Seroprevalence surveys are of utmost importance to assess the proportion of a population that has developed antibodies against a newly introduced virus and could therefore potentially exhibit immunologic protection against subsequent infection. This study aims to understand the distribution of IgM and IgG antibodies in the Dominican Republic. We surveyed a total of 12,897 participants between April and June 2020 in 10 provinces of the Dominican Republic. Survey efforts in emerging hotspots yielded a positivity for all participants of anti–SARS-CoV-2 IgM of 3.8% and IgG of 5.4%, indicating that the pathogen was in circulation before the identification of those particular communities as hotspots. We found important age differences between participants who participated in the serological study where a higher mean age is associated IgM positivity and a lower age with IgG positivity. Our results highlight the need for strategies that involve community-based seroprevalence monitoring. These should preclude syndromic case identification. Also, the higher mean age of IgM-positive participants suggests that strategies based on syndromic surveillance could identify hotspots at later phases, based on the number of cases detected at the healthcare center, as such community-based seroprevalence monitoring may be an effective intervention for future outbreaks. Decisions to loosen restrictions were made based on declining positivity rates and increases in hospital capacity to admit COVID-19 cases.2The Dominican Republic reported its first confirmed case of SARS-CoV-2 on March 1, 2020.3 Besides this, the known differences in the proportion of asymptomatic and symptomatic manifestation by varying age-groups can lead to under detection in younger populations and overestimation of severity in older communities in countries with only syndromic surveillance.6 To expand testing capacities during initial phases of the pandemic caused by SARS-CoV-2, rapid detection of antibodies by the ELISA method was implemented as a screening method for active and passive surveillance. This method was mainly based on detecting specific antibodies against SARS-CoV-2 antigens, where IgM antibodies are the first antibodies that are created in response to initial exposure to an antigen and IgG antibodies appear at a later phase and serve as immunologic memory.Because of a sudden increased demand for confirmatory diagnostic testing, mildly affected and asymptomatic individuals have limited access to laboratory testing. As a result of such circumstances, the number of confirmed SARS-CoV-2 infections can significantly underestimate the actual number of cases.7 As recommended by the WHO, monitoring seroprevalence changes over time is crucial to anticipate the epidemic’s dynamics and plan an adequate public health response to contain the spread of the pathogen or prevent its reemergence.4 In addition to this, seroprevalence studies offer the benefit of saving testing costs and time and the possibility of carrying out community-based intervention in identified emerging hotspots to stop further spread of the disease.8As seen in many countries, changing testing strategies during epidemics makes it nearly impossible to estimate the extent of the population exposed to the pathogen at a given moment. However, this information is crucial in planning evidence-based strategies for lifting physical distance and confinement measures. In this context, seroprevalence surveys are of utmost importance to assess the proportion of the population that has already developed antibodies against the virus and could potentially exhibit immunologic protection against subsequent infection.This study aimed to understand the distribution of IgM and IgG antibodies within the Dominican Republic during community-based interventions. To achieve this, we analyzed the demographic characteristics of participants who received a SARS-CoV-2 IgM/IgG rapid test in emerging hotspots within the Dominican Republic. Emerging hotspots were considered as an increased rate of new infections compared with the previous epidemiological week in a given municipality or province. Because of the inherent difference in IgM and IgG function and structure, we consider that intervened communities with an increased proportion of IgM antibody positivity indicate an early identification of community circulation of SARS-CoV-2. By contrast, a high IgG combined with a low IgM positivity proportion would suggest a late community intervention. We also consider that differences in antibody profile distribution within age-groups can help identify crucial pockets of transmission within the population and guide public health policies.9 Testing efforts consisted of consecutive weekly serological interventions between April and June in communities identified as emerging hotspots for SARS-CoV-2 infections by the syndromic surveillance program of the country’s national surveillance system. We adhered to ethical guidelines by performing each test under strict confidentiality.To assess the seroprevalence of anti–SARS-CoV-2 antibodies in communities within the Dominican Republic, we contacted individuals randomly in communities identified as emerging hotspots for SARS-CoV-2 by Ministry of Health reports and invited members of households to participate in the seroprevalence study. We collected demographic data for each participant and evaluated anti–SARS-CoV-2 IgM/IgG antibodies using a commercially available chromatographic test targeting the S1 domain of the SARS-CoV-2 spike protein . To establish the IgG/IgM concentration, sample, strips were subjected to a colorimetric assay using an automatic analyzer /specificity (98.8%), versus IgG sensitivity (56.7%)/specificity (100%) Confiscope G20, Genbody Inc.).n = 6,597) males with a mean age of 42 years  highlight the weakness of a reactionary response or symptomatic surveillance with this pathogen where time is essential. By contrast, IgM detection in asymptomatic participants by community intervention is an important component of the efforts to stop the community spread of SARS-CoV-2. This is shown by a decline in IgM positivity in continuous months of implementation. Novel approaches to detect emerging hotspots, such as viral detection in sewage wastewaters, have to be adopted to ensure a faster and earlier response to this pathogen.12Survey of all participants in emerging hotspots yielded positivity of anti–SARS-CoV-2 IgM of 3.8% and of anti–SARS-CoV-2 IgG of 5.5%, indicating pathogen circulation before the identification of the community hotspot . SignifiM = 43.1, SD = 14.5), compared with the 12,406 participants who tested negative , demonstrated significantly higher age at the time of the serosurvey . In addition, the 704 