19 severity and soluble interleukin-2 receptor (sIL-2R), which is one of the biomarkers of immunological dysregulation ,21,22. IAccordingly, the aim of the present study was to determine the predictive value of sIL-2R levels for identifying COVID-19 patients who are at risk of deterioration to severe clinical outcomes as timely initiation of targeted interventions in these patients seems crucial to prevent progression to SRF and reduce mortality.2) ≥ 94%, whereas severe pneumonia was characterised by the presence of at least one of the following: SpO2 < 94% in room air, breath rate > 30/min, respiratory ratio (pO2/FiO2) < 300, and pulmonary infiltrates in more than >50% of the lung parenchyma [sIL-2R levels were measured in the sera of 197 patients  hospitalised in our Department due to moderate or severe pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Moderate pneumonia was defined by oxygen saturation by pulse oximetry in room air , and laboratory inflammation markers  were recorded at baseline. All patients were treated with combined immunotherapies (anakinra ± corticosteroids ± intravenous immunoglobulin ± tocilizumab) based on initial ferritin levels and according to our local treatment algorithm [Demographics , smoking habits), comorbidities , cardiovascular diseases), parameters of disease severity , according to the manufacturer’s instructions. Serum samples of patients were collected and stored frozen at −20 °C until use. Samples were thawed only once to prevent repeated freeze–thaw cycles. A standard curve was constructed using 7 standards of twofold serial dilutions from 20 ng/mL to 0.31 ng/mL. To avoid a Hook effect, samples with a concentration greater than the standard of 20 ng/mL were further diluted to precisely quantitate the actual sIL-2R level. The calculated overall intra-assay and inter-assay coefficients of variations were 7.2% and 9.8%, respectively.sIL-2R serum levels were determined by using sIL-2 Receptor Human ELISA , Fisher’s exact test, Spearman’s rho correlation, and binary logistic regression analysis were used to analyse the data. The impact of baseline clinical and laboratory risk factors on our composite endpoint of intubation/mortality was evaluated by univariate and multivariate Cox regression analyses. Receiver operating characteristic (ROC) curves were constructed, and comparisons were performed by the DeLong test. Youden indices (sensitivity + specificity − 1) were calculated to establish optimal cut-offs for sIL-2R. Two-sided p-values less than 0.05 were considered statistically significant. All data analyses were carried out with SPSS version 26.0.The Kolmogorov–Smirnov test was applied to assess the normal distribution of variables, and quantitative values were expressed as median (interquartile range (IQR)). Mann–Whitney U-test, xAmong the 197 patients  with COVID-19 recruited in this study, 92 had moderate and 105 had severe disease at the time of first evaluation. Overall, 27 patients with moderate disease (29.3%) progressed to severe disease during hospitalisation.The demographic, clinical, and laboratory characteristics of patients are summarised in p < 0.001), increased BMI (p = 0.018), higher prevalence of cardiovascular disease (p = 0.001), lower lymphocytes (p = 0.005), and higher CRP (p < 0.001) and ferritin (p < 0.001) levels at the time of presentation.Patients with severe disease were characterised by older age (p = 0.017). sIL-2R levels were negatively correlated with respiratory ratio  and lymphocytes count . A positive correlation was found between sIL-2R and CRP , whereas no correlation was found with ferritin .sIL-2R levels were significantly higher in patients with severe disease (6 (6.2) vs. 5.2 (3.4) ng/mL, During follow-up, 42/197 (21.3%) were intubated or died. p < 0.001); presence of diabetes ; cardiovascular disease ; lower pO2/FiO2 ratio ; decreased lymphocytes count ; increased ferritin ; CRP ; sIL-2R levels .Univariate analysis revealed that intubation/mortality was associated with advanced age : 1.031–1.081, p = 0.030) and low pO2/FiO2 ratio , while only the increased sIL-2R levels maintained a significant negative prediction for intubation or death among the laboratory markers , while a trend was found when compared with CRP levels at baseline (AUC (95%CI): 0.683 (0.594–0.773); p = 0.065) (The AUC (95%CI) of sIL-2R for detecting patients with COVID-19 who eventually intubated or died was 0.789 (0.709–0.869). sIL-2R at presentation had higher predictive ability when compared with ferritin (AUC (95%CI): 0.612 (0.513–0.711); = 0.065) .p < 0.001), lower pO2/FiO2 ratio (p < 0.001), increased respiratory rate (p = 0.008), lower lymphocytes (p = 0.007) and higher CRP levels . Patients with sIL-2R ≥ 9 ng/mL were characterised by increased age . Serum samples were collected at baseline, in the middle of hospitalisation, and at discharge or at the time point of intubation/death. During hospitalisation, 9/23 patients were intubated or died .n = 9) and patients who were discharged from the hospital without intubation (n = 14) showed higher levels for the former group at three different time points: baseline (10.1 (4) vs. 3.7 (2.8) ng/mL; p = 0.001), middle of hospitalisation (9.4 (4.2) vs. 4.9 (4.2) ng/mL; p = 0.002), time of discharge or intubation/death (14.6 (14.2) vs. 3.9 (4) ng/mL; p = 0.002) (p = 0.013) (p = 0.001) (p = 0.109 and lymphocytes: 700 (1040) vs. 845 (670); p = 0.477) and baseline between the two groups of the patients (ferritin: 634 (1862) vs. 458 (533); p = 0.141 and lymphocytes: 900 (525) vs. 1075 (833); p = 0.250)  vs. 0.9 (1.1), p = 0.109, time of intubation/death or discharge: 0.2 (0.7) vs. 0.1 (0.2); p = 0.083) (A comparison of median (IQR) sIL-2R at three different time points between patients who were intubated or died (= 0.002) A. Furthe= 0.013) B and low= 0.001) D only at= 0.250) B,D. No d= 0.083) C.p = 0.08)) (p = 0.011)  delta sIL-2R (day of event or discharge day of admission) was higher in the group of patients that intubated or died (2.91 (10.42) ng/mL), compared with patients who did not experience an event (0.44 (2.88) ng/mL; = 0.08)) A. In add= 0.011) D, while In the current study, we evaluated the relationship between sIL-2R levels and the disease severity of COVID-19 pneumonia and outcomes of patients. Our analysis demonstrated that sIL-2R was associated with severe pneumonia and mainly that sIL-2R levels at baseline were the strongest laboratory-negative predictive factor for patient outcome. According to our results, a level of sIL-2R greater than 9 ng/mL had a PPV of 86% and an NPV of 91% for intubation or death.From the first days of the pandemic, serum concentrations of both anti-inflammatory IL-10) and pro-inflammatory cytokines  were found to be increased in severe cases of COVID-19, compared with patients with moderate disease, implying that a cytokine storm is linked to disease pathogenesis . However0 and proMoreover, several laboratory markers have been thoroughly investigated in an attempt to develop early diagnostic and prognostic biomarkers for predicting COVID-19 progression to severe disease and outcome. In this context, hyperferritinaemia, high CRP levels, and lymphopenia, along with some more sophisticated markers such as the soluble urokinase plasminogen activator receptor (suPAR) ≥ 6 ng/mL and SCOPE score ≥ 6, have already been associated with severity and survival of COVID-19 patients ,34,35,36Notably, in parallel with other reports, we found that sIL-2R levels are negatively associated with lymphocytes count, as has also been shown in previous studies ,22. SeveRegarding the outcome of patients, our data showed that increased sIL-2R levels on admission are associated with increased rates of intubation or mortality. In line with our findings, previous studies have shown that sIL-2R levels are associated with worst outcomes, as attested by longer duration of illness and time to recover, and lower survival rates ,17,18. MMoreover, sequential monitoring of sIL2R may add further diagnostic value for patients who are likely to bear poor prognoses. Indeed, according to our subanalysis, individuals who were intubated or died had consistently higher levels of sIL-2R during hospitalisation, compared with patients who did not experience an event, while ferritin and lymphocytes differ only at the time point of the event, which attenuates their predictive values. Moreover, the increase in sIL-2R levels was higher in patients with an unfavourable course of the disease.Indeed, future research is needed to evaluate the relationship between sIL-2R serum levels and immunological response to various stimuli in COVID-19 patients; however, the cross-sectional design of our study did not allow us to conduct such tests.In conclusion, the simple determination of sIL-2R on admission by an easy-to-perform, low-cost, non-invasive test method that can be performed in any laboratory, as well as its sequential measurements during the disease course, seem to reflect COVID-19 severity and predict the development of SRF and death. As a result, it could be utilised as a biomarker in risk stratification of the patients in an attempt to promptly identify those who need intensive monitoring, prompt initiation of treatment, and/or inclusion in therapeutic trials."}
{"text": "The objective of this study was to evaluate the efficacy of locking plates versus interlocking intramedullary nails in the treatment of proximal humerus fractures to provide clinical data support and theoretical guidance.P < 0.05 was considered statistically significant.Patients with proximal humerus fracture from the Third hospital of Shijiazhuang city and Third hospital of Hebei medical university and from January 2017 to June 2019 were selected, included and divided into the locking plate group and the interlocking intramedullary nail group according to the intervention received. Information pertaining to the perioperative period  of patients in both groups was collected. VAS pain scores, shoulder activity Constant-Murley scores and postoperative complications were documented. The perioperative data of the two groups were compared, and P > 0.05). However, the average operation time of the locking plate group was shorter than that of the interlocking nail group , and the intraoperative blood loss of the locking plate group (137.4 ± 16.8 ml) was higher than that of the interlocking nail group . There was no significant difference in the VAS score and Constant-Murley score between these two groups at the final follow-up.A total of 64 patients were enrolled, including 36 patients in the locking plate group, with a mean age of 61.3 ± 13.9 years, while the mean age of the interlocking intramedullary nail group was 65.6 ± 11.2 years. There was no statistical difference in gender, affected side, injury mechanism and Neer classification between the two groups (Interlocking intramedullary nails are more minimally invasive than locking plates, but fracture reduction and fixation take longer. There was no significant difference in pain and shoulder function scores between the two internal fixation strategies for the treatment of proximal humerus fracture. Proximal humerus fractures (PHFs) are common fragility fractures in the aging population, occupying about 4–5% of all fractures . The choSurgical strategies for PHFs have gone through a long evolution over the last few decades. The most widely used treatment include open reduction and internal fixation with locking plate osteosynthesis, intramedullary nail fixation, reverse shoulder arthroplasty and shoulder hemiarthroplasty. There is still controversy surrounding the treatment approaches used in displaced PHFs. The locking plate strategy is considered the gold standard treatment for PHFs, but several studies reported an association with several complications . The plaThe aim of this retrospective study was to analyze these two methods, provide clinically referable evidence on the clinical outcomes, and report the complications related to each intervention.We retrospectively analyzed all the patients diagnosed with PHFs between January 2017 and June 2019 in the Third hospital of Hebei medical university and the Third Hospital of Shijiazhuang city. The inclusion criteria were as follows: (1) patients over 18 years of age; (2) two-part, three-part, or four-part of PHFs on Neer classification; (3) fresh fracture; (4) no previous ipsilateral humeral surgery. Meanwhile, the exclusion criteria were as follows: (1) pathologic fractures or open fractures; (2) concomitant neurovascular injury; (3) fractures associated with shoulder dislocation; (4) mental illness. The committee of our institution waived the requirement for written informed consent because this present study was retrospective.The patients were operated under brachial plexus block or general anesthesia. All surgeries were conducted by the same team, which was proficient in both techniques. All patients were lying on a radiolucent operating table in a beach chair position.For the intramedullary nail group, the deltoid-splitting approach was applied. After exposing the deltoid muscle, the anterior and middle deltoid bundles were split bluntly along the direction of the muscle fibers to reach the fracture site. The entry point was revealed after temporary fixation of the fracture fragments with 2.0 mm Kirschner wires, which was 1 cm medial to the greater tuberosity. Then the Kirschner wire was used as a “joystick” to manipulate the humerus head and reduce the displacement. After the tuberosities were reduced, the three- or four-part fracture type would turn into a two-part type. Then the main nail (Targon nail) was inserted, ensuring that the end of the intramedullary nail was 2–3 mm below the cartilage of the humeral head. At last, the proximal and distal screws were locked.For the locking plate group, the classical deltopectoral approach was applied. Both the indirect and direct reductions were performed to reduce the fracture fragments with the assistance of the C-arm. The bone defect of the humeral head was evaluated, and autologous or allogeneic iliac bone grafting was used for patients with poor bone quality and larger defects to increase the stability after plate fixation. At least five locking screws were inserted proximally, with a minimum of 3 distal screws. The nonabsorbable sutures passed through the holes on the plate  and were knotted to repair the rotator cuff and increase its stability.Clinical data and radiographic materials of all the included patients were collected, including gender, age, injury type, comorbidities, operation time, time to surgery, blood loss, and Neer classification . The visShoulders were immobilized for four weeks with an abduction pillow sling. Passive movement of the shoulder began on the second day after surgery. Active movements started 4–6 weeks postoperatively.Clinical follow-up was conducted by two orthopedics attending physicians at 1 and 3 months after surgery. Postoperative plain radiographs were taken at each follow-up. Furthermore, all surgery-related complications were recorded, such as screw breakage, superficial infection, fat liquefaction, varus deformity, delayed union, and acromion impingement.t test were applied to analyze continuous variables. A value of P less than 0.05 was considered statistically significant.All statistical analyses were performed using IBM SPSS Statistics for Windows, version 20.0 . The distributions of all variables were evaluated for normality using the Shapiro–Wilk test. Data that satisfies normality were presented as the mean ± standard deviation. Those data that did not meet normality were presented as medians and quartiles. Chi-squared test were used to analyze the difference in gender distribution, Neer classification of humeral head fractures and injury mechanism between the two groups. The nonparametric test and Student P > 0.05). The average surgery time was shorter in the locking plate group compared to the intramedullary nail group . The blood loss was 137.4 ± 16.8 ml in the locking plate group, which was higher than in the intramedullary nail group . There was no difference in the VAS score (P = 0.202) and Constant-Murley score (P = 0.067) at the final follow-up, as shown in Table In total, 103 proximal humerus fracture patients were searched during this period (January 2017 to June 2019). All the patients were evaluated by two surgeons according to the inclusion and exclusion criteria. After screening, 38 patients were excluded. Sixty-five patients with locking plates and interlocking intramedullary nails were primarily included, 1 of whom was lost to follow-up. The demographic data of these two groups are displayed in Table P = 0.343), as shown in Table The incidence of general complications was 8.33% (3 patients) and 10.71% (3 patients) in the locking plate and intramedullary nail group, respectively  were used in this trial. The entry point of the second-generation nail was more medial to the greater tuberosity Fig. , which iDuring the fixation of PHFs with interlocking intramedullary nails, Kirschner wires are often used to hold the bone fragments in a limited incision for closed reduction –16. HereGardener first proposed medial support to avoid loss of the reduction . BesidesThere are several limitations to our study. Firstly, this is a retrospective, non-randomized study and all the surgeries were performed by the same surgical team. Secondly, the bone mineral density of every patient was not evaluated. However, the mean age of these two groups showed no statistical significance. Thirdly, long-term complications such as humeral head necrosis might have been overlooked since the mean postoperative follow-up was only 9 months .The interlocking intramedullary nail approach is less invasive compared to the use of locking plates but takes longer to achieve fracture reduction and repair. There was no significant difference in pain and shoulder function scores between the two internal fixation approaches for the treatment of proximal humerus fractures. Detailed preoperative evaluation and accurate intraoperative operation, combined with the clinical experience of orthopedic surgeons, can improve the postoperative satisfaction of patients by designing individualized surgical plans and enhancing postoperative functional exercise guidance."}
{"text": "The present work analyzes the influence of modified, epoxidized and maleinized corn oil as a plasticizing and/or compatibilizing agent in the PLA–PHB blend (75% PLA and 25% PHB wt.%). The chemical modification processes of corn oil were successfully carried out and different quantities were used, between 0 and 10% wt.%. The different blends obtained were characterized by thermal, mechanical, morphological, and disintegration tests under composting conditions. It was observed that to achieve the same plasticizing effect, less maleinized corn oil (MCO) is needed than epoxidized corn oil (ECO). Both oils improve the ductile properties of the PLA–PHB blend, such as elongation at break and impact absorb energy, however, the strength properties decrease. The ones that show the highest ductility values are those that contain 10% ECO and 5% MCO, improving the elongation of the break of the PLA–PHB blend by more than 400% and by more than 800% for the sample PLA. Nowadays, polymeric materials have replaced a significant part of the different materials employed in the engineering sector , as well2 emissions which directly increase global warming and destroy ecosystems [These resources, mostly of petrochemical origin, are obtained from non-renewable and limited sources which increase their depletion and the price of energy resources . Additioosystems . Anotherosystems ,12. The osystems . Therefore, one conceivable solution for this massive problem is the recycling and reuse of these materials with the aim of using less virgin material . This isOne of the most widely used biodegradable materials today is polylactic acid (PLA) ,17. PLA Due to the brittleness of PLA, it is often modified with other polymers: thanks to the compatibility of PLA, it can be blended with other polymers to improve its properties. In addition, such a combination can also have other benefits for both polymers, so it is interesting to make such modifications. Some of the petroleum-based materials with which PLA has good miscibility are polyethylene terephthalate (PET) ,24, polyBecause of the aforementioned problems, biopolymers for blending with PLA such as polyhydroxyalkanoates (PHAs) ,34,35, pPlasticizers, low-molecular-weight molecules that generally improve the ductility and processing of polymers, can be used to improve the miscibility between materials. There are two types of plasticizers, depending on their origin, namely petrochemical, such as tar pitch polymerization resins, etc. Some of these are used because they are environmentally friendly and non-toxic plasticizers, such as poly (ethylene glycol) (PEG) ,44,45,46Overall due to PLA’s compatibility, it can be blended with other polymers such as poly-B-hydroxybutyrate (PHB) ,59,60, aThus, the aim of the work was to improve the properties of PLA by blending it with PHB at 75–25%, respectively, to improve the miscibility and reduce the melting temperatures to promote its use in certain industries. For this purpose, different percentages of epoxidized corn oil and maleinized corn oil, 1, 2.5, 5, 7.5, and 10 wt%, were used as plasticizers. The processes of the maleinization and epoxidation of vegetable oils provide greater reactivity to the oil and greater thermal stability with respect to unmodified oils. Therefore, the compatibility of the oils with PLA is improved and the plasticizing effect is increased ,61. Thes−3. The PLA–PHB blend containing 25% PHB was supplied by Biomer  in pellet form with a density of 1.25 g cm−3 with commercial grade P226E. The vegetable oil used to carry out the epoxidation and maleinization process was food-grade corn vegetable oil (CO—corn oil).Poly (lactic acid), PLA commercial grade Biopolymer 2003D, was supplied by Nature Works LLC  in pellet form with a density of 1.24 g cmThe corn oil modification processes were carried out in a three-necked round bottom flask with a capacity of 500 mL in order to incorporate all the necessary elements. One of them was used to introduce the reagents, another one for temperature control and the central one to connect the reflux condenser.The epoxidation process started by adding 182.0 g of corn oil into the flask until 50–55 °C was reached with magnetic stirring. Then, 18.64 mL of acetic acid was added until the temperature was stable at 60 °C, then a mixture of 182.70 mL of hydrogen peroxide (3:1 M ratio (peroxide:oil)) with 1.46 mL of sulfuric acid was added dropwise, and the slow addition of this mixture was performed to avoid an exothermic reaction and with the heating mantle off. Once the addition of this mixture was finished and the reaction was controlled, the working temperature was increased between 80 and 85 °C for 8 h. Samples were collected at each hour of the epoxidation process according to ASTMD1652-97  and ISO c is the exact concentration of the KOH standard solution used (mol-L−1), V is the volume of the KOH standard solution used (mL), and m is the analyzed mass (g). A schematic representation of the maleinization process can be seen in The maleinization ratio used was 2.4:1, following the recommendations of previous works ,66. A toThe samples used in the different experimental techniques and tests were obtained by mixing processes in the laboratory. Before handling the PLA and PHB, both materials were dried at 60 °C for 24 h to eliminate the percentage of moisture indicated by the supplier.First, different mixtures were made with 75% PLA, 25% PHB and different percentages of epoxidized and maleinized corn oil, as shown in To achieve homogeneity in the mixtures, an extrusion process was carried out. The temperature range program used for the different zones of the extruder was the following: 175–180–180–185 °C, from the hopper to the nozzle, respectively, at constant speed of 40 rpm. At the end of the extrusion process, the material was cut until it was reduced to pellet size.Subsequently, the material in the pellets form was injected to obtain standard specimens for subsequent characterization. The chosen temperature program was 175–180–185 °C, from the hopper to the nozzle, respectively. The temperatures used in the manipulation process were those recommended by the material supplier.