The success of high-throughput sequencing has lead to an increasing number of projects which sequence large populations of a species. Storage and analysis of sequence data is a key challenge in these projects, because of the sheer size of the datasets. Compression is one simple technology to deal with this challenge. Referential factorization and compression schemes, which store only the differences between input sequence and a reference sequence, gained lots of interest in this field. Highly-similar sequences, e.g., Human genomes, can be compressed with a compression ratio of 1,000:1 and more, up to two orders of magnitude better than with standard compression techniques. Recently, it was shown that the compression against multiple references from the same species can boost the compression ratio up to 4,000:1. However, a detailed analysis of using multiple references is lacking, e.g., for main memory consumption and optimality. In this paper, we describe one key technique for the referential compression against multiple references: The factorization of sequences. Based on the notion of an optimal factorization, we propose optimization heuristics and identify parameter settings which greatly influence 1) the size of the factorization, 2) the time for factorization, and 3) the required amount of main memory. We evaluate a total of 30 setups with a varying number of references on data from three different species. Our results show a wide range of factorization sizes , factorization speed (0.01 MB/s to more than 600 MB/s), and main memory usage (few dozen MB to dozens of GB). Based on our evaluation, we identify the best configurations for common use cases. Our evaluation shows that multi-reference factorization is much better than single-reference factorization. The development of novel high-throughput DNA sequencing techniques has led to an exponentially increasing flood of data. Only one/few individuals of each species was sequenced  until recently. Decreasing costs now make it possible to sequence large samples of a given population. Examples for such projects are the 1000-Genomes project ; the UK1optimal factorization against multiple references, based on the number of pointers into reference sequences. We use this measure to evaluate a multitude of different factorization algorithms.We define the notion of an keep track of previously factorized sequences and exploit this information to speed up optimal factorization.We devise three basic algorithms for computing an optimal factorization, each with different runtime characteristics. While the first algorithm is history-free, i.e. it factorizes all sequences independently from each other, the other two algorithms factorizes against multiple references, while only an index over a single reference is needed, and thus requiring significantly less main memory.We develop a reference extension technique, which 30 configurations, which are instantiations of our general factorization framework, on six datasets from three species .We perform an exhaustive evaluation on best configurations depending on common use cases using multi-criteria decision analysis techniques.Our results show a wide range of factorization rates , factorization speed (0.01 MB/s to more than 600 MB/s), and main memory usage (few dozen MB to a dozens of GB). We identify the Compressing DNA with standard compression techniques is ineffective, since the distribution of symbols is close to random. Accordingly, standard compression schemes can often reduce the space requirements by only approx. 6:1 (one base is encoded with up to 1.3 Bits), see –10 for rsequences is a finite ordered list of characters from an alphabet Σ. The concatenation of two sequences s and t is denoted with s ∘ t. A sequence s is a subsequence of sequence t, if there exist two sequences u and v (possibly of length 0), such that t = u ∘ s ∘ v. The length of a sequence s is denoted with ∣s∣ and the subsequence starting at position i with length n is denoted with s. s(i) is an abbreviation for s. All positions in a sequence are zero-based, i.e., the first character is accessed by s(0). A referential factorization of a sequence encodes the sequence as a concatenation of subsequence-pointers into references. We extend the definition of referential factorization from a single reference  such that  ∘ m1) ∘ … ∘  ∘ mn) = s.size of a RME  is defined as l+1. The offset of a RME rmei in a referential factorization f = , denoted offset, is defined as ∑j < i∣rmej∣.The s is traversed from left to right, and at each step, the longest prefix of s which can be found in any of the references {ref1,…, refm}, is replaced with one RME. Unfolding a factorized sequence f is equally simple: We replace each RME in f with its unfolded sequence, where the unfolding of a single RME rme =  is id with the mismatch character m concatenated to the end. The factorization of s against REF is denoted with f = fact and the unfolding of f is denoted with s = unfold.The algorithm shown in REF to a single sequence ref and then compute a factorization against ref. In this case, the factorization can be computed using standard factorization algorithms, e.g. , describe a suffix of . The same observation holds when further extending to the left:  is a suffix of  although both sequences are encoded with distinct RMEs. Extending further to the left, we find thef1is a subsequence off2and that the last three RMEs off2encode a subsequence off1. These three RMEs inf2can be replaced with , where 1 is the identifier for factorized referencef1.fi accordingly to represent the longer match into the factorized reference. We call this process left-extension. In general, it is possible to combine left-extension with right-extension. However, the design of such algorithms is complicated, since one needs to factorize in both directions and possibly repeatedly replace previously factorized subsequences.We exploit the observation from Example 5 as follows: Given two identical RMEs as anchors, one in a factorized reference and one in a to-be-extended factorized sequence, we unfold the RMEs on-the-fly towards the left, until neither of the two unfolded sequences is subsequence of the other one. We find the longest possible extension  and rewrite the RME-subsequence of We used six real-world datasets in our experiments: 110 sequences from two different chromosomes of Arabidopsis thaliana (AT1 and AT5), 110 sequences from three human chromosome , and a set of 38 yeast genomes (yeast). Details of the datasets are shown in http://1001genomes.org/data/GMI/GMINordborg2010/releases/current/. For each strain, a file with SNPs with respect to the reference TAIR9 is provided. The dataset for yeast [We used the same datasets as in . The dator yeast  genomes http://www.informatik.hu-berlin.de/~wandelt/MRF for free academic use.We conducted an extensive set of experiments for the evaluation of factorization methods and heuristics, in terms of factorization size, speed, and main memory usage. All experiments were run in a server with 1 TB RAM and 4 Intel Xeon E7-4870, where all experiments were run in a single thread only. Parallelization is left for future work. Our analysis shows that single-threaded factorization algorithms already face an IO-bottleneck for highly-similar sequences. Code was implemented in C++, using the BOOST library, CST  for compThe setup for our experiments is described in Section 3.1 and parameters for experiments are analyzed in Section 5.1 In Section 3.2 we report on the results of optimal multi-reference factorization and discuss heuristic factorization in Section 3.3. We analyze the speed of multi-reference factorization techniques in Section 3.4 and report on main memory usage in Section 3.5.We compared 30 configurations: For each configuration we chose to instantiate the following: the type of index structure for reference sequences, the match finding algorithm, and the reference extension method. These components of a configuration are described as follows:CST), enhanced suffix array (ESA), and a k-mer index (KMER). The value of k is chosen in Section 5.1.We have used three index structures for references: Compressed suffix trees (BASE), RME-successor prediction (SUCC), positional RME prediction (POSI) and local match-finding (LOMA). The parameters for LOMA are chosen in Section 5.1.We compared four match finding algorithms: Basic greedy lookup in the references (L), right-extension (R) and no extension .We distinguished three types of reference extension methods: Left-extension  for each reference (N) and uses positional RME prediction (POSI). We checked all combinations except from k for a k-mer index is set to 16, δ = 10, and lmin = k = 16  for a varying number of references. We repeated the experiments with different randomly selected references and report the average values. The references were external to the datasets we used, i.e., references and to-be-factorized sequences were disjoint. The results are shown in Sequences from Human genomes have the highest absolute exponents, which means that additional references can be much better exploited than for AT and yeast. The R2-value for all six datasets is very high. Our evaluation suggests that the number of RMEs can be approximately predicted given the species/type of a sequence and a number of references. This finding should be further investigated in future research. Previous research  showed tk. For all HG, average match lengths are larger than k and thus often an optimal match is found. For the other species (AT and yeast), matches are often rather short and a RME consists of only one character, if no match longer/equal than k can be found.ESA-based local matching is always better than KMER-based local matching: This is explained as follows: if local-matching does not yield a match, then an optimal match is found with ESA. KMER only can find approximate matches, if the match length is shorter than Left/right-extension on an optimal factorization creates smaller representations than factorization based on local matching. The explanation is as follows: When using local matching for initial factorization, the algorithm is sometimes stuck in finding short local matches, while larger matches could be found with an index. These short matches are then encoded with distinct RMEs for different factorizations and thus do not provide a starting point for extension. An optimal factorization, on the other hand, produces more often identical RMEs for the same subsequence, which leads to more starting points for left/right-extension.Left-extension often creates fewer RMEs than right-extension. The reason is as follows: factorization with locality-based heuristics prefers local matches over (possibly better) matches more far away. Thus, two identical subsequences from two different sequences are sometimes encoded with different RMEs, if factorization starts in different contexts. Only once a global (large) match is found, the remaining suffixes of the two identical subsequences are encoded with the same RME(s), since the factorization context is equal for the suffixes. Thus, left-extension can uncover new kinds of sequence equalities which are hidden from right-extension.Left/right extension can even lead to (slightly) smaller representations than factorization without extension but local matching. This fact is explained as follows: When using local matching with many reference indexes, the factorizations is sometimes stuck in one reference, while much longer matches could be found in another reference. If, on the other hand, only one reference with extension is used the identical subsequences are partly identified during the extension phase. This observation is very surprising and interesting, since we will show below that factorization with extension needs less memory than factorization without extension: We obtain a better factorization, while always using less memory for references.We evaluated the quality of factorization heuristics compared to the optimal factorization. The increase in number of RMEs of each heuristic against the optimal factorization as a baseline is shown in We analyzed the factorization speed of selected configurations. First, we evaluated the factorization speed of optimal factorization techniques. The result for AT1, HG21, and yeast is shown in Factorization speed slowly decreases with an increasing number of references, since for each suffix, more lookups have to be performed. At the same time, the average match length increases .The efficiency of our RME graph-based optimizations can be seen for all datasets: With increasing similarity of sequences, SUCC and POSI perform increasingly better (speedup of approx. 30% for yeast and approx. 