surveyed participants who received a positive IgG result M = 41.9, SD = 16.2), compared with the 12,406 participants who tested negative , demonstrated significantly lower age at the time of the serosurvey . Both of these findings highlight the possibility of intense pathogen circulation before the intervention of an identified hotspot, with IgG suggesting increased immune memory in younger participants, which would lead to decreased susceptible populations, hence a decreased IgM positivity.We found important age differences between those who participated in the serological study. The 491 surveyed participants who received a positive IgM result (4 We found a lower proportion of IgM positivity, 2.1%, in children, than the 3.8% mean, and a higher IgG positivity of 5.8% versus the 5.5% mean, which might indicate cryptic transmission at community levels, especially with this age-group. This scenario has also been explored in other parts of the world, where studies found that children functioned as principal transmission foci because of a high asymptomatic proportion of infections.15 With the intense debate about whether to reopen schools and colleges in the fall, it should be a public health priority to understand the role of SARS-CoV-2 transmission in seemingly unaffected population and their role in the reemergence of the pathogen.Because of substantial differences in severity, complications, and mortality across age-groups, interventions focused on stopping the spread of SARS-CoV-2 should consider different surveillance strategies to reflect such variations.16 Because our study follows seroprevalence intervention in already identified hotspots by symptomatic surveillance and contact tracing, we can assume that the differences in antibody proportions in young populations suggest important pathogen circulation before community detection and intervention. We consider that an essential limitation to our study is the lack of pre-intervention serodata, and assay-related specificity/sensitivity; this might limit our understanding of any specific age-groups that may act as transmission pockets of the SARS-CoV-2 pathogen before the emergence of the hotspot. To counteract this limitation, we base our conclusions on prior viral circulation based on IgG positivity. By doing so, our hypothesis is supported by our findings that show a younger mean age of IgG-positive individuals than negative IgG participants. Also, the higher mean age of IgM-positive participants contributes to our suggestion that strategies based on syndromic surveillance could identify a hotspot at a later phase, based on the number of cases detected at the healthcare center.Strategies involving community-based seroprevalence monitoring should preclude syndromic case identification.


This work is concerned with fault localization for automated program repair.must location set. Intuitively, such a set includes at least one program location from every repair for a bug. Thus, it is impossible to fix the bug without changing at least one location from this set. A fault localization technique is considered a must algorithm if it returns a must location set for every buggy program and every bug in the program. We show that some traditional fault localization techniques are not must.We define a novel concept of a must with respect to commonly used schemes in automated program repair.We observe that the notion of must fault localization depends on the chosen repair scheme, which identifies the changes that can be applied to program statements as part of a repair. We develop a new algorithm for fault localization and prove that it is We incorporate the new fault localization technique into an existing mutation-based program repair algorithm. We exploit it in order to prune the search space when a buggy mutated program has been generated. Our experiments show that must fault localization is able to significantly speed-up the repair process, without losing any of the potential repairs. Fault localization and automated program repair have long been combined. Traditionally, given a buggy program, fault localization suggests locations in the program that might be the cause of the bug. Repair then attempts to change those suspicious locations in order to eliminate the bug.Bad fault localization may cause a miss of potential repairs, if it is too restrictive, or cause an extra work, if it is too permissive. Studies have shown that for test-based repair imprecise fault localizations happen very often in practice. This idmust location set. Intuitively, such a set includes at least one location from every repair for the bug. Thus, it must be used for repair. In other words, it is impossible to fix the bug using only locations outside this set. A fault localization technique is considered a must algorithm if it returns a must location set for every buggy program and every bug in the program.In this work, we define the concept of a must notion, consider the program in Fig. To demonstrate the importance of the ance in , 21 do nrepair scheme under consideration. A repair scheme identifies the changes that can be applied to program statements as part of a repair. A scheme can allow, for instance, certain syntactic changes in a condition  is returned.The setting of our work is as follows. Our approach is formula-based rather than test-based. We handle simple C-programs, with specification given as assertions in the code. Similarly to bounded model checking tools (e.g. ), the prallminimal repairs .We focus on a simple repair scheme of syntactic changes, as described above. We assume that the user prefers repairs that are as close to the original program as possible and will want to get several repair suggestions. Thus, we return must with respect to syntactic mutation schemes. The input to the algorithm is a program formula Once the notion of must fault localization is defined, we develop a new algorithm for fault localization and prove that it is v in F is computed based on dynamic dependencies among variables in v in F is a must location set that contains all assignment to the variables that v depends upon. Some assignment from F thus must be changed in order to eliminate the bug associated with For a variable P, the goal of the algorithm is to return all minimal repairs for P. The algorithm goes through iterations of generate-validate, where the generate part produces a mutated program of P and the validate part checks whether it is bounded-correct. The bottleneck of the algorithm is the size of the search space, consisting of all possible mutated programs of P. In \documentEven the method of, which sThe dynamic slicing method of, 23 alsomust be included in the result. In general, whenever a repair can be made by making changes to