−1, at room temperature. For the tensile test, the parameters defined in the ISO 527-1:2019 standard [The mechanical characterization was carried out with tensile, flexural, impact, and hardness tests. The tensile and flexural tests were performed on an ELIB 30 universal testing machine . A minimum of five different specimens were tested using a 5 KN load cell. The speed used in the tests was 5 mm minstandard  were folstandard  was usedTo determine the amount of energy absorbed by the material in the case of impact, the test was carried out with a 6 J Charpy pendulum . Five measurements have were taken, with bending type specimens and without notch. The parameters followed in the test are those defined in the ISO 179-1:2010 standard .Finally, the materials surface hardness was measured. Five measurements were taken at different points of the surface of several specimens. A Shore D scale hardness tester, JBA S.A. model 673-D , was used. The parameters followed in the test are those defined in the ISO 868:2003 standard .−1 until the penetrator penetrates 1 mm into the sample. For the HDT test, procedure B of the ISO 75-1:2013 standard [−1 until reaching 0.31 mm of deformation.To evaluate the effects of temperature on the mechanical properties of the materials, a study of softening temperature (VICAT) and heat deflection temperature (HDT) was carried out. Both tests were carried out with a Metrotec model . The parameters for the VICAT test were those of the B50 method, as defined in the ISO 306 standard . The stastandard  was used−1 in a nitrogen atmosphere with a nitrogen flux of 66 mL min−1. With this technique, the glass transition temperature (Tg), cold crystallization peak (Tcc), and the melt peak temperature (Tm) of the PLA blends formulations were identified. The degree of crystallization (X%) was calculated with the formula by Equation (2).−1) [The materials’ miscibility was evaluated through the analysis of their thermal properties, using the differential scanning calorimetry technique (DSC). The thermoanalytical tests were carried out in a Mettler Toledo 882e . The weight range of the samples was 5–10 mg, with an initial heating program from −50 to 180 °C to remove the thermal history, followed by a cooling program from 180 to −50 °C, and finally a second heating program from −50 to 220 °C, at a heating rate of 10 °C min3 J g−1) , and WPL−1, with a constant nitrogen flow of 30 mL min−1.Samples were tested by thermogravimetric analysis. The thermogravimetric analysis (TGA) was carried out in a TGA PT1000 equipment from Linseis Inc. . The analyzed samples, with an average weight between 15 and 20 mg, were subjected to a temperature program from 30 °C to 700 °C at a speed of 10 °C minThe morphology of the blends was analyzed with a ZEISS ULTRA microscope from Oxford Instruments  using a 1.5 KV accelerating voltage. The analyzed samples were the broken impact test specimens, which were sputtered with a thin layer of platinum, using a Leica Microsystems  EM MED0200 high vacuum sputter coater.D) was calculated in percentage with the following equation:im is the initial dry mass of the test material and rm is the dry mass of the residual test material recovered from the sieving.The study of the materials’ disintegration behavior, under composting conditions, was carried out according to the ISO 20200:2015 standard . The tesThe epoxidation of corn oil was performed with a peroxide-to-corn oil molar ratio of 3:1, according to previous experience in the epoxidation process of other vegetable oils . The epoAt the end of the process, at 8 h, an oxirane oxygen index of 5.82 was obtained. Comparing this result with the theoretical rate, obtained from the amount of fatty acids in the corn oil, a conversion of 85.9% was achieved. In addition, the conversion of double bonds represented by the iodine index rapidly decreased during the first 2 h from values close to 140 until the end at 1.5 after 8 h of treatment, which reflects a greater decrease in double bonds than that reflected by the oxygen oxirane. This is due to reactions parallel to the conversion of double bonds into epoxy groups . Analyzi−1 and at the end of the first hour at 180 °C, it is observed that it increases up to 24.4 mg KOH g−1, indicating that the maleinization reaction is occurring. After the second hour, at 200 °C, another large increase is observed, reaching an AV of 87.8 mg KOH g−1, and finally, after the last stage, at 220 °C, the AV reaches 109.2 mg KOH g−1. These results are in total agreement with values obtained by A. Perez-Nakai et al. with an AV of 105 for the maleinized hemp seed and 130 for the maleinized Brazil nut at the end of the epoxidation process [−1 [ process . In addicess [−1 .PLA is a brittle material, and with the intention of improving this characteristic, mixtures were made with PHB. Moreover, different percentages of MCO and ECO were used as plasticizers and compatibilizers. The tensile strength, elongation at break and impact absorbed energy values of the studied materials are represented in Both plasticizers achieved an increase in the ductility of the initial formulation, as can be seen in the values of the elongation at break, toughness modulus, and impact absorbed energy. In the case of the tensile strength, a variation was observed, with the increasing percentage of plasticizer in the formulation, decreasing tensile strength values were obtained. The values of Young’s modulus obtained a variation, although in both cases, with values above the initial formulation. Additionally, both plasticizers managed to reduce the maximum flexural strength of the initial formulation, in such a way that the higher the plasticizer percentage, the lower the flexural strength values. In the case of flexural modulus, both plasticizers increased these values, except for the formulation with 10 wt% of MCO.Regarding the thermo-mechanical properties, as shown in max), which corresponds to the peak of the first derivative curve, and the endset (T90%), which indicates the temperature at which a 90% weight loss occurs. Neat PLA possesses a thermal stability value with a T5% of 311.3 °C, a Tmax of 330.5 °C, and a T90% of 342.6 °C. However, by adding PHB to PLA, the Tmax value increases considerably (by approximately 20 °C), whilst the addition of certain amounts of ECO or MCO to blend (B) formulations causes a slight decrease in the T5%, T90%, and Tmax values. In both modified oils, these values are lower when the quantity is larger. These values are lower for the formulations with MCO. For example, the addition of 10 phr of ECO (B-10ECO) results in a T5% reduction of 15.5 °C, while with 10 phr of MCO (B-10MCO), the reduction is 21.3 °C with respect to the B formulation value (286.1 °C). Regarding the Tmax temperature, it can be observed that both plasticizers lead to a decrease in temperature, obtaining the lowest values at approximately 20 °C and 26 °C lower for B-10ECO and B-10MCO, respectively. A similar tendency was observed by Garcia-Campo et al. [max by 20 °C. For the specific case of maleinized oils, the work of Perez-Nakai et al. [max by 10 °C with respect to the value of neat PLA.The thermal stability of the PLA, PLA–PHB blend, and plasticized formulations with different contents of ECO and MCO was assessed by thermogravimetry analysis (TGA). o et al.  for PLA/i et al.  shows hog) of 61.3 °C, a cold crystallization temperature (Tcc) of 121.9 °C, and a melting temperature (Tm) of 151.2 °C. Both plasticizers—ECO and MCO—decrease the Tg of PLA, which indicates an increase in the mobility of the polymer chains at lower temperatures, evidencing the plasticizing effect of both modified oils [g of up to almost 10 °C for the specific case of B-2.5MCO and in reference to the formulation B. The same plasticizing effect was observed by Dominguez-Candela et al. [Regarding to the DSC results, ied oils . Howevera et al. , who empcc peak is observed at higher temperatures with the incorporation of ECO or MCO. In fact, the Tcc peak temperature increases from 121.9 °C (neat PLA) to 127.1 °C for the B-7.5ECO formulation. This is due to the steric impediment exerted by the ECO on the PLA chains.With respect to crystallinity, it is observed that PLA without plasticizing has a crystallinity of 7.1%, whereas as PHB is added, the PLA is totally amorphous. The PHB molecular chains intercalated between the PLA chains do not allow the PLA chains to be ordered. The incorporation of ECO or MCO shows a slight increase, however, the values are negligible. The TIn this characterization technique, the breaking surface of the specimens was observed, in this case, as those obtained in the impact test. The PLA morphology, as shown in The morphology of the fracture surface of the blends containing MCO can be seen in As was verified by the mechanical properties of the different blends, the saturation of the oil negatively affects the ductile properties of the blends. In the PLA–PHB–ECO system, the sample with the best ductile behavior is the sample with 10 phr of ECO. However, in the PLA–PHB–MCO system, saturation occurs at lower amounts of oil, at approximately 5 phr of MCO, which has a similar morphology to sample B-10ECO. Therefore, to obtain the same plasticizing effect, it is achieved with less amount of MCO than ECO.Finally, to evaluate the disintegration behavior of the developed materials, they were buried in a composting reactor carried out at a laboratory scale. The variation of the mass loss of the different materials under composting conditions can be seen in This work analyzes the usefulness of the use of modified—epoxidized (ECO) and maleinized (MCO)— corn oil as plasticizing and/or compatibilizing agents of the blend 75 wt% PLA–25 wt% PHB. Both chemical modifications of corn oil were carried out at the laboratory level and both processes were carried out correctly according to the results obtained in the analysis of the number of oxirane oxygen and iodine number for the ECO and according to the evolution of the acid number value for the MCO.The incorporation of both modified oils, ECO and MCO, allows to obtain PLA–PHB formulations with better ductile properties, requiring less MCO to achieve the same plasticizing effect as with ECO. The plasticizing effect was observed by differential scanning calorimetry due to the decrease in the glass transition temperature (Tg), decreasing by approximately 2 °C when using ECO and between 4 and 8 °C when using MCO in the PLA–PHB blend. Additionally, the plasticizing effect is observed to a greater extent in the mechanical properties, since the elongation at break and the impact absorbed energy improve considerably when using the oils in the PLA–PHB formulations, increasing the elongation at break by more than 400% when using 10% ECO and 5% MCO. The FESEM analysis reveals a particular morphology of the samples plasticized with oil, presenting spherical cavities dispersed in the matrix and that increase with the increase in oil in the formulations. Finally, the oils delay the disintegration in composting conditions with respect to the PLA–PHB formulation, delaying said degradation to a greater extent than the ECO and it is also affected by the amount of oil used.Therefore, although for this specific study, the MCO shows better plasticization and compatibilization results, two new oils with greater commercial potential were obtained by chemical modification. Compared to other oils obtained from cereals or seeds, CO presents a higher abundance and greater stability against oxidation, which makes it a very interesting raw material from the industrial point of view for applications in both bio-based and synthetic polymers."}
{"text": "Valproic acid is an antiepileptic drug used in different fields of Psychiatry. It is known mostly for its use in managing patients with bipolar affective disorder. In psychiatry of addiction, there is still no approved indications for its usage, but it is widely prescribed in treating alcohol and cocaine abuse, due to the existence of studies in these addictions.This review aims to clarify the relation between valproic acid and dependences, particularly cocaine.Non-systematic literature review using a PubMed search, using the following key words: “valproate”; “cocaine use”.Cocaine dependence can decrease GABA levels in humans. Valproic acid has multiple mechanisms that favour the synthesis of GABA, potentiating its release and postsynaptic GABAergic response. Because of this, valproic acid was found effective in promoting abstinence and in reducing the use of cocaine. There are studies that support the valproic acid’s use in alcohol and cocaine dependences. Valproic acid has been shown to be promising in relapse prevention. It has also showed efficacy in the management of impulsivity and irritability, what makes it useful in managing patients with borderline personality disorder – patients at higher risk for alcohol or substance use disorders.Cocaine addiction involves different phenomena and may respond to distinct pharmacologic approaches. Although some studies need to be confirmed by larger clinical trials, valproic acid seems a promising agent as one of some potential treatments for cocaine dependence. Further studies are required in this field to come to more reliable conclusions.No significant relationships."}
{"text": "To determine the prevalence of mouth breathing children at the santo amaro project/ esef/ upe, and study their main facial and behavior alterations.transversal study.there were 150 children in the sample, with ages ranging from 8 to 10 years. Data was collected by means of a questionnaire and clinical examinations. As for their breathing assessment, two tests were carried out: test 1- breath steam against a mirror; and test 2 - water remains in the mouth with lips closed for 3 minutes.mouth breathing prevalence was of 53.3%. There was no significant difference between gender, age and type of breathing. Facial alterations were: incomplete lip closure , fallen eyes (40.0% X 1.4%), High palate (38.8% X 2.9%), Anterior open bite (60.0% Versus 30.0%), Hypotonic lips (3.8% X 0.0%), Circles under the eyes (97.5% Versus 77.1%).high mouth breathing prevalence without significant statistical difference between genders, age and type of mouth breathing. There was no association between behavior characteristics and type of breathing. There were significant differences between physical traits and breathing pattern. Breathing is one of the vital functions of the human bodyAccording to the literature, it is rare to have exclusive oral breathing; commonly, patients have a mixed respiratory pattern: partially oral and partially nasalFew are the papers related to the prevalence of oral breathing in the literature, and they present percentages that vary from 5%Depending on how long it lasts, oral breathing may cause functional, structural, pathological, postural, occlusal and behavioral alterationsAs physical consequences, the oral breathing child has many physical traits: long face, dropped eyes, dark spots underneath the eyes, open lips, hypotonic and dry lips, narrow nostrils, hypotonic cheek muscles, high palate, narrowing of the upper arch, and occlusal relation tending to Angle's Class IIAs to behavioral alterations, we stress: restless sleep, irritability, difficulty concentration followed by a reduction in school performance and impaired sports skills, amongst othersMiddle and long run alterations, accruing from these alterations, may have harmful consequences for the individual's quality of life due to its personal, physical, psychological and social impactsKnowing the importance of epidemiological research today, working to reduce populational health problems, this paper aims at contributing to the study of oral breathing, through research about its prevalence and main facial and behavioral alterations associated to this respiratory pattern in school children. The participants in this study are enrolled in the Projeto Santo Amaro da Escola Superior de Educação Física (ESEF).This study was carried out at the Escola Superior de Educação Física (ESEF) of the University of Pernambuco (UPE) from June to August, 2005 and is a transversal and observational type of study.Our sample included children regularly enrolled in the Santo Amaro Project. Their ages varied between 8 and 10 years, because dental and facial alterations in the oral breather are already present at this age rangeThe total sample comprised 236 children; throughout a sample calculation, the necessary sample size for this study was 147 children. Thus, the number of subjects per age range was of 50 children, adding up to a total of 150.The sample was picked by randomly selecting the students in the aforementioned age ranges. Sample inclusion criteria were: children between 8 and 10 years of age, from both genders. Sample exclusion criteria were: Children who refused to participate; Children who were not properly authorized by their parents/guardians to participate and they did not sign the Informed Consent form; children with severe respiratory disorders.In order to collect data in order to fulfill the objectives of the present study, we used an identification form for the children, a questionnaire to identify the behavioral alterations of the oral breather, and another form about facial alterations observed at visual inspection, and the results of the tests applied aiming at completing the diagnosis of oral breathing. Clinical exams were carried out by the investigators, using PPE  and the information gathered was recorded in the standardized forms.Test 1:the mirror was placed underneath the child's nostrils and we checked for steam build up in the mirror face (upper or lower) due to breathing. Steam on the upper face indicated nasal breathing and on the lower or lower/upper face indicated oral breathingTest 2:we asked the child to put some water in her/his mouth and kept the lips closed, without swallowing the water, for 3 minutes, and we observed through the lips commisure if there was any effort along the time. The children who were unable to keep their lips closed were considered oral breathersIn order to analyze facial alterations, we checked to see whether or not the children had the following clinical signs: elongated face, dropped eyes, dark circles underneath the eyes, thin upper lip, dry lips, hypotonic lips, everted lower lip, narrow nostrils, high palate, inadequate lip sealing and anterior open bite. In order to assess some of these criteria, we were careful enough as to observe the children in their natural environment, without letting them see that they were being observed. The children who participated in the study underwent the mirror test and mouth moisture test to aid the diagnosis as follows:In order to consider a child as an oral breather, she/he should have at least 3 facial alterations, or steam in the lower mirror face and/or in both mirror faces, or spend less than 3 minutes with water in their mouthsFor statistical analysis purposes, the children were assigned into two groups: nasal breathers and oral breathers, the latter also comprised those children with mixed breathing or exclusive oral breathers.Calibration was carried out in four phases, intra-examiner and inter-examiner, using pictures of the children.In order to carry out