300% for HG21). Note that, as anticipated, POSI is always faster than SUCC, since more often the optimal match is predicted based on the context of factorization.We analyzed the factorization speed of selected approximate methods next. The results are shown in We analyzed the main memory usage of configurations and results are shown in With an increasing number of references, configurations with left-extension have a much lower memory footprint than their counterparts with no extension. This shows that additional overhead of RME graphs for references is small compared to the size of an index over unfolded references. The smallest overall memory footprint is obtained by CST_BASE_L. Note that all right-extension techniques use the same amount of memory as their left-extension counterparts, because they work on the same data structure (RME graphs).It is interesting to note that the ranking of techniques according to the used main memory is the same for all datasets. This observation suggests that the main memory usage of the configurations is rather independent on the degree of similarity between the sequences, but dominated by the number and length of the references. However, it can be seen that configurations with left-extension are sensitive to the similarity of the sequence, e.g., the growth of main memory usage is much steeper for (not so similar) yeast sequences compared to (highly-similar) human sequences: Dissimilar sequences yield many (short) referential match entries, which needs to be kept in the RME graph for reference extension.https://www.cs.helsinki.fi/group/pads/lz77.html. According to that webpage, ISA6r is specialized for highly repetitive inputs, KKP3 is the fastest for non-highly repetitive inputs, and KKP1s streams a suffix array from the disk. The experimental results are reported in n*∣LR∣ bytes of storage in memory , where LR is the length of the shortest reference and n is the number of references. Moreover, the factorization speed is often much slower than our own baseline implementation with enhanced suffix arrays. The size of factorizations decreases tremendously with the number of references, similarly to our own experiments.We evaluated experimentally the following other factorization algorithms: ISA6r , KKP3 3, and KKPSequence compression algorithms caught a lot of attention in the research community during the last years  problem. We solve the following MCDA-problem below: e TOPSIS , 48 to fThe first scenario  attempts to select an overall good factorization method. The best ranked methods all use a (fast) KMER index and local matching for a medium number of references. The worst method is ESA_BASE_N_10, which uses a large ESA index, non-optimized matching and ten references without extension. When we increase the weight for factorization speed (from 33% to 80%), the KMER-based methods with local matching and only a few references are ranked best. If we set the priority on small factorizations, left-extension setups with optimizations (POSI/SUCC) are ranked best, with the exception of KMER_BASE_N_10, which yields a factorization close to the optimum, with medium memory usage and medium factorization speed. Finally, methods with KMER and local-matching are also ranked best for factorization with small main memory usage. If we further increase the preference weights to 99%, we obtain the following results: KMER_LOMA are the best methods for fast factorization, ESA_POSI and ESA_SUCC are the best methods for small factorizations, and CST_BASE methods are best for a low memory footprint.We have devised and implemented referential sequence factorization against multiple references. Dissection of the framework into its components led to 30 configurations which were evaluated on six datasets from three species. Our analysis shows a wide range of factorization rates, factorization speed, and main memory usage. We identified the best configurations depending on common use cases.One direction for future work is to a develop multi-reference factorization algorithm using recently developed multi-genome indexes , 44. Thek for a k-mer index . We decided to set k = 16: then the average number of occurrences for k-mers is less than 2 for each dataset.We evaluated parameters for indexing and match finding techniques first. The choice of ribed in ) has a sδ value for our local match-finding algorithm. In general, a small δ will only identify SNPs (or very short indels), while a larger δ lead to increased verification times, since a larger neighborhood of the previous match end have to be compared to the to-be-factorized sequence prefix. The results of our analysis are shown in δ ≤ 13. For all datasets, setting δ = 0, which only identifies SNPs, saves 50% or more of the index lookups. For Human chromosomes, more than 80% of the index lookups can be saved, since many sequence deviations in Human chromosomes from the 1000 Genome Projects are SNPs. The factorization speed is increased by a factor of almost 2  to factor of 3 and more (HG). For Human genomes, δ larger than 0 further avoids index lookups (and increase factorization speed), by detecting short indels. For the other datasets, factorization speed remains stable (yeast) or is slightly decreasing (AT). Our results for AT show that local match finding can reduce the factorization speed, if no short indels can be found and neighborhoods of previous matches are verified without success. We chose δ = 10 for our remaining experiments. For the algorithm in lmin = k = 16, since this avoids spurious matches in the sequence.Next we chose a Next we analyzed the effect of RME graphs on factorization speed. Our analysis showed that it is not beneficial to store the RME graph of all previously factorized sequences for two reasons. First, the memory usage is linearly increasing with each sequence. Second, the time spent on checking RME graph predictions increases with the number of sequences in the RME graph. We measured the time spend on SUCC/POSI prediction and index lookups separately for an increasing number of factorized sequences, to find out how many sequences are actually necessary. The result is shown in


Background. A follow-up program for high risk infants was initiated in Alzahra Maternity Hospital in Tabriz city, Iran, in 2013. The aim of this paper is to give a brief report of the program. Material and Methods. Two groups of high risk neonates were studied. The first group comprising 509 infants received services in Alzahra Maternity Hospital implemented by the follow-up program. This included a full package for family to look after high risk infant and periodic clinical evaluation at two and four weeks after birth and then two, three, four, five, and six months later again. The second group including 131 infants in Taleqani Maternity Hospital received routine services after birth with no specific follow-up care. Results. Some anthropometric indices showed a significant improvement in the intervention hospital compared to control group. These included the following: head circumference at first and second months; weight in the first, fourth, fifth, and sixth months; and height in sixth month only. Clinical evaluation of infants showed an improvement for some of the medical conditions. Conclusion. Follow-up care program for a minimum of six months after discharge from maternity hospitals may help to avoid adverse and life threatening consequences in high risk infants.  According to Barker's theory, certain risk exposures few months before pregnancy, during pregnancy, and in early childhood might have a role in the occurrence of some disorders in adulthood . It is, A follow-up program for high risk infants was initiated in Alzahra Maternity Hospital in Tabriz city of Iran in 2013 . The maiThis study was carried out in Tabriz city. The city is one of the three major cities in Iran located in the northwest of Iran, a cold climate zone, with a total population of 1,398,060 in 2012 and an average annual population growth rate of 0.8 percent.High risk infant was mainly defined based on the birth weight (less than 1500 grams) and gestational age (less than 32 weeks). If an infant did not, however, have those criteria, other medical conditions  were considered for risk assessment at birth/soon after. Full details of the high risk conditions can be found elsewhere , 21.Two groups of high risk neonates were studied. The first group comprising 509 infants (282 boys and 227 girls) received services in Alzahra Maternity Hospital implemented by the follow-up program. This included a full package for family/mothers to train them to look after high risk infant and periodic clinical evaluation by fully equipped/trained medical staff at two and four weeks after birth and then two, three, four, five, and six months later again. At those time points, the pediatric nurse of the program called the family (mainly mother) to bring the child for clinical follow-up. At this regular medical program, every infant was assessed for cardiac problems, lung diseases, gastroesophageal reflux, anemia and nutritional assessment , immunization (according to the country regular plan), hypothyroidism, osteopenia, nephrocalcinosis, renal tabular acidosis, Developmental Dislocation of Hip (DDH), intraventricular hemorrhage, neurodevelopmental assessment , retinopathy, auditory assessment, a comprehensive psychological assessment and mental health , sleep disorders (using a standardized questionnaire), oral health assessment, and anthropometric indices . Bronchopulmonary Dysplasia (BPD) is routinely fully managed during the hospital stay for every high risk infant. After discharge, a detailed follow-up program is organized beginning from the fourth year onward.The second group including 131 infants (63 boys and 68 girls) in Taleqani Maternity Hospital received routine services after birth with no specific/regular follow-up or any specific nurse for this. They were medically assessed at the same time points and at the same hospital to compare them with the status of high risk infants in the first group. Infants diagnosed, as high risk infants, in two maternity centers  entered the follow-up program. These hospitals routinely provide obstetric and gynecological services in the study population under the Tabriz University of Medical Sciences. Those hospitals are both public maternity facilities in similar social/economic areas. They are located in two different places in the city covering the population in two various zones in the city. Confounding factors were considered in both groups for the purpose of data analysis. Those factors included gender and risk status .We also assessed the attitude and practice of staff and families towards the program using a questionnaire and interview and filling a check list while they are doing the job.For the data analysis, we calculated 95% confidence interval for statistical indicators.The study obtained ethics approval from the committee of ethics in Tabriz University of Medical Sciences. All personal and identity information were kept confidential. Medical staff and families were asked whether or not they are willing to participate in the study and were told that they are free to leave for any reason at any time.Of those high risk infants eligible for follow-up care in Alzahra Hospital, 33 percent  refused, even after they were called for three times, to enter the follow-up program where 84 percent  of control families with high risk infants did not participate in the hospital for follow-up services. In both groups, those refusing families either preferred to get follow-up services from private doctors and clinics or never got follow-up care at all over the study period.We compared the improvement of clinical and anthropometric indices in Alzahra and Taleqani Hospital to assess the effectiveness of this program. Some anthropometric indices showed a significant improvement in the intervention hospital compared to control group over the follow-up period. These included the following: head circumference at first and second months; weight in the first, fourth, fifth, and sixth months; and height in sixth month only . There wBefore the program begins, it is essential to appoint a neonatologist  and a trained pediatric nurse/midwife as the focal points for the program in every hospital qualified for starting the follow-up care program for high risk infants. The program nurse/midwife willcoordinate the follow-up care with family;provide the family with full package of the follow-up care ;organize the timetable of clinical visits at regular basis;arrange instruction and training sessions for family;enter the relevant data in the program online software;prepare regular reports of the follow-up program.Access to some paramedical services has to be organized/arranged for those infants needing these services after discharge. Some of those services include occupational therapy, physiotherapy, speech therapy, clinical nutrition services, and psychology and psychotherapy facilities. Some families may also need financial aids to access those services.A major attention should be made for recall and follow-up of those high risk infants living in the villages far from the program hospital. They may need several calls to come for follow-up care. The program nurse/midwife may also need to be in close contact with the local family doctor to organize the regular follow-up visit for those infants.One of our main limitations in the implementation of this follow-up program was improper infrastructure and insufficient capacity of online data handling in the country. If a program is to be implemented across the country, the online communication of the data between states, hospitals, and main office in the ministry of health has to be set up/administered properly.In this paper, we briefly reported a follow-up care program for high risk infants in Tabriz city of Iran. We showed that the program was seemingly effective in finding and managing some of the medical conditions in neonates over the six-month follow-up. Similar findings were reported before from various countries –28. HoweAs a conclusion, a follow-up care program for a minimum of six months after discharge from maternity hospitals may help to avoid adverse and life threatening consequences in high risk infants.