data analysis we obtained absolute and percentage distributions (Descriptive Statistics Method), and used the Pearson's Chi-Squared test (or Fisher's Exact test when there was no favorable condition to use the Chi-Squared test) and the equality in two proportions in independent group testing .The significance level used in the statistical test was of 5% (0.05) and the intervals were obtained considering a 95.0% confidence interval. Data were plotted in the Excel spreadsheet and the software used to obtain the statistical calculations was the SAS  version 8.Among types of breathing patterns, it is possible to see that the percentages of facial alterations were correspondently higher among children who were oral breathers when compared to the nasal breathers. It may be established that the percentage differences presented in a descending order were recorded for: inadequate lip sealing (58.8% versus 5.7%), dropped eyes (40.0% x 1.4%), high palate (38.8% x 2.9%), anterior open bite (60.0% versus 30.0%), hypotonic lips (23.8% x 0.0%) and dark circles underneath the eyes (97.5% versus 77.1%).Through the statistical analysis we see the significant difference between the two types of breathing, at the level of 5.0%, for all the items to which we could apply the comparative test (p < 0.05).Assessment of the breathing tests results: Glatzel plate and water in the mouthAs to the test about the time through which the child kept the water in the mouth and the lips sealed, we could see that most of them (86.7%) went up to 3 minutes, and this number was 25.0% higher among the nasal breathers investigated (100.0% versus 75.0%), and such difference reveals a significant relation between the two types of breathing, as far as test 2 is concerned (p < 0.05).In diagnosing a respiratory problem, it is of fundamental importance to obtain information from the parents/guardians during the medical interview. Therefore, questions about the child's sleep patterns, if he/she sleeps with the mouth opened, if there is noisy breathing, if the child lacks concentration at school, if the child feels sleepy during the day, if the pillow is wet in the morning; these questions should all be recorded, because they represent important elements in the diagnosis of oral breathingNotwithstanding, these data alone are not enough for an accurate diagnosis of those mouth breathing individuals, and that is why it is also important to carry out the Glatzel metal plate test and the time through which the child keeps water in her/his mouth with the lips sealed and without swallowing it, since we have seen that the results differ and complete each other.As to the prevalence of oral breathing, despite the few studies that have been carried out with this purpose, we have seen disagreements in the literature. In the present study we noticed that most of the children (53.3%) were considered oral breathers. The highest percentages of this alteration were seen in a study about oral suction habits in a low income population, which was of 77.78%These percentage differences may be justified by the diagnostic criteria and the different methodologies used in the studies. In this investigation, we did not record exclusive oral breathers, gathering both the exclusive oral breathers and the mixed breathing children in the same group, as advocated in the literature. However, some studies did not specify the criteria adopted in details.According to many authorsAs to gender, we noticed that despite a higher percentage seen in males (53.75%) when compared to females (46.25%), this difference was not statistically significant among oral breathers, and such data corroborate other studies in which there was a slight difference in the variable being analyzed8. On the other hand, other investigations reported that there is a slight predominance of this pathology in females when compared to malesAs to facial alterations that affect oral breathers, the highest percentages seen in the present study were: anterior open bite (60%), inadequate lip sealing (58.8%) and high palate (38.8%). These results are in agreement with those from a study in which the main craniofacial alterations seen in oral breathers were: open bite, high palate, malocclusionOther alterations such as dark spots underneath the eyes (97.5%) and dropped eyes (40%) which represented high percentages among the population studied were also mentioned by other authors as being facial alterations commonly found in patients with oral breathing syndromeAmong oral breathers and nasal breathers, respectively, the percentage differences seen in descending order were: inadequate lip sealing (58.8% versus 5.7%), dropped eyes (40.0% x 1.4%), high palate (38.8% x 2.9%), anterior open bite (60.0% versus 30.0%), hypotonic lips (23.8% x 0.0%) and dark spots underneath the eyes (97.5% versus 77.1%).In the long run, individuals who have trouble breathing may develop a number of disorders, such as: craniofacial alterations (long and narrow face), malocclusion, high palate, hypotonic lips and tongue, dry lips, sleepy face, deep dark spots underneath the eyes, greater likelihood of developing dental cavities, speech disorders, postural and gait alterations, all which interfere in school and work performance, and also in social relationshipsAnalyzing the data from the present study, it was also possible to see that of the facial alterations seen, percentages were correspondingly higher in oral breathing children than in nasal breathers, with a significant association, in agreement with the results found by aforementioned authors, despite the fact that these data support the statement that not all individuals with oral breathing patterns present these characteristicsNotwithstanding, today, oral breathing is one of the most concerning problems for public heathConsidering the high prevalence of oral breathing in the population studied, we submit that health policies should be implemented in order to improve the life quality of oral breathing children, and we stress the need for new studies with a larger number of children.1.The prevalence of oral breathing was high, without significant difference between genders.2.There were no relations between breathing pattern and behavioral alterations.3.Physical characteristics related to oral breathing were: elongated face, dropped eyes, dark spots underneath the eyes, narrow nostrils, inadequate lip sealing, dry and hypotonic lips, narrow upper lip (thin), anterior open bite and high palate."}
{"text": "Recent studies have introduced elevated lipoprotein(a) (Lp(a)) as a risk factor for coronary heart disease (CHD). This study investigated whether the addition of Lp(a) as a novel biomarker to the Framingham Risk Score (FRS) model improves CHD risk prediction.The study included 1101 Iranian subjects (443 non-diabetic and 658 diabetic patients) who were followed for 10 years (2003–2013). Lp(a) levels and CHD events were recorded for each participant.P value: 0.007).The Net Reclassification Index (NRI) after adding Lp(a) to the FRS model was 19.57% and the discrimination slope was improved (0.160 vs. 0.173). The Akaike Information Criterion (AIC), a measure of model complexity, decreased significantly after adding Lp(a) to the FRS model  to the FRS model improves CHD risk prediction in an Iranian population without making the model too complex. This could help clinicians to better identify individuals who are at risk of developing CHD and to implement appropriate preventive measures. Coronary heart disease (CHD) is the leading cause of death worldwide and a major risk factor for disability-adjusted life years, especially among young people , 2.Hypertension, dyslipidemia, tobacco smoking, diabetes, physical inactivity, and diet are known as some conventional risk factors for CHD . RecentlFramingham Risk Score (FRS) which is one of the scoring systems used for the evaluation and prediction of CHD in 10 years, categorizes patients as “high”, “intermediate,” and “low” risk. FRS variables include some conventional risk factors such as age, diabetes, smoking, hypertension, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) –8. DespiThere are already studies regarding the adaptation of the Framingham model for cardiovascular disorder in different populations, showing that the model can be adjusted slightly for different populations to better accommodate the differences , 12. AlsLp(a) is an LDL-like particle that contains a hydrophilic, highly glycosylated protein called apolipoprotein(a) bound to apolipoprotein B100 by a disulfide bridge. Lp(a) plays a significant role in atherosclerosis as an independent risk factor for cardiovascular diseases , 16.Recent studies suggested that Lp(a) levels were independently associated with an increased risk of coronary artery disease, even after accounting for traditional risk factors such as LDL-C levels. Incorporating Lp(a) measurement into risk stratification models significantly could improve risk prediction and potentially lead to identifying individuals who may benefit from more aggressive personalized preventive strategies , 18.A statistical measure used to evaluate the performance of predictive models is the area under the curve (AUC) with the range of 0.5 (no discrimination) to 1.0 (perfect discrimination) which represents the overall discriminatory ability of a model in distinguishing between individuals with and without the outcome of interest (CHD event in this study). The net reclassification index (NRI) and integrated discrimination index (IDI) are another statistical measure that assesses the improvement in risk prediction when adding a new marker to an existing model. NRI takes into account the correct reclassification of individuals into higher or lower-risk categories, as well as incorrect reclassification. It calculates the net proportion of individuals who are correctly reclassified into a higher risk category minus the net proportion of individuals who are incorrectly reclassified into a lower risk category with the range from − 2 to + 2. IDI quantifies the difference in average predicted risks between individuals with and without events and ranges from − 1 to + 1. Positive values indicate improved prediction with the addition of the new marker. NRI and IDI can be used alongside the AUC to further evaluate and compare different models in predicting CHD –21.This study aims to investigate whether adding Lp(a) to the FRS model improves its predictive value, as measured by NRI and IDI.In this retrospective cohort study, the target population was selected from the patients who were referred to the endocrinology and metabolism clinic of Vali Asr Hospital in Tehran, Iran, between 2003 and 2013. We included all the patients who underwent coronary angiography for highly suspected CHD. The patients with missing data on lipid profile and also the subjects with diabetes mellitus who were Insulin-dependent, type 1, secondary, or pancreatitis-related with a prior history of other endocrine diseases (except their diabetes status) were excluded. The diabetic patients included in the study were on oral antidiabetic agents.Informed consent was obtained from each patient and the study was reviewed and approved by the board of medical ethics at Tehran University of Medical Sciences with the code of IR.TUMS.REC.1394.412.The diabetes was diagnosed according to the American Diabetes Association criteria .Finally, 1101 patients were enrolled in this study (two sub-groups: 443 non-diabetic and 658 diabetic patients).Age, gender, weight (kg), height (cm), waist circumference (cm), systolic and diastolic blood pressure  (mmHg), and medical history of the patients were recorded.Waist circumference (WC) is defined as the midline between the lowest rib and the iliac crest.SBP and DBP were measured from both arms after 15 min of rest in the supine position and the mean value was recorded. Venous blood samples were collected after 12 h of overnight fasting.To assess fasting plasma glucose (FPG) and 2-hour post-prandial plasma glucose (2hPP), a glucose oxidase assay was used. Lipid profiles including total cholesterol, HDL-C, LDL-C, and triglyceride (TG) were measured using a direct enzymatic method .To measure fasting insulin, radioimmunoassay using separate specific antibodies was utilized . Serum creatinine was assessed by the Jaffe method. HbA1c was evaluated by high-performance liquid chromatography . Also, Lp(a) was measured by turbidometry method .2) was calculated as Weight/Height2 and Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) as [FPG (mg/dL) ×Fasting Insulin(U/L)] / 405. Metabolic syndrome was determined according to the nationally modified version of the International Diabetes Federation (Modified- IDF) criteria [Body Mass Index , improved AIC, BIC, and − 2 log-likelihood, and a small improvement in the AUC of the ROC curve. While the increase in AUC was not statistically significant (P = 0.200), it is important to note that it is a step towards improving the model. We expect that the addition of more novel biomarkers, one by one, will lead to a significant improvement in the model through these small increases for each biomarker. We also observed increases in discrimination slope and NRI. The details of the models are listed in Table We used two models to predict the risk of CHD in our subjects: the conventional FRS model and the FRS model with Lp(a) added. We found that the model with Lp(a) had a higher likelihood ratio ). Our study showed that adding Lp(a) to a model based on conventional risk factors improved NRI and IDI in the Iranian population. It also decreased the model complexity.Unexpectedly, patients with CHD had lower TG, LDL-C, and total cholesterol levels. This may be due to treatment effects, lifestyle modifications, or early detection and treatment of secondary causes of dyslipidemia, such as hypothyroidism, during CHD management.A previous study of 826 subjects aged 45 to 84 years in Bruneck, Italy, found that elevated Lp(a) predicts 15-year cardiovascular disease (CVD) outcomes, and that adding Lp(a) to the FRS model improves CVD risk prediction. The study showed that the NRI related to Lp(a) was 17.1% and 22.5% for patients with and without CVD, respectively, and 39.6% overall .Another study on 8720 Danish participants over 17 years which was in line with our study showed that the addition of extreme levels of Lp(a) to the baseline model improved CHD event classification. In other words, after adding Lp(a) to the conventional model at the levels of ≥ 80th (47 mg/dl) and ≥ 95th (115 mg/dl) percentiles, 23% and 39% of patients with myocardial infarction (MI) and 12% and 25% of patients with CHD were correctly reclassified. For these two cut points, the NRI was 16% and 23% for MI and 3% and 6% for CHD, respectively. However, Lp(a) addition over the entire concentration did not significantly change the NRI. Discrimination was improved by IDIs for MI and CHD in extreme levels of Lp(a), while C-index changes remained insignificant .A study that assessed three cohorts of women including the Women’s Health Study , Women’s Health Initiative Observational Study , and the Justification for Use of Statins in Prevention (JUPITER) trial  found that increased Lp(a) is associated with higher CVD risk only among patients with high total cholesterol (above 220 mg/dL). According to the mentioned study, prediction improvement among women also was minimal. In contrast, JUPITER showed a strong association of Lp(a) with CVD among men with low total cholesterol levels .A recent retrospective study that enrolled 1395 patients, found better capability of an existing model for predicting coronary artery disease after adding Lp(a) compared to other lipid parameters. The new model which incorporated Lp(a) achieved an NRI of 12.8% and significant improvement in accuracy, measured by IDI .One of the strengths of our study is that it was conducted in a Middle Eastern population and there are not many studies in this population on this issue. However, there are certain limitations. The study was done on retrospective data. Most of the patients with CHD were on Statin and/or other anti-lipid agents which may affect the level of Lp(a) and other lipid parameters. We only included one novel biomarker in this study. The study lacked data on the details of the major adverse cardiac events. For future studies, we will also continue enrolling patients to assess the continued effects of Lp(a) and other novel biomarkers, in a cohort with an increased sample size and increased number of events, to further improve the statistical power of our study as well.This study showed that adding Lp(a) to the FRS model compared to the conventional FRS is superior in risk stratification, discrimination, and net reclassification in a sample of the Iranian population. For future studies, we’d recommend prospective designs with a larger sample size including more novel biomarkers, in hopes of better establishing predictive models for our population."}
{"text": "E-cigarettes pose significant risks to youth, but smokers may benefit from switching to e-cigarettes by reducing their exposure to toxicants, which creates a challenge for the Food and Drug Administration (FDA) in regulating e-cigarettes to protect population health. This study aims to develop e-liquid product standards for nicotine form and concentration that reduce the appeal of e-cigarettes to young people while keeping e-cigarettes available as a safer alternative for smokers.A single-visit, double-blinded, randomized crossover design will be used to examine the effects of e-liquids with varying fractions of free-base nicotine  among a sample of 66 young adult EC users and 66 older adult smokers, across ecologically valid total nicotine concentrations (20 mg or 50 mg/mL).A 2-puff session will be conducted to test each of the 10 e-liquids in randomly assigned sequences, followed by a 10-minute washout period and participant ratings on appeal and sensory attributes such as throat hit and harshness, as well as behavioral intentions for continued use. Generalized linear mixed models will be used to determine a free-base nicotine level that has limited or no appeal to young adult e-cigarette users while remaining acceptable to smokers.This study will provide the FDA with scientific evidence regarding the effect of product standards that mandate a minimum threshold for the fraction of free-base nicotine.NCT05864586.The study is registered on clinicaltrials.gov under the identifier  Viewing ECs through this lens, the FDA needs robust evidence demonstrating how to regulate ECs based on these two competing goals that must be aligned: 1) allowing ECs to remain on the market as appealing, potentially less harmful substitutes for current smokers, and 2) reducing their appeal and addictiveness for young people.Regulating e-cigarettes (ECs) is challenging. While ECs pose significant risks to young people by increasing the risk of nicotine initiation and sustained nicotine addiction  and use The US Congress and FDA have already implemented several regulatory efforts to curb youth EC use, including: increasing the federal minimum age requirements to purchase tobacco products (including ECs) to 21 years ; banningTo protect young people and still offer smokers a potentially less harmful alternative, the FDA could establish an e-liquid nicotine product standard that sets a minimum level of FB nicotine content that is unappealing/minimally appealing to young people and non-users but still satisfies nicotine craving in adult smokers. Recent evidence suggests that young exclusive EC users prefer “smoother” e-liquids with very low levels of FB nicotine, but smokers and dual users prefer some level of throat irritation , or whatTherefore, the purpose of the present study is to determine this minimum FB nicotine level  across two ecologically valid nicotine concentrations (20 mg/mL and 50 mg/mL) among a sample of young adult EC users with minimal/no history of smoking as well as older adult smokers. We hypothesize that with increasing FB levels , ECs will be too harsh for exclusive young adult EC users but not for adult smokers, especially at the higher nicotine concentration.Using a single-visit, double-blind, randomized crossover trial design the study will examine the effects of e-liquids with varying levels of FB nicotine  at two nicotine concentrations (20 or 50 mg/mL) on appeal among young adult EC users with little or no history of smoking (n = 66) and older adult smokers (n = 66). The schedule of enrollment, interventions, and assessments is shown in Study visits will take place in the Ohio State University Comprehensive Cancer Center (OSUCCC)’s Center for Tobacco Research (CTR) in Columbus, Ohio. All study visits will be conducted in smoking/vaping rooms under negative pressure.The study team will prepare 10 tobacco-flavored e-liquids, which vary in FB nicotine  and concentration (20 or 50 mg/mL) and contain commercially available tobacco flavorings, benzoic acid, glycerol (vegetable derived), and propylene glycol see . The FB The study is planned to enroll participants from June 1, 2023 to December 31, 2023. This is a one-time visit study, and there will be no follow-up required. A sample of healthy, young adults who exclusively use ECs and have no or minimal smoking history (n = 66) and older adults (25–50 years) who smoke (n = 66) will be recruited for the study. Participants will be recruited from the general public, using targeted online and social media advertisements, flyers, and word-of-mouth marketing in the Columbus metropolitan area. Various online and social media platforms will be used to distribute advertisements. Participants will be screened initially through an online screener linked to our study advertisement, then over the telephone. Those meeting the following eligibility criteria will be asked to participate and will be compensated for their time ($125/visit). Inclusion criteria: 1) current exclusive young adult EC user (daily use) for at least the past 3 months (confirmed by urinary cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL]) between 21–24 years old inclusive, with no or minimal history of smoking cigarettes (<10 cigarettes in lifetime), or current older adult smoker (daily use and ≥100 cigarettes in lifetime) between 25–50 years old inclusive, with interest in trying an EC; 2) willing to abstain from all nicotine product use for 12 hours prior to the study visits; and 3) read and speak English. Exclusion criteria: 1) currently attempting to quit nicotine or tobacco products; 2) currently pregnant (will be verified with a urine pregnancy test), planning to become pregnant, or breastfeeding; 3) current or past use of tobacco products other than ECs or cigarettes ; 4) self-reported diagnosis of lung disease including asthma, cystic fibrosis, or chronic obstructive pulmonary disease; 5) self-reported new or unstable cardiovascular disease diagnosed within the past 12 months.Each participant will complete a single, solo laboratory visit for a maximum of 3 hours see . ParticiAfter participants complete questionnaires on sociodemographics, tobacco use history, and EC and cigarette dependence, 10 vaping sessions will take place. In each vaping session, participants will inhale two puffs from the study EC device, loaded with one of the 10 research conditions (5 freebase nicotine fractions x 2 nicotine concentrations). Participants will be told to inhale for at least 1 second per puff, while other puffing parameters  will not be controlled. Pre-filled and weighed e-liquid pods will be provided to participants in random order. The assigned randomization sequences of the study e-liquids will be generated by a statistician using SAS 9.4. Permuted block randomization, stratified by use (young adult EC users vs older adult smokers), will be employed to ensure that each research condition is sampled an equal number of times in each position. Study staff enrolling participants and collecting data will remain blinded to each participant’s assigned study e-liquids throughout the course of the study.There will be a 10-minute washout period between conditions to minimize carryover effects, where participants will be instructed to rinse their mouth and spit at least three times with room-temperature water . The wasBehavioral Intentions for continued use will be assessed by asking participants how likely they are to: 1) try the product again, 2) purchase the product for regular use, and 3) use the product regularly if this were the only e-cigarette available. Response options will range from 1 “extremely unlikely” to 7 “extremely likely” [The primary outcome measures are behavioral intentions for continued use, the intensity of sensory attributes, and the degree of liking or disliking of each e-liquid.  likely” . The int likely” , 29. Par likely”  with a vThe secondary outcome measures include the subjective appeal and overall acceptance of each e-liquid. Subjective appeal will be assessed using the Adapted Drug Effects/Liking Questionnaire  and the Statistical analysis will be conducted using SAS 9.4 . Participant demographic and other characteristics will be summarized by the study arm. Continuous variables will be reported as mean ± standard deviation and compared using t-tests, whilst categorical variables will be reported as counts and proportions and compared using chi-squared tests. Transformations will be applied to normalize the distribution and stabilize the residual variance where appropriate.To determine a nicotine fraction limit that is satisfying to smokers while minimally appealing to young adult EC users, generalized linear mixed models will be used to evaluate appeal , which include demographic and smoking history as covariates, random subject effects, main effects for usage group (smokers versus EC users), FB fraction  and concentration , and interactions between usage group and each of the condition parameter variables . A significant interaction between the usage group and a condition parameter variable would suggest that the variable influences the two user groups differently with respect to their appeal. Interactions between condition parameter variables will also be explored. Various link functions and distributions will be employed to complement the data structures for each of the outcomes, including ordinal logistic regression.Given our goal to find combinations of the condition parameter that are sufficiently satisfying for smokers but do not appeal to young adult EC users, condition combinations will also be directly compared between study arms to identify the condition parameter combinations that result in the greatest difference in appeal between the two user groups. These comparisons will be made using t-tests or Wilcoxon rank-sum tests as appropriate for the structure of the data. Lastly, carryover effects will be explored by estimating the interaction between a given condition and its preceding treatment, and period effects will be examined by testing the interaction between treatment and order. A significance level of p = 0.01 will be used, given the various measurements tested.A total of 132 participants will be recruited, including young adult e-cigarette (EC) users with minimal or no history of smoking (n = 66) and older adult smokers (n = 66). Conservatively, 10% of 132 participants are assumed to need to be replaced post hoc with another participant due to failing to abstain from nicotine use prior to the study visit. With 120 participants, a correlation of 0.5, and an alpha of 0.01, a two-sided paired t-test has 80% power to detect an effect size of 0.32 standard deviations of the measurement for the concentration main effect. For the FB fraction main effect, the model has 80% power to identify a slope of 0.31 with 120 participants with an alpha value of 0.01. The effect sizes are conservative, given that a previous study , which iStudy data will be collected, managed, and secured using REDCap, an electronic data collection tool hosted at the OSUCCC, which will be accessible to authorized study personnel who have the necessary password(s). Each participant will be granted a unique identifying number for the study (study ID) and only the research ID will be used to identify all study data. Tablets provided by OSU will be used to enter electronic data , which will be collected directly from participants. All computer systems will be password-protected to prevent unauthorized access, and all network-based inter-site communications of confidential data will be encrypted. Access to information stored on a computer will require concurrent knowledge of the data format, computer language, file name, and passwords. Participant information will be accessible only to research staff who are pledged to confidentiality and have received training in the ethical conduct of research . Physical materials will be stored in a locked file cabinet, office, or suite at the CTR.Study visits will take place in a secure laboratory setting at the CTR where access is limited to study personnel, and researchers will work with only one participant at a time to protect participants’ privacy. Since participants may perceive questions on EC use as intrusive, participants will be informed of the nature of questions in advance.Since current EC users and adult smokers will be recruited, the participants are already smoking or using ECs and will only be asked to use a fully characterized EC device and e-liquids for which an FDA ITP was submitted and will be \"approved\" . The risk of side effects and adverse events associated with the use of ECs is quite low. ECs are commercially available in the US without a prescription. Lab studies of toxicant exposure suggest that ECs incur no greater risk to health than combustible cigarettes. In general, ECs have lower levels of harmful and potentially harmful constituents than cigarettes but are not 100% safe . To dateDuring the study period, all participants will be screened for general medical precautions  and monitored for adverse events. Any serious adverse events will be reported to the study MPI and subsequently to the Ohio State University Institutional Review Board (IRB). Participants who experience a serious adverse event or have a cardiovascular or pulmonary event will be withdrawn from participation. Blood will be drawn by a trained nurse; only sterile tools will be used, and the participant’s skin will be cleansed with an alcohol wipe at the location of the needle stick. Following the completion of the study, we will encourage participants to quit smoking and/or stop using ECs. Participants will be provided with a post-study information sheet, which includes information about the Ohio Tobacco Quit Line service that offers nicotine replacement therapy resources.This study has been registered with clinicaltrials.gov under the identifier NCT05864586. This study was reviewed and approved by the Ohio State University Cancer IRB under Study Number 2022C0207 on 4/10/2023. Oral and written explanations of the study and REDCap will be utilized for receiving informed consent and a legally valid signature. During the consent procedure, the voluntary nature of the study and the participant’s right to withdraw at any time will be emphasized, and a written copy of the informed consent will be given to the participant at the moment of consent for them to keep. Personnel approved by the IRB will collect informed consent. Any protocol revisions will be reported to the OSU Cancer IRB and ClinicalTrials.gov and will be noted in manuscripts.The study is planned to enroll participants from May 2023 to December 2023.There is an urgent public health need to prevent young people from initiating EC use. However, the potential of ECs to help current smokers quit smoking makes their regulation challenging. While regulatory efforts have been implemented to curb youth use, including banning the sale of flavored cartridge-based ECs, there is a need to regulate nicotine dimensions that make youth users dependent on ECs. This study will help the FDA establish e-liquid nicotine product standards that minimize the appeal of ECs to youth while satisfying adult smokers’ nicotine craving by examining the impact of nicotine form and concentration on sensory perceptions and EC liking.There are limitations of the study to be acknowledged. First, there is a risk of carryover effect because participants will test 10 e-liquids in a single laboratory visit although a 10-minute washout period will follow sampling each e-liquid to minimize the potential carryover effects. Carryover effects will be explored by estimating the interaction of a given condition with the preceding condition. Also, the 10 test liquids will all be tobacco-flavored, thus flavor effects cannot be assessed. This design was chosen to minimize participant burden and based on the fact that the impact of nicotine concentration on sensory experiences was consistent across different flavors . Still, Strengths of the study include the use of standardized scales to assess the sensory effects of nicotine , 30, theS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file."}
{"text": "The Oxford Elbow Score (OES) and the short version of Disabilities of Arms, Shoulder and Hand (QuickDASH) are common patient-reported outcomes for people with elbow problems. Our primary objective was to define thresholds for the Minimal Important Difference (MID) and Patient-Acceptable Symptom State (PASS) for the OES and QuickDASH. The secondary aim was to compare the longitudinal validity of these outcome measures.We recruited 97 patients with clinically-diagnosed tennis elbow for a prospective observational cohort study in a pragmatic clinical setting. Fifty-five participants received no specific intervention, 14 underwent surgery (11 as primary treatment and 4 during follow-up), and 28 received either botulinum toxin injection or platelet rich plasma injection. We collected OES  and QuickDASH , and global rating of change  at six weeks, three months, six months and 12 months. We defined MID and PASS values using three approaches. To assess the longitudinal validity of the measures, we calculated the Spearman’s correlation coefficient between the change in the outcome scores and external transition anchor question, and the Area Under the Curve (AUC) from a receiver operating characteristics (ROC) analysis. To assess signal-to-noise ratio, we calculated standardized response means.Depending on the method, MID values ranged from 16 to 21 for OES Pain; 10 to 17 for OES Function; 14 to 28 for OES Social-psychological; 14 to 20 for OES Total score, and − 7 to -9 for QuickDASH. Patient-Acceptable Symptom State (PASS) cut offs were 74 to 84 for OES Pain; 88 to 91 for OES Function; 75 to 78 with OES Social-psychological; 80 to 81 with OES Total score and 19 to 23 with Quick-DASH. OES had stronger correlations with the anchor items, and AUC values suggested superior discrimination (between improved and not improved) compared with QuickDASH. OES also had superior signal-to-noise ratio compared with QuickDASH.The study provides MID and PASS values for OES and QuickDASH. Due to better longitudinal validity, OES may be a better choice for clinical trials.ClinicalTrials.gov NCT02425982 .The online version contains supplementary material available at 10.1186/s12874-023-01934-4. Elbow-specific Oxford Elbow Score (OES), and the short version of the upper limb-specific Disabilities of Arms, Shoulder and Hand (QuickDASH) both use a question-and-response format to convert a complex multidimensional health state into a single metric scale , 2. Whilthe smallest difference in score in the outcome of interest that informed patients or informed proxies perceive as important, either beneficial or harmful, and that would lead the patient or clinician to consider a change in the management”  – [mean change of ‘unchanged’]) (3) ROC curve method (closest point to the upper left corner) dichotomizing between ‘no change’ and ‘little better’ and excluding participants with responses ‘little worse’ or ‘much worse’ [We determined the MID using three different anchor-based methods: h worse’ .For PASS, we used 75th percentile method and ROC method from the 6-month and 12-month time points . In the To assess the signal-to-noise ratio, we calculated standardized response mean (SRM) for both outcomes as mean score change (between baseline and 1 year follow-up) divided by the SD of the change. SRM is the ratio of signal to noise. A SRM of 0.2 can be considered to indicate low responsiveness; 0.5 moderate and 0.8 as large responsiveness .We recruited 97 participants with clinically diagnosed tennis elbow Table . At baseMost n = 55; 61%) participants did not receive any specific intervention initially; four participants received surgery during follow-up. Smoking was fairly common (27%) among the participants, and most (71%) had received at least one corticosteroid injection before enrolment (range 1 to 6 injections) . Both QuickDASH and OES captured the perceived change adequately, but OES change correlated more strongly with the transition anchor compared with QuickDASH change , and accordingly, OES discriminated better Table . CorrelaFor both OES and QuickDASH change scores, the correlations with the transition anchor (GRC) increased at longer follow-up. After three months, the change scores correlated more strongly with the current state than with change score . The change scores correlated with the baseline score only for OES at 24 months .MID = minimal clinically important difference, ROC = receiver operating characteristics, OES = The Oxford Elbow Score, DASH = Disabilities of Arms, Shoulder and Hand, CI = Confidence interval.MID and PASS estimates varied depending on the calculation method Tables  and 5. TIn people with tennis elbow, the elbow-specific OES better captured the change in the perceived health than did the upper extremity-specific QuickDASH. The OES better discriminated people who improved from people who reported no change, and the MID and PASS values are therefore more credible for OES than QuickDASH. OES also had superior signal-to-noise ratio, indicating that smaller sample sizes may be used in clinical trials to identify or exclude clinically-relevant differences when OES is used compared to QuickDASH.The MID reflects the difference in score that informed patients perceive as important , and that would lead the patient or clinician to consider a change in management. The methods impacted the MID values adding a layer of uncertainty to the interpretation of the results. If we determine the MID using the mean change in people reporting being ‘little better’, approximately half of the participants who reported feeling better will be classified as ‘not improved’— a relatively high rate of misclassification. ROC analysis optimizes the cut off, therefore MID from ROC analysis is more valid for discrimination purposes. Due to the impact of outliers, the ROC method is also more robust to skewed distributions compared to the mean change method or the mean difference of change method. However, ROC analysis is affected by the prevalence of people who report improvement. With larger prevalence, the MID values tend to be larger . There iWe propose that differences that are smaller than the lowest values from our study  are judged as not meaningful in people with tennis elbow. For example, authors of a trial or meta-analysis should conclude that the intervention does not provide clinically-important benefits only if the treatment effect confidence intervals are below the smallest provided values. Conversely, higher differences than the highest values  should be considered indicating relevant benefits. When the difference is within the range of possible MID or PASS values, some uncertainty regarding the conclusions is appropriate.Our MID estimates correspond reasonably well with the previous studies. The developers of the OES found MID values of 9, 18, and 18 points with pain, function, and social-psychological scale respectively in a heterogenous population undergoing surgery for elbow complaints. One in ten patients in their sample received surgery for tennis elbow . AnotherPatient accepted symptom state is a cut off for PROM beyond which people consider being well . In our Recall bias affected the patient global improvement assessment beyond three months. When participants were asked to report whether they were better compared to baseline, their responses were more closely associated with their current status than how their condition had changed. This is typical with musculoskeletal conditions . CliniciThis study determined credible MID and PASS values that can be used to interpret trial results. Comparison of longitudinal validity of OES and QuickDASH in tennis elbow patients suggests that although the performance of QuickDASH is acceptable, OES has better signal-to-noise ratio and better longitudinal validity. The OES may be better option as an outcome measure in clinical trials.Below is the link to the electronic supplementary material.Supplementary Material 1"}