The TGF-β transforming growth factor is the most pleiotropic cytokine controlling a broad range of cellular responses that include proliferation, differentiation and apoptosis. The context-dependent multifunctional nature of TGF-β is associated with complex signaling pathways. Differential models describe the dynamics of the TGF-β canonical pathway, but modeling the non-canonical networks constitutes a major challenge. Here, we propose a qualitative approach to explore all TGF-β-dependent signaling pathways.Using a new formalism, CADBIOM, which is based on guarded transitions and includes temporal parameters, we have built the first discrete model of TGF-β signaling networks by automatically integrating the 137 human signaling maps from the Pathway Interaction Database into a single unified dynamic model. Temporal property-checking analyses of 15934 trajectories that regulate 145 TGF-β target genes reveal the association of specific pathways with distinct biological processes. We identify 31 different combinations of TGF-β with other extracellular stimuli involved in non-canonical TGF-β pathways that regulate specific gene networks. Extensive analysis of gene expression data further demonstrates that genes sharing CADBIOM trajectories tend to be co-regulated.As applied here to TGF-β signaling, CADBIOM allows, for the first time, a full integration of highly complex signaling pathways into dynamic models that permit to explore cell responses to complex microenvironment stimuli. TGF-β binding to TβRII induces the recruitment and phosphorylation of TβRI, which transduces signals to downstream intracellular substrates, the first of which are the R-Smad proteins. Once phosphorylated, R-Smad proteins hetero-dimerize with a common partner, CoSmad, and heterodimeric complexes move into the nucleus where they regulate the transcription of TGF-β target genes. Alternatively, non-Smad pathways involved in TGF-β signaling include several MAP kinase pathways, the Rho-like GTPase signaling pathways, and phosphatidylinositol-3-kinase/AKT pathways → B. A and B are respectively the origin and target places of the transition. The guard of the transition is composed by an event h and a condition Cond, a logical formula with places as variables and ∨, ∧ and ¬ as logical operators.CADBIOM formalism is based on guarded transitions and the introduction of discrete signals called events. A guarded transition is graphically represented by: A ⊥). A realization of a finite set of events (hi)Ii∈ is a sequence of elements of {T, ⊥}I \{ ⊥I }. To handle integrated models containing large amount of data, we combine events and states. Operations on events are well known in computer science. The two basic operators are a merge that corresponds to multiplexing and a selection of occurrences corresponds to under-sampling. CADBIOM borrows the default operator and the when operator from the Signal language [h1 and h2. The event h = (h1 default h2) is present when either the event h1 or the event h2 is present, h is absent otherwise. The when operator is an operator between events and logical combinations of state variables and selects occurrences of an event when the propositional formula evaluates to True on its right-hand side. The event h = (h1 when B) is present when h1 is present and B is True, it is absent otherwise.Places are state variables that take Boolean values and represent biomolecules such as proteins or complexes. An event is the mathematical concept that denotes an occasional occurrence. An event has a name h and a singleton domain to denote the occurrence of the event. At each step, an event can occur (denoted by the symbol T) or not . The transition event htr is present if and only if h is present and (A ∧ C) is True, that is, when the input place is True and the condition C is verified. When a transition is fired, the source is inactivated and the target is activated. The evolution function of a state A relies on the current value Ak+1 at step k to the next value Ak+1 at step k + 1.The system evolves according to transition firings that change the value of places, which is either The state of a place changes if either an in-transition or an out-transition is fired. In the case of simultaneous firing, we postulate that activation prevails over inactivation.We define two events associated with a place A by:hin event is present if at least one of the in-transitions is fired. The hout event is present if at least one of the out-transitions is fired. The mathematical formalization of the rules is given by:The A’ is the value of A at next step.where The initial condition for simulation analyses requires the design of activated places  and a specific timing. CADBIOM proposes either a manual selection or an automatic selection of “frontier” places. Frontier is the set of places which cannot be activated from inside the model. In guarded transition models, places without input transitions belong to the frontier. The script for events timing is provided during reachability analyses as a txt file that can be loaded in simulation toolThe temporal properties of models are explored using methods based on model checking. The aim of these methods is to determine an allocation of state variables and event timing that verify a property, that is, any logical formula composed of model places and logical operators ∨, ∧ and ¬. To investigate the reachability of biological properties such as "how to express a given gene?", we search for the allocation of states variables and event timing that lead to the activation of the place symbolizing the gene. We focus on frontier places because signaling networks are usually polarized from the extracellular environment to the nucleus. A frontier is the set of places without any in-transitions that cannot be activated from inside the model. All other places are initialized in an inactivated state.i and i + 1. We then unfold the trajectory from step 0 to n. The whole formula is then given to a SAT solver to find an allocation of state variable and event timing that satisfies the formula.We first generated the formulas that describe the evolution of the system. Using the evolution rules above, dynamic models are translated into propositional logic formulas using the Tseitin translation. The resulting formulas are under conjunction normal form (CNF) and qualify the dynamics between steps h. This reachability property is then used to search for minimal scenarios such that the property is not achieved as soon as one component is removed in the initialization places or as soon as a component of event timing is disabled. A scenario is said to be minimal if, for any place A ∈ F,  is not an activation condition and for any i < n and any h ∈ Hi, ) is not an activation condition. Any scenarios can be simulated to retrieve all the activated places that lead to reachability of the property. These activated places compose a trajectory.We next define a scenario as the  pair, where F is a set of places which are activated at the initialization step and T is a sequence of sets of events CADBIOM software includes several compilers sharing the same back-end. The front-ends compile PID XML files, CADBIOM XML files and Cadlang files into an intermediate representation of guarded-transition models. The common back-end generates logical constraints in propositional clause form. During this step, constant propagation and common sub-expression elimination optimizations are performed. Combined with many peephole optimizations, these techniques allow the back-end to generate reduced sets of constraints that facilitate the work of the SAT solver.http://pid.nci.nih.gov/). See Additional file http://www.msoos.org/cryptominisat2/). Graph representations of solutions are displayed using Gephi, an open-source platform for graph visualization (https://gephi.org/).The CADBIOM model is a translation of the PID database content  and 1 (present) in trajectories.Click here for fileGenes with non_Smad-independent trajectories identified by CADBIOM. The Table lists genes with non-Smad-dependent trajectories. Gene names (ID) are given according to their nomenclature in the HUGO and PID databases, respectively, and are followed by their description.Click here for fileTarget genes regulated by combinations of extracellular stimuli identified by CADBIOM. Proteins that serve as extracellular stimuli and their combinations thereof are listed together with their target genes, listed according to the PID nomenclature.Click here for fileList of the 649 pairs of genes randomly chosen to evaluate the association between co-expression and trajectories.Click here for file


To promote appropriate antimicrobial use in bloodstream infections (BSIs), we initiated an intervention program consisting of electronic alerts and automated infectious diseases consultations in which the identification and antimicrobial susceptibility test (ID/AST) results were reported.We compared the appropriateness of antimicrobial prescriptions and clinical outcomes in BSIs before and after initiation of the program. Appropriateness was assessed in terms of effective therapy, optimal therapy, de-escalation therapy, and intravenous to oral switch therapy.There were 648 BSI episodes in the pre-program period and 678 in the program period. The proportion of effective, optimal, and de-escalation therapies assessed 24 hours after the reporting of the ID/AST results increased from 87.8% , 64.4% (95% CI 60.8–68.1), and 10.0% (95% CI 7.5–12.6) in the pre-program period, respectively, to 94.4% (95% CI 92.7–96.1), 81.4% (95% CI 78.4–84.3), and 18.6% (95% CI 15.3–21.9) in the program period, respectively. Kaplan-Meier analyses and log-rank tests revealed that the time to effective (p<0.001), optimal (p<0.001), and de-escalation (p = 0.017) therapies were significantly different in the two periods. Segmented linear regression analysis showed the increase in the proportion of effective (p = 0.015), optimal (p<0.001), and de-escalation (p = 0.010) therapies at 24 hours after reporting, immediately after program initiation. No significant baseline trends or changes in trends were identified. There were no significant differences in time to intravenous to oral switch therapy, length of stay, and 30-day mortality rate.This novel form of stewardship program based on intervention by infectious disease specialists and information technology improved antimicrobial prescriptions in BSIs. In the treatment of bloodstream infections (BSIs), administration of appropriate antimicrobials without delay is crucial for improving clinical outcomes –3. HowevThere have been several trials of interventions to improve the appropriateness of antimicrobial therapy in BSIs, some of which led to improvements in antimicrobial prescribing and clinical outcomes ,8,9. HowTo promote appropriate antimicrobial use in BSIs, we initiated an intervention program in August 2011 which consisted of electronic alerts and automated infectious diseases consultations, and evaluated the impact of the program on the appropriateness of antimicrobial prescriptions.Our institution is a tertiary teaching hospital with 900 beds. We planned an intervention program taking advantage of our health information system for electronic medical records (EMR) in our hospital. The key feature of this program is a pop-up message, which gives the ID/AST results with a note about whether a consultation with an infectious disease specialist (IDS) is needed. Attending physicians can issue the consultation according to the automated process. The only action needed to make a consultation is to click on the message. However, the consultation is not