{"text": "Bariatric surgery has emerged as a highly effective option for individuals with obesity, offering significant and sustainable weight loss outcomes. This surgical approach involves various procedures that alter the anatomy of the gastrointestinal tract, leading to reduced food intake and nutrient absorption. Established procedures such as sleeve gastrectomy, gastric bypass, adjustable gastric banding, and biliopancreatic diversion with duodenal switch have proven track records. In contrast, emerging options like intragastric balloons, AspireAssist devices, and endoscopic sleeve gastroplasty show promise but require further investigation. Numerous studies have highlighted the remarkable benefits of bariatric surgery, not only in weight loss but also in the resolution of obesity-related comorbidities and significant improvements in quality of life. However, successful outcomes rely on a multidisciplinary approach, encompassing preoperative evaluation, patient selection, comprehensive postoperative care, nutritional support, and psychological counseling. Regular follow-up and adherence to postsurgical recommendations are crucial for sustained weight loss and positive long-term results. As bariatric surgery continues to evolve, tailored procedures based on individual needs and ongoing research hold the potential for even more refined and effective approaches. Through this ongoing advancement, bariatric surgery is poised to offer improved patient outcomes, transforming lives for those grappling with the challenges of obesity. HighlightsBariatric surgery has proven to be highly effective in providing substantial and sustainable weight loss outcomes for individuals struggling with obesity.Bariatric surgery not only aids in weight loss but also resolves obesity-related comorbidities and significantly improves patients’ quality of life. Numerous studies have highlighted the remarkable benefits of improving overall health and well-being.Successful outcomes in bariatric surgery depend on a comprehensive multidisciplinary approach. This involves preoperative evaluation, patient selection, comprehensive postoperative care, nutritional support, and psychological counseling. Regular follow-up and adherence to postsurgical recommendations are crucial for sustaining weight loss and achieving positive long-term results.1. These conditions are characterized by abnormal or excessive fat accumulation, which can adversely affect an individual’s overall health and quality of life1. To classify overweight and obesity, the Body Mass Index (BMI) has emerged as a widely used tool, calculated by dividing an individual’s weight in kilograms by the square of their height in meters (kg/m2). In adults, a BMI falling within the range of 18.5–24.9 kg/m2 is considered normal, while a BMI equal to or greater than 25 kg/m2 is categorized as overweight, and a BMI of 30 kg/m2 or higher indicates obesity2. For children and adolescents aged 2–18, overweight is defined as a BMI between the 85th and 94th percentiles3. At the same time, a BMI represents obesity at or above the 95th percentile for the individual’s sex and age3. Over the past four decades, the prevalence of obesity has witnessed a worrisome escalation, affecting both the young and the adult populations alike4. Childhood obesity rates have surged from less than 1% to 6–8%, while adult obesity has risen from 3% to 11% among males and 6% to 15% among females4. Currently, an estimated 1.5 billion adults worldwide grapple with the burdens of being overweight or obese, and this number is projected to soar to 3 billion by 2030, underscoring the urgent need for effective interventions2.The prevalence of overweight and obesity has reached alarming proportions globally, posing significant global challenges to global health systems2. In addition to the immediate health impacts, children with obesity face a heightened risk of long-term adverse outcomes, including premature mortality, disability, respiratory complications, fractures, and early-onset chronic medical conditions like hypertension, cardiovascular disease, insulin resistance, and psychological disorders2. The cumulative impact of obesity has spurred considerable concern among healthcare professionals and policymakers, necessitating comprehensive and innovative strategies to curb this growing epidemic3.The health consequences of obesity are profound and far-reaching, encompassing an increased risk of cardiovascular diseases , type 2 diabetes mellitus, musculoskeletal disorders, and certain cancers5. While previous reviews have explored the efficacy of bariatric surgery in addressing obesity, this study seeks to provide an up-to-date and comprehensive analysis of the latest advancements in this field. By consolidating evidence from recent research, we aim to identify novel approaches and emerging techniques that promise to optimize surgical outcomes and improve long-term patient well-being.The escalating prevalence of overweight and obesity has become a pressing global health challenge, demanding effective interventions to address its significant impact on public health. Bariatric surgery has emerged as a promising therapeutic avenue for severely obese individuals, offering substantial weight loss and improvements in associated comorbiditiesWe conducted a comprehensive narrative review to explore recent advances in bariatric surgery as a weight loss procedure. Our primary objectives were to identify and analyze the latest developments in bariatric surgery techniques, evaluate their efficacy, safety, and impact on patient outcomes and quality of life, and propose potential areas of future research to enhance the field.We conducted an exhaustive literature search to identify relevant studies and publications using databases such as PubMed, MEDLINE, Scopus, Web of Science, and Google Scholar. We used combinations of keywords and Medical Subject Headings (MeSH) terms related to bariatric surgery, obesity, weight loss, surgical techniques, patient outcomes, and quality of life.They should be published in peer-reviewed journals and written in English.The studies should focus on recent advances in bariatric surgery, that is, studies published within the last 10 years.Studies reporting original research, clinical trials, systematic reviews, and meta-analyses.Studies providing data on patient outcomes, safety, or quality of life after bariatric surgery were included.For inclusion in the review, articles must meet the following criteria:Were published more than 10 years ago.Are written in languages other than English.Primarily focus on nonsurgical weight loss interventions or non-bariatric surgical procedures.Comprise case reports or letters to the editor without original data.On the other hand, studies were excluded if they:Two independent reviewers extracted data using a predefined data extraction form. Findings from the selected studies were synthesized and presented descriptively. Our review emphasized identifying common trends, gaps in the literature, and areas of innovation in bariatric surgery.7. It involves vascular attachment division, gastric fundus detachment, posterior gastric artery ligation, and stapling near the pylorus, followed by leak testing and omental suturing for leakage prevention9. LSG induces weight loss by reducing gastric volume, altering hormonal levels (particularly ghrelin and peptide YY), and changing gastric motility11. Studies by Durmush et al.12 reported percentage of excess weight loss (%EWL) of 67.1% at 6 months, 81.2% at 1 year, and 83.8% at 2 years, with improved comorbidities. Radu Neagoe et al. reported %EWL of 41.8% at 3 months and 64.1% at 6 months, with favorable outcomes for type 2 diabetes mellitus (T2DM) and hypertension13. Complications include bleeding, leakage, abscess formation, and late complications like gastric stenosis, nutrient deficiencies, pouch migration, and exacerbated gastroesophageal reflux disease (GERD)14.Laparoscopic Sleeve Gastrectomy (LSG) is a prominent bariatric procedure that reshapes the stomach into a narrow, tubular structure using five or six upper abdomen ports16. Roux-en-Y gastric bypass (RYGB) results in significant body weight loss due to reduced eating, increased energy expenditure, nutrient limitations, and potentially altered metabolic efficiency17. Weight loss averages around 77% of excess body weight at 1 year, with high-resolution rates for preoperative comorbidities, especially diabetes mellitus17. However, long-term weight loss varies between 34% and 80.2% %EWL 10 years post-surgery, and complications include wound infections, respiratory issues, hemorrhage, leakage, obstruction, hernia, gallstones, ulcers, and more19.Gastric Bypass (Roux-en-Y) is a multistep surgical procedure involving balloon sizing of the upper stomach, tract creation for lesser curvature dissection using hook cautery, and stomach division with an endoscopic stapler. It includes the use of a circular stapler anvil, enteroenterostomy with linear staplers, and retrocolic passage of the proximal Roux limb. A Penrose drain is used for this passage, and air leakage is checked during distension20. The adjustable gastric band induces weight loss by promoting early satiety and reducing appetite, impacting esophageal and proximal gastric functions through the vagus nerve21. Postoperative %EWL varies between 25.3 and 71.7%, with mean %EWL of 30% at 6 months, 40% at 12 months, 50% at 24 months, and 50–60% at 48 months in one study22. Long-term follow-up shows a mean BMI below 30 kg/m2 in patients followed for over 5 years, compared to a mean preoperative BMI of 42 kg/m2. Complications include gastric perforation, bleeding, port infection, conversion to open surgery, and death as early complications, and late complications such as band erosion, food intolerance, and access port problems22.In the Adjustable Gastric Banding (Lap-Band) procedure, a Nathanson liver retractor is inserted through a 5-mm incision below the xiphoid to retract the left hepatic lobe. Two atraumatic graspers are used to manipulate the stomach and create a passage for the band around the stomach, with gastric-to-gastric sutures to align it above and below the band11. Initially, a SG is performed, followed by transection of the small bowel 250 cm proximal to the ileocecal valve. The alimentary limb is anastomosed to the transected portion of the duodenum to form the duodenoileal anastomosis, and an ileoileal anastomosis is created 100 cm proximal to the ileocecal valve. The procedure concludes with suturing the surgical wounds23. Biliopancreatic diversion results in early weight loss due to SG and long-term weight loss from fat malabsorption. Hormonal changes, including reduced ghrelin and increased peptide YY levels, lead to early satiety. Gastrectomy affects ghrelin changes, while rapid nutrient entry into the ileum and jejunum after the distal bypass increases peptide YY levels11. Despite being less common than other bariatric procedures, biliopancreatic diversion achieves impressive long-term weight loss, ~70% %EWL24. Common complications include anastomotic leak, hemorrhage, and nutritional deficiencies. Other complications involve abdominal wall hematoma, wound infections, atelectasis, cholecystitis requiring cholecystectomy, and trocar site hernia, which can be surgically repaired25.The biliopancreatic diversion procedure involves three main components: creating a stomach tube with pylorus preservation, performing distal ileoileal anastomosis, and establishing proximal duodenal-ileal anastomosiset al.12 reported impressive average %EWL figures: ~67.1% at 6 months, 81.2% at 1 year, and 83.8% at 2 years post-surgery. Even in the long run, SG showed promise, with %EWL ranging from 41.8% among 179 patients to 64.1% among 14 patients observed at the 72-month follow-up13. Gastric bypass also demonstrated significant efficacy, achieving an average %EWL of 77% at 1 year and maintaining the results throughout the 60-month follow-up16. However, the average %EWL at 10 years after surgery showed a decreasing trend, ranging between 34 and 80.2%18. On the other hand, adjustable gastric banding yielded more modest outcomes compared to other procedures, resulting in a %EWL of 30% at 6 months and 50–60% at 48 months during the follow-up period22. Conversely, BPD/DS emerged as one of the most effective options, with an impressive average %EWL of ~70%, sustained even at long-term follow-ups24.The reviewed literature compared various weight loss procedures; among them, SG was a highly effective option for significant weight reduction Table . Durmush26. Similar outcomes were observed in another study with BPD/DS demonstrating the highest % total weight loss, followed by RYGB and SG27. Further analyses revealed that BPD/DS outperformed other procedures regarding weight loss outcomes28. Contrarily, a systematic review showed no significant differences in midterm and long-term weight loss outcomes between Laparoscopic Roux-en-Y gastric bypass (LRYGB) and LSG29. Another study compared laparoscopic adjustable gastric banding (LAGB) and laparoscopic gastric bypass, showing better weight loss results favoring gastric bypass30. Other studies reported consistent findings, with laparoscopic gastric bypass demonstrating superior weight loss compared to LABG31. In a comprehensive study encompassing various hospitals, laparoscopic gastric bypass showed the greatest average BMI reduction compared to LSG and LABG32.Comparative studies have also been conducted on various weight loss procedures. One study compared SG to RYGB and BPD/DS. BPD/DS showed the greatest BMI change after a year, followed by RYGB and SGet al.12, SG contributed to the complete resolution and significant improvement of type 2 diabetes in 71 and 18% of patients, respectively. It also resulted in the resolution of hypertension in 39% and improvement in 33% of patients. Similar results were found in the study by Neagoe et al.13, with 65.8% experiencing resolution or significant improvement in type 2 diabetes. In contrast, gastric bypass showed clinical reversal of diabetes mellitus in an impressive 98% of patients16. Although adjustable gastric banding can improve comorbidities, the outcomes may not be as significant as SG and gastric bypass. BPD/DS stands out as a highly effective procedure, improving obesity-related comorbidities and providing substantial health benefits to patients.BPD/DS and RYGB demonstrate the most favorable outcomes when examining the impact on obesity-related comorbidities. In the study by Durmush et al., with probabilities 1.88 times greater than SG. Even after BPD/DS, the probabilities of GERD resolution were 1.57 times greater, possibly due to a larger pouch and acid and bile diversion further downstream. They also noted that BPD/DS and RYGB were best for resolving type 2 diabetes and hypertension, outperforming SG. When comparing the chances of resolving obstructive sleep apnea (OSAS), RYGB, and BPD/DS showed equivalent probabilities, 1.46 and 1.76 times higher, respectively, compared to SG26. In the Bariatric Surgery Network study comparing comorbidities, they reported resolution or improvement rates of 44%, 68%, and 79% after 1 year for LAGB, LSG, and LRYGB, respectively, in terms of hypertension. For diabetes, the rates were 44%, 55%, and 83% for LAGB, LSG, and LRYGB, respectively. In patients with hyperlipidemia, the rates were 33%, 35%, and 66% for LAGB, LSG, and LRYGB, respectively. For patients with OSAS, the rates were 38%, 62%, and 66% for LAGB, LSG, and LRYGB, respectively. For GERD, the rates were 64%, 50%, and 70% for LAGB, LSG, and LRYGB, respectively. They concluded that LRYGB had the most significant outcomes for diabetes, hypertension, sleep apnea, and hyperlipidemia, followed by LSG and then LAGB. However, for GERD, LSG appeared less effective compared to LAGB, and LRYGB32.Regarding GERD, RYGB demonstrated the highest resolution, according to Sudan 2 and without obesity-related comorbidities  for arthritis may need to be more suitable candidates. Additionally, daily multivitamin intake is necessary, so patient commitment is essential. The gastric bypass carries the risk of severe perioperative complications, such as anastomotic or staple line leaks, occurring in ~4% of cases and may require re-operation. Other potential adverse effects include stomal stenosis, nutrient deficiencies, and internal hernia formation, all of which must be considered during patient selection30. BPD/DS is highly effective but typically recommended for patients with severe obesity and significant health risks. Careful evaluation is necessary to assess the patient’s suitability for this complex procedure, as it requires vitamin replacements due to potential malabsorption. Performing BPD/DS also demands specialized surgical skills, and it carries the highest incidence of adverse effects26.Adjustable gastric banding may be more suitable for patients who prefer a less permanent solution or have concerns about more extensive surgeries. It is less invasive, reversible, and associated with less serious side effects, leading to faster postoperative recovery and shorter hospital stays. However, it may be more appropriate for individuals with a lower BMI and fewer obesity-related health issues26. BMI is a crucial consideration, with higher BMI potentially indicating the need for more extensive procedures like gastric bypass or BPD/DS27. Surgical risks and patient preferences, including the desire for a reversible or permanent solution, also play a crucial role in the decision-making process28. Furthermore, the patient’s lifestyle and commitment to long-term changes, such as dietary modifications and regular exercise, are essential factors regardless of the procedure29. The experience and expertise of the bariatric surgeon are equally critical in selecting the most appropriate procedure for each patient, ensuring optimal outcomes and minimizing potential complications30. A comprehensive evaluation of these factors is essential to tailor the weight loss procedure to the individual’s needs and health circumstances.Several factors influence the choice of weight loss procedure for each patient. Medical history and obesity-related health conditions are significant determinants in selecting the most suitable option33. It can remain for up to 6 months, requiring endoscopic removal, and is effective when combined with lifestyle modifications34. In a study of adults with BMIs from 30 to 40 kg/m2, one group received the IGB and lifestyle advice, while the other received lifestyle intervention alone. The IGB group had significantly greater short-term weight loss at 3 and 6 months after removal, but some experienced adverse gastrointestinal events, leading to early balloon removal. A small percentage showed gastric abnormalities at removal35. In a post-marketing clinical trial, 8.9% of adults with BMIs of 30–40 kg/m2 experienced device and procedure-related serious adverse events (SAEs) with the IGB, below the 15% threshold. Combining the IGB with lifestyle intervention led to significant weight loss, supporting its use as an adjunct for weight reduction, with lower SAE rates in real-world clinical settings36. IGB therapy for super-obese patients showed short-term weight loss of 9.04% and a BMI reduction of 3.8 at 60 months postoperatively. However, more durable weight loss was achieved when IGB was combined with definitive bariatric surgeries, though three deaths (1.4%) were reported in the study37.The Intragastric Balloon (IGB) is a minimally invasive weight management tool – a single silicone sphere filled with saline, endoscopically placed in the stomach38. It is generally well-tolerated for the first 6 months but can pose technical challenges during insertion and removal. A study by Giuricin found that the Heliosphere BAG led to a mean weight loss of 12.66 kg and an overweight loss of 24.37% during a 6-month treatment period for severely obese patients. At 18 months post-removal, the mean BMI was 37.28 kg/m2, with a mean weight loss of 9.8 kg or 18.2%. The study suggested using the balloon as a ‘bridge treatment’ before major surgery to mitigate preoperative risks for severely obese patients39. Another study by Lecumberri et al. showed significant weight loss and BMI reduction with the Heliosphere IGB in 84 patients before bariatric surgery. Mean weight loss was 14.5 kg, BMI loss was 5.3 kg/m2, and 70.4% achieved a body weight loss of over 10%. The %EWL reached 33.2%. Age and initial BMI inversely affected weight loss outcomes40.The Heliosphere balloon is an air-filled polyurethane IGB designed to reduce side effects like nausea and vomiting2 who have not succeeded in losing weight through supervised programs41. It is implanted endoscopically and can be adjusted during its 8-month usage period before endoscopic removal. In a clinical study, 92% of Spatz3 Adjustable Balloon recipients experienced at least a 5% reduction in total body weight, with an average weight loss of 15.0%41. However, it is not suitable for individuals with specific medical conditions. It should be part of a long-term supervised diet and behavior modification program to enhance weight loss chances after removal. A study by Russo et al. compared the Spatz Adjustable Balloon System (ABS) with the BioEnterics Intragastric Balloon (BIB) for temporary obesity treatment. Both procedures resulted in similar median weight loss and BMI reduction at the end of therapy. Complications during the procedure and removal were minimal, with some cases of intolerance, gastrectasia, vomiting, and bowel migration. Weight gains were observed in the Spatz ABS group compared to the BIB group during follow-ups42. In addition to the Spatz balloon, other IGB devices like the ReShape Duo dual balloon system, Obalon system, and Elipse balloon are effective for promoting weight loss45. These balloons reduce food intake, promote fullness, delay gastric emptying, and influence hunger and satiety hormones. The choice of balloon depends on individual characteristics and medical advice.The Spatz balloon is an adjustable IGB filled with saline, designed for adults with a BMI between 30.0 and 40.0 kg/m2 who have not succeeded in losing weight through nonsurgical means46. It involves a tube attached to a Skin-Port port outside the abdomen. After each of their three daily meals, patients open the port valve, connect tubing, and allow gravity to drain stomach contents into a toilet or container. In a 52-week trial, the AspireAssist group achieved a significant 12.1% body weight loss at 52 weeks compared to 3.5% in the Lifestyle Counseling group, with a difference of 8.6%. This procedure also led to better excess weight loss, improved cardiometabolic risk factors, and enhanced quality of life. It demonstrated a favorable safety profile and high procedural success. More research is needed for long-term effects and broader applicability47. Another study involved 201 participants who underwent aspiration therapy between 2012 and 2016, resulting in promising weight loss outcomes , improved cardiometabolic parameters, and minimal complications. However, some participants discontinued the therapy for various reasons, necessitating further research to understand its benefits and drawbacks48. In a shorter 4-week study, 25 obese individuals achieved a considerable weight loss of 16.5 ± 7.8 kg, suggesting that the AspireAssist device may be a valuable tool for managing obesity49.The AspireAssist device is an option for adults with a BMI between 35.0 and 55.0 kg/m51. Abu Dayyeh et al. studied ESG’s efficacy and safety for treating classes 1 and 2 obesity. They found that ESG and lifestyle modifications led to significant and sustained weight loss up to 104 weeks and improved metabolic comorbidities. ESG is considered a safe and effective weight loss intervention for individuals with class 1 or 2 obesity, but more research is needed to assess its long-term durability and effectiveness in larger populations52.Endoscopic Sleeve Gastrectomy (ESG) is a minimally invasive weight loss procedure that uses an endoscope to reduce the size and reshape the stomach into a tubular structure. This procedure can lead to calorie intake reduction and weight loss. It offers advantages like fewer complications, shorter hospital stays, and quicker recovery compared to traditional bariatric surgerieset al.’s study supported the short-term effectiveness and safety of ESG using the Overstitch system for patients with classes I and II obesity. They observed significant weight loss at 6 months and 12 months, along with substantial excess BMI loss. The safety profile was favorable, with minimal complications53. A systematic review comparing LSG to other bariatric procedures, such as LGB and LAGB, found that LSG was well-tolerated, had a lower complication rate than LGB, and showed promising short-term weight loss outcomes. LSG also demonstrated potential in treating T2DM54.Neto 2 or 35 kg/m2 with obesity-related comorbidities55  or total  weight loss64. This highlights the importance of regular follow-up in supporting the patient’s weight loss journey and achieving successful outcomes after bariatric surgery.Studies have shown that adherence to postoperative follow-up significantly impacts the clinical outcomes of bariatric surgery56. Before bariatric surgery, candidates undergo a psychological evaluation to assess their readiness for the procedure and to address any potential psychosocial issues. Additionally, they are counseled to lose weight before surgery56. Registered dietitians play a crucial role in working with patients to develop personalized meal programs that meet their nutritional needs, facilitate weight loss, and promote overall health.Psychological and nutritional support are essential for an effective bariatric multidisciplinary team65. Educating patients about nutrition and its impact on successful weight loss is also vital to preoperative and postoperative care. Guiding food choices, portion control, and eating techniques empowers patients to make sustainable changes to their eating habits65.As obesity is considered a form of high-energy malnutrition, patients are often placed on a very low-energy diet (VLED) for 2–4 weeks before surgery. This approach helps reduce the size of the liver by 25%, thereby decreasing the risk of operative complications66. The postoperative follow-up serves as a platform to address psychosocial issues that may arise after bariatric surgery67. While the primary goal of bariatric surgery is to improve the quality of life for obese patients, not all patients who achieve weight loss experience significant improvements in their overall well-being67.Following the surgery, the multidisciplinary team continues to offer psychological support to ensure effective weight loss67, some common psychosocial issues patients encounter after bariatric surgery include concerns about excess skin, social support changes, struggles with illicit drug use disorders, and severe depression and suicidal thoughts. Addressing these psychological challenges is crucial to supporting the patient’s long-term success and well-being after the surgery.According to Kalarchian and Marcus5. However, it is not without risks, with complications falling into early (immediate postoperative) and late (after 30 days) categories68. Early complications include infections, bleeding, anastomotic leaks, pulmonary embolism (PE), and gastrointestinal issues like obstructions or perforations68. Timely medical attention and potential surgical intervention are crucial to manage these. Late complications manifest beyond the immediate postoperative period and include nutritional deficiencies, gallstones, gastrointestinal ulcers, hernias, and internal herniation68. Nutritional deficiencies stem from reduced nutrient absorption in some procedures, requiring ongoing monitoring and supplementation.Bariatric surgery is a popular and effective option for obesity, modifying the digestive system to promote weight lossWhile complications can occur, bariatric surgery is generally safe, with varying risks based on the procedure, patient health, and surgical team expertise. Preoperative assessments, patient selection, and postoperative care are vital for minimizing risks and optimizing outcomes. Patients considering bariatric surgery should undergo a thorough evaluation, receive counseling, and understand potential risks and benefits. Postoperative care and follow-up are essential for addressing complications and supporting the patient’s weight loss journey.69. The prevalence of SSI is a matter of concern due to its association with increased nosocomial morbidity and mortality rates69. These infections can range in severity from relatively mild surface incisional infections to more serious complications, such as intra-abdominal abscesses involving deep organ spaces and the development of incisional hernias71.Over the years, the field of surgery has made significant advancements in medical knowledge and infection control measures. However, surgical site infections (SSI) continue to be a persistent and concerning issue in healthcare institutions74. For instance, research by Kushner et al. found that patients with class I obesity have a 50% higher risk of SSI compared to individuals with a normal BMI. Patients classified as class II obese face an 86% higher risk, and patients with class III obesity experience a risk exceeding 100% higher than those with a normal BMI72. Several factors contribute to the risk of SSIs, including changes in respiratory physiology, modifications in the skin and soft tissue integrity, the presence of comorbidities like T2DM and cardiovascular disease, drug therapies, and antimicrobial resistance68.The rise in SSI has been linked to the increasing prevalence of higher BMI in patients undergoing surgery, with a progressive increase in infection risk as BMI categories increaseet al.71 revealed that the incidence of wound infection following bariatric surgery varies, with reported rates ranging from as low as 1% in the International Bariatric Surgery Registry to as high as 16.5% in several published studies. The bacterial isolates found in the study encompassed a range of microorganisms, with Staphylococcus aureus accounting for 39% of the isolates, alpha-hemolytic Streptococcus at 26%, Enterococcus at 16%, Proteus mirabilis at 9%, and various other bacteria collectively comprising 10% of the isolates71.Another study conducted by Christou Addressing the risk of SSIs in bariatric surgery and other procedures requires a comprehensive approach. Healthcare institutions and surgical teams must implement strict infection control measures, adhere to evidence-based guidelines, and tailor preventive strategies to address the unique risks associated with each patient. Proper preoperative evaluation, optimization of patient health, and meticulous postoperative care are essential to reducing the incidence of SSIs and improving overall surgical outcomes for patients.76. UTIs can occur at any point along the urinary tract, including the urethra, bladder, ureters, or kidneys. During bariatric surgery, the use of urinary catheters is often necessary to facilitate urine drainage and monitor output. However, this practice also introduces potential risks, as catheters can serve as a pathway for nosocomial bacteria to enter the urinary tract, leading to UTIs76.Urinary tract infection (UTI) is a common postoperative complication of bariatric surgery, affecting over 40% of patients and accounting for a significant portion of nosocomial infections, with ~1.7 million patients affected annuallyEscherichia coli (E. coli) is the primary causative agent, responsible for over 90% of cases. Similarly, ~50% of nosocomial catheter-associated UTIs (CAUTIs) are attributed to E. coli infections77. The duration of urinary catheterization also plays a crucial role in UTI risk. Patients with catheters in place for more than 2 days are twice as likely to develop a UTI compared to those with catheterization lasting 2 days or less76. Apart from urinary catheter use, several other risk factors have been identified in previous studies. These factors include age, with older patients being more susceptible to UTIs, longer operative times during surgical procedures, and prolonged hospital stays, which increase exposure to healthcare-associated infections79.In ambulatory patients diagnosed with a UTI, To reduce the risk of UTIs following bariatric surgery, healthcare providers must adhere to strict infection prevention protocols. This may involve minimizing the duration of urinary catheterization whenever possible, using aseptic techniques during catheter insertion and maintenance, and employing prophylactic antibiotics when indicated. Additionally, healthcare professionals should be vigilant in monitoring patients for signs and symptoms of UTIs and promptly initiate appropriate treatment when infections are identified. By addressing these risk factors and implementing effective preventive measures, healthcare teams can help reduce the incidence of UTIs in bariatric surgery patients and improve overall postoperative outcomes.81.Gastrointestinal leaks, also known as anastomotic leaks or gastric leaks, are serious and potentially life-threatening complications that can occur after certain gastrointestinal surgeries, including bariatric surgeries. While the incidence of postoperative gastrointestinal leaks has decreased over the years, it remains a relatively infrequent complication that carries a significant risk of morbidity and mortality82. It is important to note that the location of the anastomosis and the type of surgery performed can significantly impact the risk of leaks.The rates of leaks after gastrointestinal surgeries vary depending on the specific site of the anastomosis. For instance, leak rates are observed in the range of 2–16% for the esophagus, 1–9% for the stomach, 9–16% for the pancreas, 10–16% for the bile ducts, 1–3% for the small intestine, 3–29% for the colon, and 8–41% for the rectum82.Gastrointestinal leaks can lead to various complications, including intra-abdominal abscesses, sepsis, and other infections, which can further contribute to morbidity and mortality. The severity of these leaks can range from mild and self-limiting to severe and life-threatening. Mortality rates associated with gastrointestinal leaks can be as high as 35%, emphasizing the importance of early detection and prompt interventionTo minimize the risk of gastrointestinal leaks, surgeons follow meticulous surgical techniques and employ advanced technologies, such as intraoperative leak testing, to ensure the integrity of the anastomosis. Additionally, postoperative monitoring and early recognition of signs and symptoms of leaks are crucial for timely intervention and management.Patients undergoing gastrointestinal surgeries, including bariatric procedures, should be educated about the potential risks and signs of complications, including gastrointestinal leaks, so that they can seek medical attention promptly if needed. By adopting comprehensive preventive measures and maintaining a high index of suspicion for leaks in the postoperative period, healthcare teams can work together to reduce the incidence of gastrointestinal leaks and improve patient outcomes after bariatric and other gastrointestinal surgeries.84. A study carried out by Stein and Matta83 revealed that the in-hospital prevalences of PE, DVT, and VTE following bariatric surgery were found to be 0.9%, 1.3%, and 2.2%, respectively.The reported incidence of venous thromboembolism (VTE), which includes PE or deep vein thrombosis (DVT), following bariatric surgery exhibits significant variability, ranging from 0% in some studies to as high as 3.5% in others85. The mechanism behind this increased risk is attributed to factors such as venous stasis caused by abdominal adiposity, endothelial dysfunction, and the pro-inflammatory and prothrombotic state associated with obesity87.Obesity itself is a significant risk factor for VTE, particularly symptomatic DVT. The combination of obesity and abdominal surgery that necessitates general anesthesia for ~30 min or more further increases the risk of thrombosis, leading to a 10-fold increased risk for the development of VTE86. As patients undergoing bariatric procedures may include older individuals, advanced age can contribute to an increased risk of postoperative complications, including VTE.Age is also a notable risk factor for adverse events following bariatric surgery, including VTETo mitigate the risk of VTE following bariatric surgery, several preventive measures are implemented. These may include the use of pharmacological prophylaxis (such as anticoagulant medications) and mechanical prophylaxis (such as pneumatic compression devices) to enhance blood flow and prevent clot formation. Early ambulation and patient mobilization after surgery are also essential to reduce the risk of venous stasis.It is crucial for the multidisciplinary team caring for bariatric surgery patients to identify those at higher risk for VTE and tailor the prophylactic measures accordingly. By implementing comprehensive VTE prevention strategies, healthcare providers can reduce the incidence of postoperative complications and optimize patient outcomes following bariatric surgery. Regular monitoring and prompt detection of any signs or symptoms of VTE can further contribute to early intervention and improved patient care.87.Long-term nutritional deficiencies and monitoring are essential in managing patients who have undergone bariatric surgery. Weight loss procedures, such as gastric bypass, SG, and biliopancreatic diversion, can alter the gastrointestinal anatomy and the absorption of nutrients, causing deficiencies in iron, calcium, and various vitamins, including vitamins D, B1, B9, and B1288.These deficiencies can lead to specific health conditions such as anemia due to iron deficiency, osteopenia (reduced bone density), and neurological symptoms resulting from vitamin B12 deficiency. Other malnutrition-related complications have been observed following gastric bypass surgery (GBP), such as protein malnutrition and vitamin A deficiency, which may cause ocular complications89. Among these deficiencies, iron, vitamin D3, and folic acid deficiencies were notably more frequent in the first year compared to the second year after the surgery89.Monitoring for postoperative deficiencies is crucial as they can arise anytime. However, most deficiencies are commonly observed between 12 and 15 months after the surgery, except for vitamin D3, which may occur earlier90. Early postoperative deficiencies often correspond with the most common preoperative deficiencies, suggesting that nutritional imbalances existing before the surgery may persist after the procedure. This highlights the importance of preoperative nutritional assessments and interventions to optimize the patient’s nutritional status before bariatric surgery89.Various factors can contribute to postoperative nutritional deficiencies, including preoperative deficiencies, intolerance to certain foods after surgery, alterations in taste and eating patterns, and non-adherence to dietary and supplement recommendations post-surgeryTo address and prevent nutritional deficiencies, bariatric surgery patients should receive ongoing nutritional counseling and follow-up care from registered dietitians as part of the multidisciplinary team. The dietitians work closely with patients to develop personalized meal plans and supplement recommendations to ensure adequate intake of essential nutrients. Regular blood tests are typically conducted to monitor the patient’s nutritional status and detect deficiencies early, allowing prompt interventions.Long-term nutritional monitoring and follow-up are crucial to ensuring bariatric surgery patients’ overall health and well-being and to minimize the risk of complications related to nutritional deficiencies. By actively addressing and managing nutritional issues, healthcare providers can enhance the success and safety of bariatric surgery as a weight loss and metabolic health intervention.91. The evaluation typically includes psychological testing, nutrition evaluation, and medical assessment. Medical evaluation involves laboratory testing, including complete blood counts, metabolic profiles, coagulation profiles, ferritin levels, thyroid function testing, and lipid profiles91. For those undergoing malabsorptive procedures, vitamin B12 and fat-soluble vitamin levels may be considered92.During the preoperative evaluation of patients considered for bariatric surgery, healthcare providers conduct a comprehensive assessment to determine their candidacy and identify potential factors that may impact the procedure’s success92.Pulmonary and cardiac assessments are also essential components of the preoperative evaluation. This may involve conducting chest radiographs, arterial blood gas measurements, pulmonary function tests, electrocardiograms, and stress tests to assess coronary artery disease. Given the prevalence of obesity-related sleep apnea, a sleep study may also be part of the evaluationHelicobacter pylori infection is recommended for patients undergoing bariatric surgery. Positive H. pylori testing has been associated with a higher likelihood of abnormal endoscopy and postoperative marginal ulcers. If H. pylori infection is detected, preoperative therapy for eradication is advised92.Preoperative testing for 93.Furthermore, liver histology is assessed as obesity often presents with nonalcoholic fatty liver disease. Gastric bypass surgery has shown significant improvement in liver histology for most patients. Preoperative evaluation involves blood testing and imaging studies, such as ultrasonography, with a liver biopsy performed if cirrhosis is suspected91. A thorough preoperative evaluation by bariatric behavioral clinicians is essential to identify risk factors affecting surgical success and weight loss goals. This evaluation fosters a trusting relationship between the clinician and the patient, allowing for tailored interventions and support throughout the weight loss journey93.Psychosocial factors significantly impact the long-term outcomes of bariatric surgery, affecting adherence to postoperative lifestyle recommendations, emotional adjustment, and weight loss outcomes94. However, adherence to these postsurgical recommendations has been closely linked to the procedure’s success in terms of weight loss95.Adherence to professional recommendations is a critical aspect of post-bariatric surgery care, and it encompasses following dietary guidelines, engaging in physical activity, attending medical follow-ups, and participating in support group sessions. Despite its importance, studies have shown that preoperative and postoperative noncompliance with these recommendations is relatively common89. During the first year after surgery, patients experience rapid weight loss, which puts them at risk for deficiencies in vitamins and minerals, dehydration, and gastrointestinal symptoms88. Proper dietary management and supplementation are essential to prevent complications arising from these deficiencies and to ensure overall patient safety and well-being88. Regular screenings for anemia, clotting abnormalities, and nutrient deficiencies are also vital to detect any potential imbalances or deficiencies that could negatively impact the patient’s health95.Nutritional care after bariatric surgery is paramount for successful wound healing and overall well-being95. Addressing these concerns early can improve outcomes and ensure patients achieve their weight loss goals.Monitoring weight loss progress is an essential part of post-bariatric surgery care. Unsuccessful weight loss or significant weight gain may signal potential issues that require additional support and prompt intervention from the healthcare teamEngagement in physical activity is another crucial aspect of post-bariatric surgery care. Regular exercise can enhance weight loss, improve cardiovascular health, and improve the patient’s overall well-being. Healthcare providers should encourage patients to adopt an active lifestyle and provide guidance on suitable exercise routines based on their needs and capabilities.Participation in support group sessions can also benefit patients undergoing bariatric surgery. Support groups offer a valuable platform for patients to share their experiences, challenges, and successes with others who have undergone similar procedures. These groups can provide emotional support, practical advice, and a sense of community, positively impacting the patient’s overall experience and long-term success.et al. demonstrated that many participants who underwent bariatric surgery could maintain their weight loss long-term, with almost half of the individuals maintaining their weight within 20% of the lowest weight achieved after the surgery. This long-term weight maintenance was superior to the outcomes observed in a nonsurgical comparison group of individuals with severe obesity96. This highlights the effectiveness of bariatric surgery as a long-term weight control strategy.The study conducted by Ryder 97 In a study by Svetkey et al., participants who received personalized contact through monthly telephone or face-to-face interactions with healthcare professionals demonstrated significantly better weight loss retention compared to those in self-directed or interactive technology intervention groups98. This emphasizes the importance of ongoing support and guidance from healthcare professionals in helping patients sustain their weight loss efforts.Regular and continuous communication between patients and healthcare providers is essential to ensure successful long-term weight maintenance.et al.99, which focused on patients who underwent RYGB, revealed that those who engaged in at least 150 min per week of moderate-intensity physical activity experienced greater weight loss at 6 and 12 months after the surgery. Physical activity contributes to weight loss, offers numerous health benefits, and supports overall well-being.Consistent engagement in physical activity is also recognized as a key strategy for ensuring long-term weight maintenance. The study by Evans 100Bariatric surgery has been shown to promote significant weight loss and lead to substantial improvements or even resolution of obesity-related comorbidities. Conditions such as T2DM, hypertension, and cardiovascular