mandatory and can be canceled . Pop-up We designated the period before the initiation of the program as the “pre-program period”, and the period of the program as the “program period”. Excluding the month of initiation of the program (August 2011), the total duration of the study was 2 years, comprising a 1-year pre-program period (from August 2010 to July 2011) and a 1-year program period (from September 2011 to August 2012).Corynebacterium species or coagulase-negative staphylococci were considered pathogens only when two or more sets of blood cultures were positive within 48 hours, and when clinically related infection existed.During the study period, all episodes of BSI in adult (≥18 years) patients were reviewed. A BSI episode was defined as growth of pathogenic bacteria or fungi from one or more sets of blood culture samples in a patient with systemic inflammatory response syndrome. When present, common skin commensals, such as BSI episodes were included in the analysis only when blood cultures were performed for hospitalized patients or those in the emergency department. Because the program started to operate after the reporting of the ID/AST results, the episodes involving patients discharged from the hospital before the reporting were excluded. We considered that the impact of the program on antimicrobial prescription and clinical outcomes could not be assessed appropriately in cases involving patients who died within 24 hours after the reporting and patients with polymicrobial infection, defined as the isolation of two or more pathogens from the same blood culture set. Furthermore, the appropriateness of the use of antimicrobial agents for polymicrobial infections was not estimated solely on the basis of the ID/AST results. Therefore, the episodes that involved early death and polymicrobial infections were excluded from the study.BSI onset was defined as the time when the initial blood culture was ordered. If there was a new positive culture two weeks after BSI onset, that episode was considered a separate one. However, if an obviously different infection developed, it was counted as a separate episode even when it developed within two weeks of the initial episode.Primary outcomes were the time to appropriate antimicrobial administration and the frequency of appropriate antimicrobial administration after the ID/AST results were reported. The appropriateness of antimicrobial use was assessed in terms of effective therapy, optimal therapy, de-escalation therapy, and intravenous to oral switch. Secondary outcomes were length of stay, and all-cause and infection-related mortalities 30 days from BSI onset. Infection-related mortality was defined according to the following criteria, adapted from Harbarth , (1) posReference time: the time when the ID/AST results were reported .Type of acquisition: (1) community-onset BSI: BSI that occurred <48 hours after admission ; (2) healthcare-associated BSI: BSI that occurred <48 hours after admission with risk of healthcare exposure ; (3) hosEmpirical therapy: antimicrobial in use at the reference time. Antimicrobials were classified into ineffective, effective and optimal, and effective but non-optimal. Superfluous broad-spectrum antimicrobial therapy was considered effective but non-optimal.Effective therapy: Effective therapy was defined as the administration of an active antimicrobial for a confirmed pathogen for each ID/AST result. Time to effective therapy was defined as the interval from the reference time to effective therapy.Superfluous broad-spectrum antimicrobials: if the antimicrobials of interest were used as empirical therapy and the ID/AST results revealed that their spectra were excessive, we classified them as superfluous. For gram-negative BSI, the antimicrobials of interest were piperacillin/tazobactam, ceftazidime, cefepime, carbapenem, and polymyxin E. Glycopeptide and oxazolidinone were the antimicrobials of interest for gram-positive BSI .Optimal therapy: Optimal therapy was defined as the administration of an effective antimicrobial which was not one of the superfluous broad-spectrum antimicrobials and adequate in relation to the site of infection, route of administration, and dosing. Likewise, time to optimal therapy was defined as the interval from the reference time to optimal therapy.De-escalation therapy: De-escalation therapy was defined as the discontinuation of use of a broad-spectrum antimicrobial and the administration of an antimicrobial with a narrower spectrum. We assumed the time of discontinuation of an antimicrobial to be the time of the next scheduled administration of the antimicrobial. The antimicrobial with a narrower spectrum was not necessarily the one with the narrowest spectrum of those available.Intravenous to oral switch: Only the cases involving the switch to antimicrobials that were active in vitro were included in the analysis. We considered the switch as appropriate in cases of (1) absence of clinical indication for intravenous therapy, such as CNS infection and endocarditis, (2) normalization of body temperature, (3) decrease in the level of inflammatory markers, (4) improvement in the patient’s symptoms and signs, and (5) normal gastrointestinal absorption  ,12.t-test or the Mann-Whitney median test for continuous variables. Kaplan-Meier analysis and log-rank tests were performed to compare the time to effective therapy, optimal therapy, de-escalation therapy, and intravenous to oral switch. All data were censored at 10 days (240 hours) from the reference time. We considered the data as censored when a patient died within 10 days after reporting, or if the patient was discharged from the hospital within 10 days and was not monitored after this period.To compare general characteristics between the two periods, the chi-square test or Fisher's exact test was used for categorical variables, and Student’s Segmented linear regression analyses were performed to evaluate the trends of outcomes over time before and after the initiation of the intervention program . AnalyseAll analyses were performed using SAS software version 9.4 (SAS Institute Inc.). All tests were two-tailed and adopted an alpha level of 0.05.The study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (IRB No. B-1412/278-113). The study was conducted with a waiver of informed consent from individual patients. Patient data were de-identified and anonymized before analysis.A total of 879 episodes in the pre-program period and 899 episodes in the program period were identified as BSIs of clinical significance. After that, 231 episodes in the pre-program period and 221 episodes in the program period were excluded according to the exclusion criteria. Therefore, 648 episodes in the pre-program period and 678 in the program period were included in the analysis . The corThe time from BSI onset to the reporting of the ID/AST results was shortened in the program period by 9.0 hours . The automated consultations were canceled by the attending physicians in 22.0% of all episodes in the program period (149 of 678). In the remaining 529 episodes, in which the consultations were not canceled, the mean interval from automated consultation forwarding to signed reply was 6.8 hours (95% CI 5.8–7.8).The clinical characteristics of the episodes in the two periods are compared in Escherichia coli . The proportion of extended-spectrum beta-lactamase (ESBL) producers among the isolates of E. coli was larger in the program period . The pathogen distribution is shown in There were also no significant differences in the distribution of pathogens between the two periods except for cases of coagulase-negative staphylococci  and We compared the time to effective therapy between the two periods in two steps according to the target group using Kaplan-Meier methods as shown in The proportions of optimal therapy at 24 and 48 hours increased after the reporting of the ID/AST results from 64.4% (95% CI 60.8–68.1) and 74.3% (95% CI 70.9–77.6) in the pre-program period to 81.4% (95% CI 78.4–84.3) and 91.4% (95% CI 89.3–93.5), respectively, in the program period for all episodes . For nonAmong the effective episodes, the proportions of de-escalation therapy at 24 and 48 hours after the reporting of the ID/AST results increased from 10.0% (95% CI 7.5–12.6) and 17.0% (95% CI 13.8–20.3) in the pre-program period to 18.6% (95% CI 15.3–21.9) and 26.6% (95% CI 22.8–30.4) in the program period, respectively . Among tThe proportion of switch therapies performed on days 2, 4, and 8 increased from 8.5% (95% CI 6.4–10.7), 14.1% (95% CI 11.4–16.8), and 22.2% (95% CI 18.9–25.4), respectively, in the pre-program period to 9.1% (95% CI 7.0–11.4), 16.1% (95% CI 13.3–18.9), and 24.4% (95% CI 21.1–27.7), respectively, in the program period. However, Kaplan-Meier analysis and the log-rank test showed that there was no statistically significant difference between the times to intravenous to oral switch in the two periods (p = 0.447).The results of segmented linear regression analyses are shown in The number of episodes in which the outcome of the admission treatment was death was 85 in the pre-program period and 89 in the program period. These cases were excluded from the estimation of length of stay. There was no significant difference in the median length of stay between the two periods (16.6 [interquartile range (IQR) 8.8–29.8] versus 16.0 [IQR 9.5–28.9], p = 0.715). There was also no significant difference in all-cause 30-day mortality rate between the two periods . The infection-related 30-day mortality rate decreased (39/648 versus 27/678), but there was no statistical significance (p = 0.089).When we compared the 22.0% (149/678) whose automatically generated consultations were canceled to the remaining 78.0% (529/678), log-rank tests revealed there were no significant differences in time to effective, optimal, de-escalation, and intravenous to oral switch therapies . Length However, the proportion of subjects on effective  and optimal  antimicrobials at the reference time was higher in the canceled subgroup. The proportion of subjects on superfluous broad-spectrum antimicrobials at the reference time was not significantly different . The proportion of cases in which infectious diseases consultations were issued between 24 hours before the BSI onset and the reference time was higher in the canceled subgroup (51.0% [76/149] versus 45.0% [238/529]) without statistical significance (p = 0.193).This study showed that the intervention program in our institution promoted appropriate antimicrobial prescription in the treatment of BSI. Kaplan-Meier analyses and log-rank tests revealed that the probabilities of receiving effective, optimal, and de-escalation therapies were higher in the program period. Furthermore, time to effective, optimal, and de-escalation therapies was shortened in the program period. Segmented linear regression analysis revealed that the proportion of effective, optimal, and de-escalation therapies increased after the implementation of the program. No significant trends or changes in trends were identified before or after the implementation of the program. We did not detect improvements in time to intravenous to oral switch, reduction in length of stay and 30-day mortality rate.The program consisted of alerts and automated consultations. Alerts coupled with antimicrobial stewardship intervention , and rouIntervention by IDS is a key element in optimizing antimicrobial therapy, and previous studies have shown that IDS services and consultation improved antimicrobial prescription and clinical outcomes ,8,9,18. Another remarkable feature of our program was the use of