diseases, which are often associated with obesity, have been found to improve after bariatric surgery.et al. in 2012 investigated the impact of gastric bypass surgery on adults with uncontrolled or medication-controlled type 2 diabetes. Over 13 years, 4434 adults underwent gastric bypass, and within 5 years after surgery, ~68.2% of them experienced complete remission of diabetes101. This study demonstrated the significant potential of bariatric surgery in achieving diabetes remission and improving glycemic control.A retrospective cohort study conducted by Arterburn et al.102 conducted a study that showed that achieving the resolution of hypertension through weight loss reduced the risks of coronary arterial disease (CAD) by ~39% in men and 25% in women. This highlights the cardiovascular benefits of weight loss achieved through bariatric surgery.Hypertension is another common comorbidity associated with obesity, and weight loss-induced resolution of hypertension can significantly reduce cardiovascular risk in patients. Vogel Bariatric surgery has been associated with decreased prevalence and improvement in the severity of various mental health conditions, focusing on improvements in depression.et al.103 found that among individuals seeking and undergoing bariatric surgery, the most prevalent mental health conditions were depression, with an estimated prevalence of 19%, and binge eating disorder, with an estimated prevalence of 17%. However, the study also revealed that bariatric surgery consistently correlated with postoperative reductions in the prevalence of depression, with improvements ranging from 8% to 74%. Notably, the severity of depressive symptoms also showed improvement following the surgical procedure103.A study conducted by Dawes 103.The observed improvements in depression and other mental health conditions after bariatric surgery can significantly impact patients’ overall well-being and quality of life. Weight loss, changes in hormonal factors after surgery, and increased self-esteem and body image perception have been suggested as potential factors contributing to improving mental health outcomes104. While this diversity aptly mirrors the intricate nature of the field, it simultaneously introduces complexities when attempting direct comparisons and generalizing findings across disparate studies104. Therefore, a promising direction for future research involves adopting standardized protocols and rigorous, expansive trials to enhance the reliability and quality of the presented evidence.In the context of this review on recent advancements in bariatric surgery, it is crucial to acknowledge certain limitations arising. Bariatric surgery research presents a diverse landscape encompassing various study designs, patient groups, and chosen outcome measures105. However, the critical factor of long-term sustainability necessitates deeper exploration. Attaining a comprehensive understanding of the durability of weight loss, metabolic enhancements, and potential complications over extended periods is crucial to guide informed clinical decision-making. Beyond conventional clinical endpoints, the incorporation of patient-centered outcomes assumes heightened significance. While achieving weight loss and metabolic improvements remains pivotal, an equally vital facet involves comprehending the broader impact of these procedures on aspects such as overall quality of life, psychological well-being, and patient satisfaction. This holistic perspective promises a deeper understanding of the intricate implications of bariatric surgery on individuals.The immediate benefits observed in several modern bariatric procedures are undeniably compellingFuture research endeavors would benefit from robust comparative studies to inform tailored and personalized surgical decisions. Directly comparing different techniques can offer a nuanced understanding of distinct advantages and disadvantages, aiding practitioners in making informed choices based on patient profiles. Similarly, recognizing that the effectiveness of bariatric surgery extends beyond the operating room, the integration of multidisciplinary care gains prominence. Collaborative efforts involving nutritionists, psychologists, and exercise specialists stand poised to optimize patient outcomes. Therefore, further research investigating the seamless integration of these components into the overarching bariatric care framework is warranted.106. The potential of technology to reshape procedural success rates and patient recovery experiences beckons further exploration, making the study of these technological innovations and their tangible impact a compelling avenue for future research. In addition, the persistent challenge of health disparities demands continuous attention in bariatric surgery107. To this end, dedicated research aimed at dissecting and mitigating factors contributing to differential access, treatment outcomes, and overall experiences among diverse patient populations remains imperative.Technological advancement holds considerable promise for refining bariatric procedures, encompassing innovative surgical tools, advanced imaging methods, and minimally invasive approachesBariatric surgery encompasses a range of well-established weight loss procedures, each with distinct mechanisms and outcomes. Additionally, emerging options like IGBs, AspireAssist devices, and endoscopic sleeve gastroplasty show promise but require further research to solidify their effectiveness. Nevertheless, bariatric surgery has consistently demonstrated substantial benefits in weight loss, comorbidities resolution, and quality of life improvements. By tailoring procedures to individual needs and continuing research efforts, the field is poised for advancement, leading to improved patient outcomes and transformative changes in the lives of those seeking effective weight loss solutions.Ethics approval was not required for this review.Informed consent was not required for this review.None.N.A.: conceptualization. All authors were involved in writing the manuscript.All authors declare no conflicts of interest.Nicholas Aderinto.Data sharing is not applicable to this article.Not commissioned, externally peer-reviewed."}
{"text": "Obesity is a complex, multifactorial and chronic disease. Bariatric surgery is a safe and effective treatment intervention for obesity and obesity-related diseases. However, weight loss after surgery can be highly heterogeneous and is not entirely predictable, particularly in the long-term after intervention. In this review, we present and discuss the available data on patient-related and procedure-related factors that were previously appointed as putative predictors of bariatric surgery outcomes. In addition, we present a critical appraisal of the available evidence on which factors could be taken into account when recommending and deciding which bariatric procedure to perform. Several patient-related features were identified as having a potential impact on weight loss after bariatric surgery, including age, gender, anthropometrics, obesity co-morbidities, eating behavior, genetic background, circulating biomarkers , psychological and socioeconomic factors. However, none of these factors are sufficiently robust to be used as predictive factors. Overall, there is no doubt that before we long for precision medicine, there is the unmet need for a better understanding of the socio-biological drivers of weight gain, weight loss failure and weight-regain after bariatric interventions. Machine learning models targeting preoperative factors and effectiveness measurements of specific bariatric surgery interventions, would enable a more precise identification of the causal links between determinants of weight gain and weight loss. Artificial intelligence algorithms to be used in clinical practice to predict the response to bariatric surgery interventions could then be created, which would ultimately allow to move forward into precision medicine in bariatric surgery prescription. Obesity is a complex, chronic and multifactorial disease . Weight 2 with co-morbidity  are associated with %EWL at 3 and 6 months after SG. In addition, serotonin and serotonin/5-hydroxytryptophan (5-HTrp) ratio were shown to depict a superior performance at predicting weight loss 3 months (ROC AUCs for serotonin: 0.78 and serotonin/5-HTrp ration: 0.81) and 6 months (ROC AUCs for serotonin: 0.79 and serotonin/5-HTrp ration: 0.80), after SG [Metabolomics recently emerged as a powerful tool to provide new insights on the pathological processes. Multiple studies have focused on the identification of metabolite profiles that could be used as biomarkers for diagnosis, disease monitoring and prognosis , 179. Meafter SG , with loThe anatomical gut rearrangement induced by bariatric surgery yields alterations in patients’ gut hormone profile , 183. Inet al found that preoperative responses of glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) to a mixed meal tolerance test (MMTT) do not correlate to short-term postoperative weight loss, after RYGB surgery [Werling  surgery . Another surgery . Higher  surgery .Moreover, SADI-S and RYGB with a biliopancreatic limb (BPL) of 200 cm seem to elicit a favorable post-prandial glucose, GLP-1 and insulin profile, with a potentially lower risk of protein malnutrition when compared to BPD-DS and lower risk of post-prandial hyperinsulinemia as compared to RYGB with a shorter BPL . The posDespite there are universally accepted indications for considering bariatric surgery and a vast array of surgical techniques is presented, there are no defined criteria for the use of each procedure nor guidelines for a patient-tailored decision among different technical procedures in order to optimize bariatric surgery outcomes. The decision to conduct a given procedure is empirical taking into account the individuals’ phenotype, the peri-operative risk and the surgeon experience, without any robust evidence based approach. This remains one of the most frustrating shortfalls in bariatric practice, both for clinicians and patients since weight loss after bariatric surgery is heterogeneous and can be highly unpredictable.Although the pre-operative factors with a greater impact on post-surgical weight loss are BMI, age and T2D, these are not sufficient to explain the diversity of bariatric surgery outcomes. In addition, genetic factors and food addiction were also demonstrated to be major determinants of weight loss after surgery; while miRNAs, metabolomics and hormonal profiling, although promising are far from being robust preoperative biomarkers to be implemented in clinical practice. Consequently, there is the unmet need for a better understanding of the socio-biological drivers of weight gain, weight loss failure and weight-regain after bariatric interventions.Machine learning models targeting preoperative factors and effectiveness measurements of specific bariatric surgery interventions, could enable a more precise identification of the causal links between the determinants of weight loss and weight gain. Artificial intelligence algorithms grounded on preoperative factors could then be created to be used in clinical practice to predict the response to bariatric surgery interventions Fig. . In a fu"}
{"text": "Obesity is an increasingly widespread disease worldwide because of lifestyle changes. It is associated with an increased risk of cardiovascular disease, primarily type 2 diabetes mellitus, with an increase in major cardiovascular adverse events. Bariatric surgery has been shown to be able to reduce the incidence of obesity-related cardiovascular disease and thus overall mortality. This result has been shown to be the result of hormonal and metabolic effects induced by post-surgical anatomical changes, with important effects on multiple hormonal and molecular axes that make this treatment more effective than conservative therapy in determining a marked improvement in the patient’s cardiovascular risk profile. This review, therefore, aimed to examine the surgical techniques currently available and how these might be responsible not only for weight loss but also for metabolic improvement and cardiovascular benefits in patients undergoing such procedures. Obesity is a chronic disease, characterized by an excess of adipose tissue, whose etiology is complex and multifactorial, resulting from the interaction of numerous genetic and environmental factors . AccordiThe pathophysiological mechanisms linking obesity and the risk of cardiovascular diseases (CVDs) and adverse cardiovascular events are multiple and different . Obesity2 ..33].The AGB consists of a laparoscopic positioning of a silicone prosthesis (the band) around the stomach creating a proximal gastric pouch of approximately 20–30 mL. The prosthesis is adjustable, i.e., it has the possibility of tightening or widening the passage between the pouch above and the remaining stomach (outlet) below the banding; this occurs because the banding consists of an insufflation chamber that is connected to a valve, positioned in the subcutaneous tissue, through a catheter .The SG consists of the vertical partition of the stomach reaching a reduction of its size of about 25%.Originally, the SG was the first step to execute either a gastric by-pass (GPB) or biliopancreatic diversion (BPD) with duodenal switch (DS) but since most patients showed to successfully achieve satisfactory weight loss without the full procedure, it is now a standing-alone surgery which trend is constantly increasing . Even ifCurrently, the two most frequent malabsorptive procedures performed worldwide are BPD and DS  Figure .These procedures are thought to achieve weight loss through a controlled malabsorption of nutrients.BPD consists of the resection of approximately two distal thirds of the stomach with the closure of the duodenal stump followed by an intestinal bypass. The procedure allows the creation of two tubes: food passes through one and biliopancreatic secretions from the liver and pancreas pass through the other. The meeting of pancreatic secretion and food only takes place for a short distance, approximately 70 cm from the colon, thus resulting in reduced digestion and less food absorption . The gasTo avoid or reduce several complications related to BPD , a SG is executed instead of a distal gastric resection in the so-called BPD/DS .Roux-en-Y gastric bypass (RYGB) is a mixed restrictive and malabsorptive procedure. Weight loss is achieved through gastric restriction and decreased intestinal absorption, which is the greater the further downstream the outlet of the bilio-pancreatic secretions. It consists of the definitive separation of the stomach with the creation of a small gastric pouch of 25–30 mL. This pouch is anastomosed with the alimentary tract of a digiunal loop, while the biliopancreatic tract is anastomosed between 100 and 150 cm downstream of the gastrodigiunal anastomosis .Today, one-anastomosis gastric bypass (OAGB) is the third most commonly performed MBS surgery. It is a modification of the mini-gastric bypass that originated in 1997  and now 2), OAGB was shown to be non-inferior to RYGB with regard to weight loss in a 2 years follow-up [OAGB has been associated with a greater weight loss than RYGB . Furtherollow-up . Interesollow-up . Howeverollow-up ,48.2 or in patients with a BMI ≥ 30 kg/m2 and T2DM. In addition, MBS should be considered in patients with a BMI of 30–34.9 kg/m2 who do not achieve satisfactory weight loss despite optimal lifestyle and medical therapy [According to the 2022, American Society for Metabolic and Bariatric Surgery (ASMBS) and International Federation for the Surgery of Obesity and Metabolic Disorders (IFSO) guidelines ,50, bariNo recommendation is listed to address a patient to a specific surgical technique. Hence, local expertise, patient individual profile, and preference are the main factors to take into account during the decision-making process .There are no absolute contraindications to BS but severe heart failure, unstable coronary artery disease, end-stage lung disease, active cancer treatment, portal hypertension, drug/alcohol dependency, and impaired intellectual capacity are considered relative contraindications . MoreoveT2DM accounts for 90–95% of diabetic patients  and it iThe number of T2DM patients is dramatically grown worldwide with 151 million adults affected twenty years ago and 462 million in 2019 with a trend destined to increase in the coming years .In 1990 the prevalence of the disease was 3.9% in men and 3.5% in women, in 2019 it reached 6% in men and 5% in women . The proIn a survey conducted in China in 2007–2008, the prevalence of T2DM was higher than in other countries of the world reaching 9.7%, and has been predicted that in 2030, 2 of 10 people with T2DM in the world will live in China.Obesity is one of the major modifiable risk factors of T2DM, but it has been observed that BMI does not correlate with T2DM because it is not representative of the body fat distribution being the android model of obesity with visceral and abdominal fat deposits to have a role in the physiopathology of T2DM rather than the absolute individual weight . This asIn the last twenty years, BS has become the first-choice treatment for severe obesity with an acceptable risk of morbidity and mortality and high efficacy on T2DM control. In a recent study that included 1111 diabetic patients treated with BS, 74% of patients had diabetes remission at 1 year . The effBariatric surgery reduces the incidence of CVD through multiple hormonal mechanisms ,63,64,65The primary change after BS is weight loss, which results in several benefits . A previAfter bariatric surgery, blood levels of triglycerides and glucose are significantly reduced, while postprandial levels of adiponectin, glucagon-like peptide 1 (GLP-1), insulin, and serum insulin-like growth factor-1 (IGF-1) are significantly increased. Elevated adiponectin levels are associated with changes in total fat mass and reduced risk of atherosclerosis . IncreasWeight loss after bariatric surgery may help blood pressure control in obese patients. Previous observations have shown a remission rate of HTN between 60% and 70% in the year following weight loss surgery reaching a peak as high as 90% in a medium follow-up ,79.In this regard, the prospective, observational, unicenter BARIHTA study , has reported that patients with severe obesity scheduled to undergo bariatric surgery  showed aBariatric surgery also reduces the rates of T2DM because of better glucose control that may lead to a remission of diabetes in up to 95–100% of patients . InitialWeight loss after bariatric surgery modulates GLP-1 levels ,92,93,94Peptide YY (PYY) is a peptide released by enteroendocrine L cells in the distal small intestine and colon in response to feeding . It is mAlthough patients with T2DM openly outnumber patients with T1DM, bariatric surgery has been associated also with benefits on T1DM subjects and associated biomarkers. One study indicated that comparable benefits might be achieved by bariatric surgery on complications associated with T1DM as well as T2DM . Other eThe impact of bariatric surgery on the course of Nonalcoholic Fatty Liver Disease (NAFLD) in obese individuals has been also reported ,104,105.Bariatric surgery has been proven to achieve benefic results on cardiovascular risk, by short- and long-term effects on diabetes mellitus, HTN, dyslipidemia, and inflammation, leading to a better quality of life .Several, large cohort studies comparing BS to conventional obesity management have confirmed that patients undergoing bariatric surgery achieve diabetes remission more frequently than those on conventional therapy alone, over a follow-up period ranging from one to 5 years ,109,110.Glycemic profile improvement after bariatric surgery is largely due to the weight loss and the subsequent increase in insulin sensitivity, as it happens to patients who lose an equivalent amount of weight by using caloric restriction . A substWithin 3 months from surgery, the gut microbiome appears markedly altered, with increased diversity ,114,115.Bile acid signaling is also altered after some bariatric surgery interventions. In particular, serum bile acid concentration and composition change following RYGB and SG, but not following laparoscopic AGB. Bile acids act as hormones that bind to the farnesoid X receptor (FXR), improving glucose tolerance ,118. TheRecent meta-analyses have highlighted how different surgical techniques produce different effects on glucose homeostasis, being more drastic procedures  more effective than less drastic ones , and having laparoscopic AGB  the lowest remission rate ,120.Two hypotheses could explain the gut rearrangement that occurs early after bariatric surgery: (1) the hindgut hypothesis, under which the rapid transit of nutrients into the distal bowel, due to proximal small bowel bypass, would cause increased secretion of gut hormones; and (2) the foregut-exclusion hypothesis, by which the levels of an unidentified anti-incretin factor would decrease after exclusion of nutrients from the duodenum and the proximal jejunum ,122.Bariatric surgery leads to improvement or resolution of HTN. A systematic review and meta-analysis of prospective studies have shown that a BMI reduction of 5 corresponds to an HTN reduction of 27% after 12–24 months from surgery .Decrease in vasoconstrictors , increase in vasodilators, and natriuresis  are key factors which mediate blood pressure reduction ,125.HTN improvement after bariatric surgery is also due to obstructive sleep apnea resolution. Obstructive sleep apnea syndrome in obese people is characterized by intermittent hypoxia/hypercapnia, which causes the activation of the sympathetic nervous system and renin-angiotensin-aldosterone system, contributing significantly to HTNBariatric surgery-induced weight loss results in significant improvement of atherogenic lipid profile, in terms of reduction in total cholesterol, low-density lipoprotein cholesterol (LDL-C), and triglycerides, and increase in cardioprotective HDL-C ,127. HowBariatric surgery has been proven to reduce obesity-related inflammatory states. A meta-analysis of 52 studies reported a decrease in serum inflammatory markers such as CRP levels, measured by high-sensitivity assay (hs-CRP), of 61.7%, up to 34 months after bariatric surgery . SimilarThe amelioration or resolution of comorbidities typically associated with obesity, including T2DM, HTN, obstructive sleep apneas, dyslipidemia, and gastroesophageal reflux disease, after bariatric surgery, improves significantly patients’ quality of life. A meta-analysis of more than 2000 patients across 21 studies reported a marked improvement in mental health, assessed by using the Short-Form 36 (SF-36) questionnaire, after surgery . SignifiMBS is now considered the most effective treatment for obesity and its complications even in the long term ,136,137.Twelve randomized controlled trials, with a total of 874 patients with T2DM comparing surgical vs. medical therapy with follow-up from 1 to 5 years ,144,145 In these studies, the following surgical procedures have been used: RYGB (9 studies), AGB (5 studies), SG (2 studies), and BPD (1 study) . All of n = 2010) indicate that up to 44% of patients who experience initial remission had a recurrence and need to restart antihypertensive medications within 10 years. However, it should take into account that aging and weight regain might be responsible for this HTN [However, the effect of bariatric surgery on HTN in the long term is not well established yet. Data from an observational study (this HTN .p < 0.05), but with comparable benefits in a 3-year follow-up. Hence, longer follow-up is needed to better evaluate procedure-specific differences in dyslipidemia outcomes.Another study of long-term evaluation, including 1738 RYGB and 610 AGB patients, has shown that the prevalence of dyslipidemia was reduced at 7 years follow-up . In a meMBS was linked to a significantly lower incidence of Major Adverse Cardiac Events (MACE) in patients with severe obesity, T2DM, and HTN up to 10 years after surgery ,150,151.Another analysis including 14 studies with up to 29,208 patients who underwent bariatric surgery and 166,200 matched controls , surgical patients showed more than 50% reduction in mortality compared to control group . In the p < 0.001) compared with nonsurgical group (35717432). The risk of MI , stroke , cardiovascular death , and all-cause death  was significantly lower in bariatric surgery group [Also, Tang et al., in a recent review of the literature and meta-analysis of population-based cohort studies  showed MACE relative risk (RR) in the bariatric surgery group was 0.53  = 0.45–0.62, ry group . These rry group .The current literature, mainly related to observational studies, suggests that patients undergoing bariatric surgery have a lower risk of cardiovascular events and mortality compared to controls. However, future randomized studies are needed to confirm these benefits.“Globesity” and “diabesity” are increasing worldwide with a high social and medical impact.Metabolic surgery is an evolution of obesity surgery. As reported in this article, it represents a valid and feasible therapeutic option for reducing cardiovascular risk, in this class of patients, even compared with medical and lifestyle change interventions.Several studies have shown that MBS acts not only by reducing body weight but also by interacting with complex molecular and hormonal systems which dysfunction correlates closely with the onset of CVDs: in perspective, this means that MBS will have the potential of therapy for dysmetabolism rather than obesity per se.Understanding the molecular and hormonal changes that follow MBS can be useful to deepen the knowledge of the pathophysiology of cardiovascular complications in obese patients, allowing a better definition of the individual profile that can benefit the most from this kind of treatment approach. Indeed, short and long-term studies have demonstrated that MBS can control the major modifiable cardiovascular risk factors including dyslipidemia, HNT, and diabetes which severity should be taken into account in the decision-making process to improve long-term outcomes.Being the new goal of MBS the treatment of metabolic illness, a multidisciplinary approach including cardiologists, surgeons, anesthesiologists, endocrinologists, psychologists, and nutritionists seems necessary to define the most effective therapeutical strategy for each patient, making the field of bariatric surgery of great medical and surgical interest."}
{"text": "Bariatric surgery is one of the most effective interventions for treating obesity and weight-related complications. Since the early days of bariatric surgery, several innovations in surgical technique and periprocedural management have improved overall outcomes and reduced the risk of short and long-term complications. Bariatric surgery continues to be the treatment of choice for many patients with medically complicated obesity in whom lifestyle interventions, with or without anti-obesity medications, have been insufficient.Weight regain is a recognized complication of bariatric surgery that affects about 20 to 25% of patients and is more prevalent after the second postoperative year . Weight Ames et al.). Recognizing problematic eating behaviors and the complexity of eating behaviors offers an opportunity to tailor a behavioral treatment strategy that is effective for the patient.‘Behavioral Interventions to Attenuate Driven Overeating and Weight Regain after Bariatric Surgery’ focuses on identifying and classifying eating behaviors after bariatric surgery that lead to over-consumption of calories  This is an area of immense growth, and as more patients undergo bariatric surgery and the number of available anti-obesity medications increases, we will learn more about the efficacy, safety, and scope of these medications for this understudied patient population.Anti-obesity medications are effective therapies for treating post-bariatric weight regain. In ‘Pharmacologic Management of Weight Regain Following Bariatric Surgery‘, the authors discuss the evidence supporting the use of anti-obesity medications for weight regain in patients who have undergone bariatric surgery . This review highlights short and long-term weight loss outcomes and safety data associated with these emerging procedures. As with other treatment modalities, endoscopic therapies show superior outcomes when paired with a multidisciplinary approach to weight regain, which reinforces the importance of a comprehensive treatment strategy for patients with weight regain after bariatric surgery.‘Endoscopic Management of Weight Recurrence Following Bariatric Surgery’ highlights the role of minimally invasive, endoscopic options for managing weight regain in select patients . Understanding the contributors to weight bias internalization can help improve the patient-provider relationship and may reduce delays in patients with weight regain seeking care.A patient’s lived experience with obesity, including their interactions with the healthcare system, influences how and whether they choose to seek treatment for weight regain following bariatric surgery. This is complicated by the high level of attrition to follow-up after bariatric surgery, which may be impacted by experienced weight stigma and bias. In ‘The Role of Weight Stigma in Weight Regain in Bariatric Surgery’ the authors discuss the critical role of societal messaging, as well as messaging within the medical community towards postsurgical patients that can affect adherence and follow-up (Bariatric surgery is a safe, effective, and durable therapy for obesity and its complications. Beyond improving health, bariatric surgery also increases quality of life and life expectancy. However, obesity is a chronic disease and bariatric surgery is not curative. Significant weight regain is a reality for a small proportion of patients who undergo bariatric surgery and may be challenging to treat. The optimal management of post-bariatric weight regain acknowledges the complexity of the contributing physiological, behavioral, and anatomical factors, and that a multidisciplinary team approach can increase the likelihood of optimizing weight, health outcomes, and quality of life.AS: Writing – review & editing. MS: Writing – original draft. JA: Writing – review & editing."}
{"text": "Obesity is a global health concern, necessitating effective weight-loss interventions. This study aimed to compare the efficacy and safety of semaglutide, a pharmacotherapeutic option, with bariatric surgery, a commonly utilized surgical intervention, for weight reduction. A systematic review of clinical trials, including the STEP (Semaglutide Treatment Effect in People) trials, sustain trials, pioneer trials, and the STAMPEDE  trial, was conducted to evaluate the outcomes of these interventions. The analysis of the clinical trials revealed that semaglutide demonstrated significant weight reduction in participants. However, adverse effects such as gastrointestinal (GI) disturbances, increased pulse rate, and rare cases of thyroid cancer were observed. Long-term effects showed partial weight regain and a return of certain cardiometabolic variables to baseline levels after semaglutide withdrawal. Comparatively, bariatric surgery, as demonstrated in the Longitudinal Assessment of Bariatric Surgery (LABS) consortium and supported by the STAMPEDE trial, exhibited higher efficacy in weight reduction and the management of obesity-induced complications such as diabetes. The STAMPEDE trial demonstrated that bariatric surgery, specifically Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG), led to a significantly higher percentage of patients achieving desired diabetes treatment targets compared to medical therapy alone. While bariatric surgery showed superior efficacy, it also carried a higher risk of complications. In contrast, semaglutide presented a noninvasive alternative with significant weight reduction and lower incidences of adverse effects. In conclusion, this study highlights that bariatric surgery, such as Roux-en-Y gastric bypass and sleeve gastrectomy, remains a highly effective intervention for weight loss and management of obesity-induced complications. However, semaglutide represents a valuable noninvasive alternative, offering significant weight reduction and lower risks of adverse effects. The choice between these interventions should be based on individual patient characteristics and a comprehensive assessment of the risk-benefit profile. In 1975, a mere 5% of the global population fell into the category of obesity, whereas by 2014, this figure had more than doubled to 13% [The prevalence of obesity worldwide, characterized by a body mass index (BMI) of 30 kg/mAccording to some studies, gastrointestinal surgery offers unparalleled efficacy and enduring weight-loss outcomes for individuals grappling with obesity . BariatrSemaglutide, an investigational human glucagon-like peptide-1 analogue , is being developed as a potential treatment for type-2 diabetes (T2D). It shares a similar structure to liraglutide and exhibits a high degree of structural similarity (94%) to native human GLP-1 . The motIn recent years, there have been significant advancements in the field of weight loss interventions, specifically focusing on the efficacy of semaglutide treatment and bariatric surgery. This review aims to compare the effectiveness of these two treatment modalities in achieving weight loss outcomes and will acknowledge the practical limitations of using either of these approaches for long-term weight management. While both approaches have shown promise in promoting weight loss, it is crucial to consider factors such as accessibility, cost-effectiveness, sustainability, and potential adverse effects when evaluating their practicality as weight loss interventions. By critically examining the recent developments and limitations of semaglutide treatment and bariatric surgery, this review aims to provide a comprehensive understanding of their respective efficacies and practical implications for individuals seeking long-term weight management solutions.Efficacy of bariatric surgery for weight lossIn the United States, the number of bariatric procedures performed annually was estimated to be around 252,000 as of 2018. Among primary bariatric procedures, approximately 61% were sleeve gastrectomy procedures, while 17% were RYGB procedures. The AGB and biliopancreatic diversion procedures each accounted for less than 2% of the total . AccordiBariatric surgery has shown long-term effectiveness in sustaining weight loss. The mechanisms contributing to sustained weight loss include changes in appetite regulation, altered gut hormone secretion, and modifications in the gut microbiota. A study published in the New England Journal of Medicine in 2017 reported that five years after bariatric surgery, patients maintained an average weight loss of 25% of their initial body weight . The STAIn a clinical cohort study involving 1787 veterans, the effectiveness of bariatric surgery was evaluated for patients who underwent Roux-en-Y gastric bypass (RYGB) and other forms of metabolic bariatric surgeries. The results are summarized in Table As evident from the aforementioned data, at the four-year mark, veterans who underwent RYGB surgery experienced significantly more weight loss compared to those who had SG or AGB.Effectiveness and mechanism of weight loss using semaglutideGLP-1, a hormone crucial for regulating appetite and digestion, is produced in two primary locations within the body: L-cells in the gastrointestinal tract and specialized cells in the hindbrain's nucleus tractus solitarius. The effects of native GLP-1 are mediated through GLP-1 receptors present in various tissues throughout the body. GLP-1 orchestrates a wide array of physiological actions, including enhancing the feeling of fullness, diminishing appetite, and decelerating the process of gastric emptying .Semaglutide, having 94% structural homology with native human GLP-1, exhibits three important modifications. An amino acid substitution at position 8 reduces susceptibility to degradation by the dipeptidyl peptidase-4 inhibitor (DPP-4). Lysine acylation at position 26, along with a spacer and C-18 fatty di-acid chain, facilitates strong and specific binding to albumin. Additionally, an amino acid substitution at position 34 prevents unintended binding of the C-18 fatty di-acid chain. These structural modifications enhance semaglutide's stability, duration of action, and dosing convenience . SemagluThe outcomes of the STEP 1-3 trials revealed significant weight loss of 15% to 16% in participants without T2D and 9.6% in those with T2D, achieved through once-weekly subcutaneous semaglutide 2.4 mg in adults with overweight or obesity -35. In SMultiple studies have been conducted to assess the effects of semaglutide on weight loss. Some of these are summarized in Table Based on the data presented in the table, the clinical trials consistently demonstrate that semaglutide is effective in promoting weight loss across various patient populations. Semaglutide, whether administered orally or subcutaneously, consistently leads to statistically significant reductions in body weight compared to placebo or other antidiabetic agents. The weight loss achieved with semaglutide is dose-dependent, with higher doses generally resulting in greater weight reductions. Additionally, semaglutide has been shown to be effective in reducing body weight when added to insulin therapy or used in combination with other medications. These findings support the conclusion that semaglutide is an effective treatment option for weight management in patients with type 2 diabetes, obesity, or overweight individuals.Complications of metabolic surgery vs. semaglutide for weight reductionMetabolic surgery has remained one of the preferred choices for weight loss; however, despite having a plethora of benefits, it also has certain complications . These cOne of the most common complications of bariatric surgery is nutritional deficiency . MalabsoHepatobiliary complications can also occur after bariatric surgery. The formation of gallstones is a common complication . GallstoDumping syndrome is another significant complication that can occur after bariatric surgery, particularly gastric bypass . This syGastrointestinal complications can also arise after bariatric surgery. Gastric ulcers may develop due to changes in stomach acid production and the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for pain management -46. BoweNeurological complications, although rare, can occur after bariatric surgery -49. PostGynecological complications are also worth considering. Obesity itself can cause infertility due to hormonal imbalances . After bIn conclusion, bariatric surgery can lead to a range of complications affecting different systems in the body. Nutritional deficiencies, hepatobiliary complications, dumping syndrome, gastrointestinal issues, neurological complications, and gynecological complications are among the potential risks. Close monitoring, regular follow-up appointments, and appropriate management strategies are necessary to detect and address these complications effectively, ensuring the best possible outcomes for patients undergoing bariatric surgery.On the other hand, there are patients who tend to lean towards noninvasive techniques such as pharmacotherapy for weight loss, and semaglutide has proven to be markedly successful in this domain; however, like most of the other therapeutic drugs, this comes with a sideline of adverse effects. The clinical trials mentioned above for semaglutide also precipitated certain side effects, some of which are summarized in Table Based on the available information, the gastrointestinal (GI) side effects associated with semaglutide, such as nausea, diarrhea, vomiting, and constipation, are generally self-limiting. This means that they typically resolve on their own without the need for permanent treatment discontinuation. Most of these GI side effects were reported to be mild-to-moderate in severity and transient in nature.In the STEP and sustain trials, there have been reports of thyroid cancer cases in participants treated with semaglutide. However, it is important to note that the incidence of thyroid cancer in these trials was relatively low and overall not statistically significant compared to the control groups -37. In tThe main finding from the STEP 1 trial extension is that after a significant reduction in body weight during 68 weeks of treatment with once-weekly subcutaneous semaglutide plus lifestyle intervention, most of the weight loss was regained within one year after treatment withdrawal. Additionally, some cardiometabolic variables showed a similar change back to baseline levels . This fiThe choice between surgery and semaglutide for weight reduction should be based on individual factors and preferences. Bariatric surgery has demonstrated high efficacy but carries a higher risk of complications, while semaglutide offers notable weight reduction benefits with a lower risk profile. Long-term considerations, patient preferences, and multidisciplinary care should be taken into account when making a decision.While several effects and adverse effects of surgical and pharmacologic interventions for weight loss have been discussed, continuous research is required to understand the long-term outcomes of the treatment modalities. For example, the participants of the STEP trials were roughly between one and two thousand in number, which limits the trial’s ability to showcase semaglutide’s variable effects on larger populations over a longer period of time. In addition to this, the participant number for the pioneer trials remained ever lower than 1000 in instances, further perpetuating the need to have ongoing research in this field with regular follow-ups. As a result, even though research has shown promising effects of semaglutide on weight loss, further studies, including a more diverse variety of ethnicities, are required to know the precise effects of the drug before it develops this niche role for weight loss in the market.In conclusion, semaglutide shows promise as a noninvasive pharmacotherapeutic armamentarium for weight loss, demonstrating notable efficacy. However, it is important to acknowledge the potential adverse effects, such as gastrointestinal disturbances, increased pulse rate, and rare cases of thyroid cancer. While the incidence of these side effects is relatively low, ongoing research is needed to better understand their relationship with semaglutide. The long-term effects of treatment withdrawal indicate partial weight gain and the need for continued intervention. When choosing between bariatric surgery and semaglutide, individual factors and preferences should be considered. Bariatric surgery offers high efficacy but carries a greater risk of complications, while semaglutide provides significant weight reduction benefits with a lower risk profile. A comprehensive approach involving long-term considerations, patient preferences, alternative treatment modalities, and multidisciplinary care is crucial. Further research and longer follow-up studies are necessary to optimize the use of semaglutide and fully comprehend its long-term outcomes and safety profile in clinical practice. This review has gathered information from clinical trials and other forms of previously published data to provide a comparison between bariatric surgery and semaglutide for effective weight loss, which will allow readers to access updated developments in this field of medicine."}