information technology resources. Interventions based on electronic health records (EHRs) and clinical decision support systems (CDSSs) have been predicted to increase the efficiency of ASP ,20. In oThere has not been any unified definition of appropriate antimicrobial use ,8. OtherHowever, our approach had no significant impact on the intravenous to oral switch. We suspected that this was due to the relatively low cost of hospitalization and the lack of economic incentives to reduce the duration of hospitalization in our health care system. This reasoning could also be applied to the issue of length of stay. With respect to the extended average length of stay in hospitals, the Republic of Korea had ranked the second among OECD member countries recently . Since aStaphylococcus aureus BSI [In the program period, the interval from the BSI onset to the reporting of the ID/AST results decreased. We assumed that this finding was the result of placing dedicated staffs in our laboratory since June 2011. Although the decrease in the reporting time was not directly related to the program, it might influence the overall time to appropriate antimicrobial therapy. Despite this subsidiary change and the promotion of appropriate antimicrobial prescriptions, there was no significant improvement in the 30-day mortality rate after the initiation of the program. The possibility of improvement in the mortality rate might be small because approximately 80% of the study subjects were already on effective antimicrobials at the reference time. In addition, the impact of our intervention on the mortality rate might be limited owing to the considerable portion of older individuals with a high comorbidity index. First, it is possible to infer that the critical time point may be some time before the reference time of our study ,22. Nonereus BSI ,17, may S. aureus PCR [Despite its limitations, the combination of electronic alerts and automated consultations aimed at BSI treatment was found to improve antimicrobial prescription in the current study. Our approach offers an efficient form of stewardship intervention and demonstrates the possibility of exploiting informatics. However, additional efforts to identify more effective and efficient forms of stewardship program using information technology are needed. Also studies to establish the most appropriate time for intervention in terms of efficiency and practicality are warranted. The point in time when the results of the Gram stain are reported may represent the appropriate time for an intervention. However, an earlier intervention will require more resources and intensive work. The introduction of novel diagnostic tools such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and reus PCR ,24 offerS1 FigaEmergency department. bIdentification and Antimicrobial susceptibility test.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Serious and long-lasting psychiatric consequences can be found in children and adolescents following earthquake, including the development of posttraumatic stress disorder (PTSD). Although researchers have been focused on PTSD recently, its prevalence and risk factors after a huge natural disaster are still unclear because of limited sample size. The purpose of this study is to explore the prevalence of posttraumatic stress disorder (PTSD) in adolescent survivors three years after the Wenchuan earthquake, describe PTSD symptoms, and to find out risk factors of PTSD.A total of 4,604 adolescents from three middle schools which located in earthquake-stricken areas were recruited in this study. Instruments included the demographic questionnaire, questionnaire about earthquake exposure, the Social Support Appraisal Scale (SSA), the Posttraumatic stress disorder Checklist-Civilian Version (PCL-C), and the structured clinical interview for DSM-IV Disorders (SCID).The prevalence rate of PTSD was 5.7% (frequency: n = 261), and the most commonly occurring symptoms of PTSD were distress at reminders (64.5%), difficulty concentration (59.1%), and being easily startled (58.6%). Loss of houses and property, being injured, deaths of family members, and witness of death are positive risk factors of PTSD, and physical exercise and social support are negative risk factors of PTSD.Professional and effective interventions are needed to reduce the development of PTSD among adolescents after the Wenchuan earthquake, especially for these who lost their houses or property and lost their family members, witnessed death, and lacked of social support in the earthquake. Moreover, injured adolescents and adolescents who lacked of physical exercise also need intervention due to high risk. There were almost 100,000 persons dead and around 370,000 people injured, and at least 14,000 schools were destroyed in the earthquake. Lots of adolescents lost their homes and family members, and suffered great physical and psychosocial damage.At 14: 28 pm  on 12Serious and long-lasting psychiatric consequences can be found in children and adolescents following earthquake, including the development of posttraumatic stress disorder (PTSD) –3. PTSD It has been shown that earthquakes might cause PTSD, 4.5% to 95% children and adolescent who experienced the earthquake was reported to develop this disorder –11. ManyPrevious study showed that the prevalence of PTSD was 45.5% in heavily damaged county and 9.4% in moderately damaged county in Wenchuan earthquake . Liu andPTSD can last a long time even many years later. Previous surveys reported that PTSD would not appear in a short time after stressors but a long time after that –7. GoenjThese findings from literatures have important implications for preventing and treating stress disorders in adolescents after earthquakes. For the service provider, educations and interventions could be used to help adolescent survivors to deal with PTSD. Although researchers have been focused on PTSD recently, its prevalence and risk factors after a huge natural disaster are still unclear because of limited sample size. To our knowledge, only a few studies reported the rate of PTSD in the Wenchuan earthquake, with the absence of description of PTSD in adolescents. A survey with a large sample was required to be undertaken to find long-lasting mental influence of earthquake in adolescent survivors. We hypothesize that the prevalence of PTSD is not low and PTSD symptoms are serious in adolescent survivors three years after the Wenchuan earthquake, and risk factors such as physical exercise, earthquake exposure, and social support are associated with PTSD. So, the purposes of this study were: 1) to explore the prevalence of PTSD and describe PTSD symptoms in adolescent survivors three years after the Wenchuan earthquake; and 2) to find out associated risk factors of PTSD among adolescent survivors.Ethical approval was obtained from Sichuan University. In addition, informed consent was obtained from each adolescent and their guardians. They were assured that anonymity and confidentiality would be guaranteed and the right to withdraw from the study at any time. No pressure or inducement of any kind was applied to encourage adolescents to participate in the research.Subjects were recruited from three most severely damaged middle schools among the 2008 Wenchuan earthquake, which located in three most severely earthquake-stricken counties in Sichuan Province. These schools were totally destroyed and students were studied in newly-built schools. Participants were considered for inclusion in this study if they were between 12 and 20 years old and experienced the Wenchuan earthquake. And the exclusive criteria included: 1) with problems in hearing, verbal communication, and vision; 2) refused to participate in this study. Finally, data were collected from 4805 adolescents, and data for 4604 respondents were completed and effective, businesslike for 95.8%.Among these adolescents, there were 2617 girls of an average age of 15.2  and 1987 boys of an average age of 15.1 . Most of adolescents were the Han ethnic group (99.6%) and the only-child in their families (79.2%); more than half of them (57.0%) were senior middle school students; 72.5% of them took physical exercise frequently; and 26.2% of them had ever been student cadres. The data about earthquake exposure showed that 2.8% of them buried in this earthquake and 8.8% injured; some of them were with family members dead (7.7%) and handicapped (3.2%); over fifty percent (53.4%) lost their houses and property; 27.4%, 56.3%, and 29.9% of them witnessed burial, injury, and death in the earthquake respectively , and the Structured Clinical Interview for DSM-IV Disorders (SCID). Based on scores of PCL-C, PTSD-positive cases (score of the PCL-C ≥50), partial PTSD cases (score of 38-50), and PTSD-negative cases (score of the PCL-C <38) were evaluated. Next, PTSD-positive cases were screened by psychiatrists using SCID to exclude the false positive cases. Finally, adolescents who were diagnosed as PTSD-positive by both the PCL-C and the SCID fitted into PTSD cases.With the help of school staff who came from villages, townships and county-level cities, we identified the children who were present at school from Monday to Friday. Written informed consent was obtained from each subject and their guardians because they were adolescents and then carefully recorded by the interviewers after standard and complete descriptions of the study. Then each participant completed the questionnaire independently, and was presented a small gift upon its completion.Demographic data; demographic variables included age, gender, the ethnic group, grade, being only-child or not, took physical exercise frequently (track and field sports three times or more in a week) or not.Earthquake exposure; variables included buries, physical injuries, and handicaps in adolescent themselves, injuries and/or deaths in the family members, injuries and/or deaths in friends and teachers, witness of bury, injury, or death, and loss of house and property.The Social Support Appraisal Scale (SSA); was revised by Professor Xin Ziqiang [2/df <5, NFI, NNFI and CFI above 0.95) in Xin’s study [ Ziqiang  into Chi Ziqiang  to asses’s study , and theThe PTSD checklist-civilian version; a 17-item self-report checklist of PTSD symptoms developed by the Behavioral Science Branch of American PTSD research center according to DSM-IV in 1994 for evaluating experience of ordinary people after trauma in normal life. The PCL-C is composed of three dimensions: the first dimension is re-experiencing of the traumatic events, which includes intrusive thoughts, bad dreams, fears recurrence, and distress at reminders; the second dimension is nominated avoidance of trauma-relevant stimuli and numbing of general responsiveness which includes feeling detached or estranged from others, and deriving markedly less pleasure from previously enjoyable activities; the third dimension includes symptoms such as irritability, hyper-arousal, and difficulties in sleep and concentration. Individuals would rate each item from 1  to 5 (“extremely”) to indicate the degree to which they have been bothered by that particular symptom over the past month. Total possible scores range from 17 to 85. It is recommended that when the instrument is used as a continuous measure, a cut-off score of 50 is optimal for making the diagnosis of PTSD, and the score of 38-50 is diagnosed as partial PTSD [ial PTSD . In the The structured clinical interview for DSM-IV Disorders (SCID); it was used to evaluate PTSD which is a clinician-administered diagnostic interview, corresponding to diagnostic criteria for PTSD in the DSM-IV [e DSM-IV . The SCIe DSM-IV . The asse DSM-IV .All statistical procedures were performed using the SPSS 13.0 software . Continuous data were described as means and standard deviations and as quartiles where appropriated. Multiple logistic regression analyses were performed to identify independent predictors of PTSD status. The level of significance was set at 0.05 (two-tailed).After measured by the PCL-C and the SCID, the findings showed that 5.7% (n = 261) of the respondents were PTSD patients, 11.3% (n = 519) of them were partial PTSD patients.The mean scores of PLC-C, re-experiencing, avoidance and hyper arousal were 28.11 ± 10.25 (range: 17-85), 8.37 ± 3.30 (range: 5-25), 10.59 ± 4.34 (range: 7-35), and 9.16 ± 3.91 (range: 5-25) respectively. Figure Table To our knowledge, this was the first study to investigate the prevalence of PTSD in a large number of adolescent samples who experienced earthquakes in China. In this study, the prevalence of PTSD was 5.7%. It was lower than other earthquake studies among adolescents –3, 5–11,Besides, there was 11.8% of partial PTSD as a result of the current study, which means that 17.5% (5.7% + 11.8%) of the adolescent survivors in this earthquake have had PTSD or had a great possibility of getting PTSD even three years after the Wenchuan earthquake, and an appropriate post-disaster mental health intervention program is needed to prevent the development of PTSD.Previous study showed a high percentage of PTSD symptoms among adolescents after earthquake, especially distress at reminders (74.5%), being easily startled (68.8%), and intrusive thoughts (60.9%) and physiologic reactivity (66.7%) . AnotherCharacteristic risk factors of PTSD symptoms in adolescents after disasters were widely studied. It has been shown that girls exposed to earthquakes are more likely to develop PTSD than boys –7. HowevIn this study, the physical injury caused by earthquake and deaths of family members were found as risk factors of PTSD. This is understandable and similar with most previous studies about adolescents after earthquakes , 9. SimiSimilar with former studies , loss ofThis study also examined the predictive factors for PTSD. Social support was found to be major positive factor. Prior studies indicated that low levels of social support after the traumatic events were related to PTSD symptoms –12. In oSome limitations of this study are important to be highlighted. Firstly, most adolescents are the Han ethnic group and therefore the results may not be generalizable to other Chinese ethnic group populations. Secondly, data on important demographic variables such as history of psychiatric illness and family psychiatric illness were not collected. Finally, owing to the cross-sectional design, the present study only described the PTSD symptoms of adolescents three years after the earthquake. Longitudinal study is suggested to follow up with the changes.Despite these limitations, this study is the first study reporting the prevalence of PTSD among adolescent survivors after the Wenchuan earthquake and its associated risk factors in a large sample (n = 4604) study. Current findings provided important information that education and mental health intervention are needed to help adolescent survivors to deal with PTSD after earthquakes.Professional and effective interventions are needed to reduce the development of PTSD among adolescents after the Wenchuan earthquake, especially for these who lost their houses or property and lost their family members, witnessed death, and lacked of social support in the earthquake. Moreover, injured adolescents and adolescents who lacked of proper physical exercise also need intervention due to high risk.


Based on the evaluation findings of the B.E.S.T. Teen Program which aimed at promoting behavioral, emotional, social, and thinking competencies in primary school students, it is argued in this paper that promotion of psychosocial competence to prevent addiction in primary school students is a promising strategy. A total of 382 Primary 5 (Grade 5) and 297 Primary 6 (Grade 6) students from five primary schools in Hong Kong participated in the program. Different evaluation strategies were adopted to evaluate the program. First, objective outcome evaluation adopting a non-equivalent group pretest–posttest experimental-control group design was conducted to examine change in the students. Second, to gauge students’ perceptions of the program, subjective outcome evaluation was conducted. The evaluation findings basically converged to tentatively suggest that young adolescents benefited from participating in the program. Implications on the development, implementation, and evaluation of addiction prevention programs for teenagers are discussed. In their “Handbook of Adolescent Health Risk Behavior”, DiClemente et al.  painted Mak et al.  conducteStudies have also reported relatively widespread problem of gambling among young people. In Wong and So’s  recent sMasten and Coatsworth  concludeBased on the above mentioned findings, Lam et al.  argued tIn fact, there is ample evidence to support the association between different positive youth development qualities  and closely examined existing programs especially in the Hong Kong context to ensure that the present program was distinctive, suitable for the local culture and for the target group of Hong Kong primary school students. Based on the findings of the review, it was decided that promotion of psychosocial competence of young people would be the best strategy to prevent addiction.The multi-addiction prevention program adopts a positive youth development approach and aims to introduce knowledge of addiction behaviors to targeted students and promoting students’ intrapersonal  and interpersonal  competencies, which serve as protective factors against risk behaviors. The B.E.S.T. Teen Program consists of five components related to positive youth development: 1) equip students with knowledge on the nature of addiction and remove their misconceptions about addiction; 2) promote cognitive competence; (3) promote emotional competence; (4) promote social competence; and (5) promote behavioral competence. Because of differences in students’ cognitive development, there are different curriculum materials for Primary 5 and 6 students. The curriculum is composed of five parts, which are then further divided into two units. Each unit takes 30 min, totaling to 5 h. The curriculum, teaching goals, and intended learning outcomes for Primary 5 and 6 students are presented in Tables  equip st promote When the curriculum was developed, two primary school teachers and two social workers were invited to attend a focus group where they shared comments on the lesson plans of the curriculum. On the whole, the teachers and social workers viewed the curriculum positively. They saw the content as relevant, the curriculum appropriate for the students and the activities as engaging. They also provided feedback on how to improve the curriculum  and informed the researchers on activities that students usually find engaging. The curriculum development team then further revised the curriculum based on these comments.The program was implemented for Primary 5 and 6 students in the randomly selected schools. The duration of implementation varied across schools ranging from two-day intensive implementation to one month gradual implementation. Implementation team of the B.E.S.T. Teen Program included registered social workers, psychologists with Bachelor’s and postgraduate degree and researchers who had ample training and experience in teaching and implementation of positive youth development and addiction prevention programs. The programs were implemented during students’ life education lessons or unstructured periods. A teacher from the school introduced to the students the implementer who was then responsible for leading the lesson. Table Recruitment of participants to the B.E.S.T. Teen Program was conducted in the 2013–2014 school year. Over 50 local primary schools in Hong Kong were contacted including government schools, as well as government aided schools operated by the Tung Wah Group of Hospitals. A total of 37 primary schools in Hong Kong expressed their interest in joining the Program. Among the interested parties, five primary schools were randomly selected to participate in the program as the experimental group and five primary schools with similar background  as the experimental schools were selected as the control group. Selected primary schools were all from districts in the New Territories. The program was implemented in the 2013–2014 school year. A total of 382 Primary 5 and 297 Primary 6 students participated in the program , a 1 h workshop was delivered to the students after the data collection had been completed. The workshop offered basic knowledge of addiction, common misconceptions about addiction, and a brief introduction of the intrapersonal and interpersonal competencies.Different methods were used to examine the effectiveness of the program. At the first stage, in order to examine whether students changed after taking part in the program, analyses of the data collected from the non-equivalent group pretest–posttest experimental-control group design was carried out. For the experimental group, pretest questionnaires were administered to students before the first session of the program, and posttest questionnaires were handed out after the completion of the final session. For the control group, questionnaire surveys were conducted twice at the same time with the experimental group. Second, to find out students’ perceptions of the program, a subjective outcome evaluation form was given to participants after completing the final session. Qualitative evaluation methods were also used in the study. However, because of space limitation, related qualitative findings will be reported elsewhere.Part A: Addictive Behavior (eight items) Participants were invited to indicate their frequency of showing different types of addictive behavior in the past month, including smoking, drinking alcohol, using cough mixture without medical condition, inhaling the fumes from organic solvent, using Ketamine, using Heroin, gambling, and uncontrollable Internet use. Respondents rated the frequency of these behaviors in the past month on a 6-point Likert scale . It should be noted that while there are controversies regarding the definition and classification criteria of Internet addiction, it was defined as one’s uncontrollable use of the Internet or computer games even the use has negatively affect one’s life  Respondents were asked to assess on a 4-point Likert scale  the likelihood that they might engage in the above eight addictive behaviors in the next 2 years. Example items are “would you smoke cigarettes in the coming 2 years?” and “would you spend most of your time on the Internet or computer games even it will negatively affect your life?” Cronbach’s alpha for the scale ranged from .36 to .37 for both the experimental group and the control group at pretest and posttest. Similar to Part A, individual item scores instead of the scale scores were used for data analysis.Part C: Psychosocial Competencies (12 items) Participants were invited to indicate their agreement on items describing skills relevant to different positive youth development competencies . For example, one question assessing the behavioral competence was “I will consider all choices before I make decisions”. Respondents rated their perceptions on a 6-point Likert scale ranging from 1 = strongly disagree to 6 = strongly agree. Cronbach’s α of pretest was = .68 and for posttest it was .69.Part D: Knowledge about Addiction (16 items) Students were given 16 claims to assess their knowledge on addiction. Response options were true, false, and unsure. Some examples included: “There are many different types of addiction” and “There are many factors that may lead to addiction”. While students in the control group did not participate in the Program, they were provided with the correct answers after the posttest.Part E: Beliefs about Addiction (13 items) Students were asked how much they agreed with statements describing different beliefs about addiction. Example statements included, “It is fine for children of my age group to smoke” (reverse coding); and “People shall control their time spent on the Internet or computer games”. Respondents rated the items on a 6-point Likert scale ranging from 1 = strongly disagree to 6 = strongly agree. Cronbach’s α for pretest was .73 and for posttest it was .76.Based on existing literature, validated questionnaires  Program attributes included program objectives, content, student interaction, classroom atmosphere, and encouragement received during lessons.Part B: Implementer Attributes  Implementer attributes included implementers’ preparedness, professional attitude, knowledge, and interaction with participants.Part C: Program Effectiveness  Items evaluating program effectiveness included promotion of different psychosocial competencies, overall personal development, and achievement of the intended learning outcomes.The subjective outcome evaluation comprised of 38 items evaluating participants’ perceptions regarding the following three attributes:strongly disagree to 6 = strongly agree). For Program Effectiveness, participants indicated the extent to which the program had helped to enhance their skills and competencies (ranging from 1 = no help to 5 = great help).For Program and Implementer Attributes, participants were invited to rate on a 6-point Likert Scale the extent to which they agreed with the items (ranging from 1 = highly dissatisfied) to 6 (highly satisfied).In addition, two items were included in the subjective outcome evaluation form to examine whether (1) students would recommend the program to their friends with similar backgrounds/needs; and whether (2) they would participate in similar programs in the future. Moreover, students’ overall satisfaction towards the program was measured using a single item with ratings ranging from 1  addictive behaviors; (2) behavioral intentions; (3) psychosocial competencies; (4) knowledge about addiction, and (5) beliefs about addiction.First, experimental group and control group were compared at pretest in terms of the five categories. For “addictive behavior”, percentages of students who reported “never” showing the behavior were compared between the two groups based on Chi square tests. Independent samples t-tests were conducted to examine the differences in item scores of “behavioral intention” and scale scores of “psychosocial competencies”, “knowledge about addiction”, and “beliefs about addiction” between experimental and control groups.never; 1 = at least once in the past month) served as the dependent variable in one model. For the multiple regression models, the mean scores of each “behavioral intention” item , the “knowledge about addiction” scale, “psychosocial competencies” scale, and “beliefs about addiction” scale served as the dependent variables. For independent variables, gender  and age were entered at the first block; students’ pretest score was entered at the second block, and group  was entered at the third block.Second, a series of regression analyses were performed to evaluate whether participation in the program predicted students’ posttest objective outcome indicator scores. A total of eight logistic regression models and 11 multiple regression models were estimated. For the eight logistic models, each “addictive behavior” . In other words, students who participated in the program were 45 % less likely to display uncontrollable Internet use than did students who didn’t participate in the program. No significant effect of group was found on other addictive behavior indicators. For multiple regression analyses, after controlling for the effects of gender, age, and pretest scores, group significantly predicted participants’ intention to use Internet excessively in the coming 2 years  and predicted smoking intention with borderline significance . Experimental group students reported a lower intention to use Internet excessively than did control group students, and there was a tendency that students participating in the program had a lower intention to smoke in the future than control group. For other objective outcome indicators, no significant group effect was identified. In sum, objective outcome evaluation provides partial support for the effectiveness of the program.Results of logistic regression analyses showed that group significantly predicted the occurrence of uncontrollable Internet use in the participants after controlling for the effects of gender, age, and pretest scores , program implementers , and program effectiveness (78.1–86.2 %). Students reported that their overall personal development was improved (81.3 %). The findings from the subjective outcome evaluation from both Primary 5 and 6 students showed that the program was well-received and that the intended learning outcomes of the curriculum were achieved. The findings from the subjective outcome evaluation illustrate the important roles of curriculum content and implementers in successful implementation of prevention programs.The experiences and findings that are gained through implementing and evaluating the B.E.S.T. Teen program have led us to make several recommendations regarding the development and implementation of future youth addiction prevention programs. First, it is suggested that a positive youth development approach should be adopted in addiction prevention. Recently, researchers and practitioners agreed that adolescent problem behaviors including addictive behaviors share common antecedents and that prevention science should adopt a broader focus to address both risk and protective factors  were well-received by students and helped to benefit prevention efforts. Lastly, in a review of school-based prevention programs, Durlak and Wells  noted thWhile the findings from both objective and subjective evaluation provided support to the effectiveness of the B.E.S.T. Teen program, the present study has several limitations. First, due to limited resources, timetabling of different schools and other reasons, not all students who took part in the program joined the evaluation . It would be desirable to recruit a larger sample in the different areas of evaluation. Second, addictive behaviors were measured by single items. Psychometrically, sound measures must be adopted in future to provide a more accurate estimation about teenagers’ addictive behaviors. For example, it would be helpful to use Young’s Internet Addiction Test Young  to measuFinally, it is noteworthy that the Cronbach alphas for adolescent past addictive behaviors and intention to engage in addictive behavior subscales suggested low internal reliability. Interestingly, such scales have been found to be valid and reliable in previous studies on early adolescent population in Hong Kong (Shek and Yu As few evaluation studies have been conducted on multi-addiction prevention programs targeted at primary school students, especially in Hong Kong, the B.E.S.T. Teen Program and the evaluation study were developed. The program and the related evaluation study can be regarded as pioneer but the evaluation findings should be regarded as preliminary. Based on the objective outcome and subjective outcome evaluation findings, the present study provides some initial support for the effectiveness of the program, although it is noteworthy that there are several limitations of the study. Obviously, recommendations put forward in the present paper may serve to inform program developers and practitioners on how to design and implement effective prevention programs. Furthermore, more studies should be conducted to replicate and extend the findings arising from the present study.


In June 2012, the Chicago Department of Public Health passed ordinances to legalize mobile produce vending throughout the city, provided at least 50% of produce carts operate in designated underserved areas. In response, the Neighbor Carts program emerged to promote the opportunity for economic success and healthful food access through an unconventional retail structure. Neighbor Carts are independent produce carts selling fresh fruit, vegetables, and nuts. Part food access enterprise and part workforce initiative, this program employs people who have previously experienced homelessness, addiction, or other barriers to employment.AM to 4 PM, weather permitting, from April through November. Customers from 6 carts were surveyed as part of the larger evaluation  data to define and visualize program effectiveness. The Northwestern University institutional review board reviewed and approved the project. Data for these maps were generated via a customer intercept survey that was conducted immediately after a purchase was made (n = 98). Twelve customer surveys were from residents outside of Chicago, from mostly suburban areas, and are not included on these maps. Questions focused on dietary habits, suggestions for program improvement, overall purchase satisfaction, and geo-demographic information. Carts typically operate Monday through Friday from 7 aluation . These mCart 1 is outside an underserved zone (as defined by city ordinances), in a centrally located commercial hub just a few blocks west of major shopping and tourist attractions. The map for Cart 1 shows that it reached customers from many areas of Chicago, including those from underserved zones on the south and west sides of the city. Cart 2, in an underserved zone, experienced much higher use from customers living in closer proximity to the cart. Results from the larger evaluation demonstrated additional differences between the 2 carts, showing variation in customer age, sex, employment status, and Supplemental Nutrition Assistance Program (SNAP) participation . Both carts had a broader service area than a typical 0.5-mile walking radius, as was originally anticipated.In an effort to prevent chronic diseases associated with poor nutritional intake and increase healthful food access, mobile produce vending programs have been identified as a promising strategy. These maps add to the growing body of evidence that the food environment is highly complex. Carts close in proximity but in different neighborhoods demonstrated unique consumer trends. Future evaluation must encompass a more nuanced look at program effectiveness, taking place on a microlevel to take neighborhood context into account. Factors including community engagement, proximity to public transportation lines, and local business support shape the food landscape and contribute to cart success in varying degrees, depending on the neighborhood. Additionally, it may be necessary to incorporate different evaluation metrics to determine cart success. Although sales volume is a key metric for individual cart sustainability, customer volume should also be considered. More specifically, if carts reach a broad service area that goes beyond a walking radius to provide access to fruits and vegetables to residents of underserved zones, then one of the over-arching program goals, to increase access to fresh produce to all Chicagoans, is accomplished.


Plasmodium falciparum malaria takes years to develop, probably reflecting the ability of the parasites to evade immunity. A recent example of this is the binding of the Fc region of IgM to VAR2CSA‐type PfEMP1. This interferes with specific IgG recognition and phagocytosis of opsonized infected erythrocytes (IEs) without compromising the placental IE adhesion mediated by this PfEMP1 type. IgM also binds via Fc to several other PfEMP1 proteins, where it has been proposed to facilitate rosetting . To further dissect the functional role of cF‐mediated IgM binding to PfEMP1, we studied the PfEMP1 protein HB3VAR06, which mediates rosetting and binds IgM. Binding of IgM to this PfEMP1 involved the cF domains Cμ3‐Cμ4 in IgM and the penultimate DBL domain (DBLζ2) at the C‐terminus of HB3VAR06. However, IgM binding did not inhibit specific IgG labelling of HB3VAR06 or shield IgG‐opsonized IEs from phagocytosis. Instead, IgM was required for rosetting, and each pentameric IgM molecule could bind two HB3VAR06 molecules. Together, our data indicate that the primary function of cF‐mediated IgM binding in rosetting is not to shield IE from specific IgG recognition and phagocytosis as in VAR2CSA‐type PfEMP1. Rather, the function appears to be strengthening of IE–erythrocyte interactions. In conclusion, our study provides new evidence on the molecular details and functional significance of rosetting, a long‐recognized marker of parasites that cause severe P. falciparum malaria.Acquired protection from  Plasmodium falciparum infections in areas of stable parasite transmission produce only relatively mild symptoms or are asymptomatic. Nevertheless, about 600 000 people, mainly children, die from severe malaria complications annually  surface  can act as ‘bridges’ between the IE and the surrounding erythrocytes, and IgM‐containing fibrillar strands have indeed been observed at apposed IE and erythrocyte membranes   Fig. K. No oth+ IEs bound non‐specific IgM , with rapid association (ka = 2.3 × 106 and 1.8 × 106 M–1 s–1) and slow dissociation kinetics (kd = 3.0 × 10−4 and 5.1 × 10−4 s−1)   furthermore indicated that the HB3VAR06 ectodomain is rigid as reported for other PfEMP1 molecules , and by the Regional Research Ethics Committees, Capital Region of Denmark (Protocol H‐4‐2013‐083). All donors were adults and provided written informed consent. All the animal experiments were conducted according to Danish Law and approved (permit 2012‐15‐2934‐00567) by the Danish Animal Procedures Committee (‘Dyreforsøgstilsynet’).2+ metal chelate agarose HisTrap HP columns  followed by dialysis of eluates . Proteins used for analysis by SPR, analytical ultra‐centrifugation or small‐angle X‐ray spectrometry (SAXS) were further purified on Superdex 200 16/60 size exclusion chromatography columns . Domain boundaries for all the recombinant HB3VAR06 constructs are shown in Fig. et al., et al., The entire ectodomain of the PfEMP1 protein HB3VAR06 (FV6) was codon optimized (GenBank accession number KP203835) for insect cell expression by GeneArt . Single‐, double‐, triple‐domain and full‐length constructs were cloned into the transfer vector pAcGP67‐A  with a C‐terminal hexa‐histidine tag. The transfer vector was then co‐transfected with linearized BakPak6 Baculovirus DNA (BD Biosciences) into Sf9 insect cells to generate recombinant virus particles. Secreted proteins were purified from the supernatants of transfected high‐five insect cells using Ni+ erythrocyte reactivity. Pre‐immunization serum was used as negative control. Mouse monoclonal antibodies specific for human Fcμ2, Fcμ3 and Fcμ4  and rabbits . One (FV6) or two  animals were used per construct for immunization. Antisera were collected on day 49 (rats) or 70 (rabbits), pooled and depleted of non‐specific O RhP. falciparum‐exposed adults  or pooled serum from non‐exposed blood bank donors; NHS) was used with or without IgM depletion by incubation with biotinylated donkey anti‐human IgM‐Fcμ5 (Jackson ImmunoResearch) coupled to streptavidin‐conjugated Dynabeads . Absence of IgM after depletion was confirmed by immunoblotting with horse radish peroxidase‐conjugated rabbit anti‐human IgM .Pooled plasma from 10 anonymous healthy, 2, 2 mM MgCl2, pH 7.4), blocked  with TSMB (TSM buffer with 1% Ig‐free BSA and 0.05% Tween‐20), washed with TSM buffer supplemented with 0.05% Tween‐20 and incubated  with purified human IgM . After washing away unbound antibody, IgM was detected using rabbit anti‐human IgM HRP . After washing unbound secondary antibody, bound HRP was reacted with OPD  according to manufacturer's instructions and absorbance detected (492 nm). In assays to determine which Fc domains were involved in IgM binding to HB3VAR06, IgM (10 nM) was pre‐incubated overnight (4°C) with mouse anti‐human Fcμ domain‐specific monoclonal antibodies or IgA  in TSMB buffer.Binding of IgM to recombinant HB3VAR06 constructs was quantified by ELISA. Flat‐bottomed 96‐well MaxiSorp plates (Thermo Scientific) were coated overnight with recombinant protein  in Tris saline magnesium (TSM) buffer  first with non‐specific IgM (10 nM), then rat or rabbit antisera or EHP (1:20), and finally appropriate secondary antibody , goat anti‐rat IgG‐FITC , goat anti‐rabbit IgG‐FITC  and ethidium bromide (2 μg ml−1). In some assays, the antibody order was reversed, i.e. first antisera or EHP, then IgM. In antibody competition assays, the competing antibodies were applied in separate steps. In experiments assessing IE surface labelling by non‐specific IgM and antigen‐specific IgG at the same time, the donkey anti‐human IgM‐PE and donkey anti‐rabbit PerCP  were used as secondary antibody reagents and ethidium bromide was omitted. IEs were washed three times between incubation with primary and secondary antibody. Antibody surface labelling of IEs was quantified by flow cytometry using a Beckman Coulter FC500 instrument (Beckman Coulter) followed by analysis of list mode data files using FlowJo software v.7.6 (Treestar).The binding of IgM and antigen‐specific IgG to IEs was detected by flow cytometry essentially as described ‐supplemented Roswell Park Memorial Institute (RPMI) medium and a controlled atmosphere, essentially as described  with NHS (10%), IgM‐depleted NHS, IgM‐depleted NHS plus human IgM , Albumax II (10%) or Albumax II (10%) plus IgM (4 mg ml−1). The following day the relative rosetting frequency of triplicate wells was assessed as described above. To examine the involvement of different HB3VAR06 domains in rosetting, synchronous HB3VAR06+ IEs were grown from the ring to the late trophozoite stages in 10% NHS in the presence of 1:20 dilutions of domain‐specific antisera followed by assessment of rosetting rates as above.Rosetting assays were performed using cultures with high rosetting rates (60–95%) and frequencies were assessed by counting 200 ethidium bromide‐stained IEs and noting those that had two or more erythrocytes adhering using wet slide preparations and fluorescence microscopy. To determine the role of F−1 for 60 s followed by buffer for 120 s) IgM (750 RU) immobilized on a CM4 biosensor chip  by amine coupling. After each run, the chip was regenerated (1 M NaCl). Specific binding response to IgM was calculated by subtracting the response to an uncoupled chip and to a buffer injection. The kinetic sensorgrams were fitted to a global 1:1 interaction model to allow calculation of ka, kd and KD using BIAevaluation software v.4.1 .We used a Biacore 2000  for affinity measurements by SPR. All experiments were performed in N‐[2‐hydroxyethyl]piperazine‐N′‐[2‐ethanesulfonic acid] (HEPES) buffer  at pH 7.4 and 20°C. FV6 or FV2 were flowed over , washed four times in PBS2 and resuspended in RPMI supplemented with 2% FCS. Human monocytic leukaemia line THP‐1 cells  (4 × 105 per well) and IEs (2 × 105 per well) were incubated  in 96‐well plates . Non‐phagocytosed IEs were lysed , and phagocytosis assessed by flow cytometry as above. In some experiments, IEs were pre‐incubated with 10 nM IgM or IgA before addition of anti‐ or immune‐serum.Phagocytosis of antibody‐opsonized IEs was measured as described previously (Tippett Rg) and maximum particle dimension (Dmax) . The beam line was equipped with a PILATUS 2M detector set 3.1 m from the sample using a 0.13–0.14 nm beam wavelength. Concentration series of different constructs were prepared in order to eliminate structure factor effects. All measurements were performed at 10°C, collecting 20 frames of 0.05 s exposure time of each construct. The pair distance distribution function [P(r)] was obtained by indirect Fourier transformation of the scattering data giving the estimated radius of gyration . Reference and sample were loaded into a double‐sector centrepiece and mounted in a Beckman An‐60 Ti rotor. Results from multiple scans (monitored at 280 nm) at molar ratios 1:1, 1:2 and 1:3 of IgM and FV6 (where the IgM concentration was kept constant at 0.2 μM to give an absorbance of 0.296 in a 1.2 cm path length) were fitted to a continuous size distribution using SEDFIT v.14.1 software , viscosity (0.01020 P) and the partial‐specific volumes of IgM (0.727 cm3 g−1) and FV6 (0.727 cm3 g−1) were calculated using SEDNTERP software (v.20120828)  . All the size exclusion chromatography‐purified protein samples were prepared in a PBS buffer  and experimental scattering data (points and error bars) (A) and distance distribution functions (B) for full‐length HB3VAR06 , the N‐terminal domains D1–D4 (red) and the C‐terminal domains D7–D9 (green) (B). The P(r) functions were calculated from the scattering intensity I(q). Kratky plot of full‐length HB3VAR06 (FV6) (C).Click here for additional data file.