a set of lines, at least one of them must be included in the result.In this example, we have seen how many different fault localization techniques fail to include a statement that is relevant, i.e., where a modification could be made for the bug to be fixed. In contrast, the set of locations returned by our technique for this example is program is a sequential program composed of standard statements: assignments, conditionals, loops and function calls, all with their standard semantics. Each statement is located at a certain location (or line) For our purposes, a assume statements of the form assume(bexpr), and assert statements of the form assert(bexpr). In both cases bexpr is a boolean expression over assertion violation, and it is an indication that an error has occurred.In addition to the standard statements, a program may also contain bug on input correct for error trace, which is the sequence of statements visited during the execution of A program mentclass2pt{minimmodel) of the program formula represents an execution of the program that satisfies all assumption but violates at least one assertion. Such an execution is a counterexample.In this section we explain how a program is translated into a set of constraints, whose conjunction constitutes the program formula. In addition to constraints representing assignments and conditionals, such a formula includes constraints representing assumptions and a constraint representing the negated conjunction of all assertions. Thus, a satisfying assignment  form, which means that each variable in the new program is assigned at most once. This is done by replacing all variables with indexed variables, and increasing the index of a variable whenever it appears on the left-hand-side of an assignment. In the example, the first assignment to t is replaced by an assignment to t0 and the second, by an assignment to t1. Since t is assigned inside a conditional statement and is used after the statement, the if-then-else assignment t2 := g0?t1:t0 is inserted in order to determine which copy of t should be used after the conditional statement. These special if-then-else assignments are called Conversion to SMT constraints: Once the program is in SSA form, conversion to SMT is straightforward: An assignment x:=e is converted to the constraint encodes the statement it came from and we partition constraints into three sets, If the program includes several assertions, then they are converted to one constraint, representing the negation of their conjunction. In the example, the two assertions are converted to the following constraint: program constraint set. The program constraint set we get from a program program formulaThe triple ([). A program . A progFor simplicity of notation, in the rest of the paper we omit the superscript Since the program formula is the result of translating an SSA program, the formula is defined over indexed variables. Further, each constraint in In this section, we precisely define when a location should be considered relevant for a bug. This definition is motivated by a repair perspective, taking into account which changes can be made to statements in order to repair a bug.repair scheme-patch for a program defined over the set of locations -patch-patched program of -patched programs created from a program -search space of In order to define the changes allowed, we use repair schemes. A -repair for -repairable set is a set of locations minimal if removing any location from it makes it no longer an -relevant if it is a part of a minimal Let entclass1pt{minimaIn this paper, we focus on two repair schemes that are frequently used for automated program repair: the arbitrary scheme . An -must location set is a set of locations that contains at least one location from each minimal . An -must fault localization algorithm is an algorithm that for every program Note that, an not an Also note, that the set of all locations visited by Continuing the previous example, the set Consider again the absValue procedure of Fig. entclass1pt{minimaentclass1pt{minimaIn this section we formally define the notion of slicing. Based on this, we present an algorithm for computing must fault localization for slicing. But, we do not define slicing in terms of the program directly, but in terms of the program formula representing it, instead. The input to the slicing algorithm is a program formula slice of the variable A central building block in our fault localization technique is input variables, and denote the set containing them by computed variable, and the (unique) constraint encoding the assignment to it is denoted e.Similar to traditional slicing, it is easy to define the slice as the reflexive-transitive closure of a dependency relation. But, unlike traditional slicing, which defines dependencies between statements, our dependency relation is between variables of the formula. These variables are indexed. Each originates from a variable of the underlying SSA program, where it was assigned at most once. We refer to variables never assigned as (Static Dependency). The static dependency relation of a program formula The left-hand-side of Fig. (Dynamic Dependency). The dynamic dependency relation of a program formula Note that, dynamic dependency includes only dependencies that coincide with the specific model The bold arrows on the right-hand-side of Fig. (Influencing Variables). Given a program formula The circled nodes on the right-hand-side of Fig. . Given a program formula Thus, intuitively, version .For every Continuing with our example of foo procedure,The computation of the program formula slice is composed of two steps. In the first step, we build a graph based on the static dependency relation, Static Dependency Graph (SDG) of true. See the left-hand-side of Fig. true annotations are omitted.The graph built during the first step is called the The algorithm for the second step is presented in Algorithm 1. This algorithm gets a program formula Our fault localization algorithm is presented in Algorithm 2. The input to this algorithm is a program formula origin of c, and denote it by As before, we assume to know the origin of constraints in partial model of As a first step, the algorithm computes a set of variables version .The formula passed to entclass1pt{minimanot remain the same, we use slicing: The algorithm proceeds by computing the program formula slice for each of the variables in S. This set represents all constraints that if remain the same, then all the variables in S must be included in any repair.To make sure that their values do Note that, by first applying S back to statements of S may belong to either S, for Finally, we need to translate the constraints in version .Algorithm FOSFL is an incremental, which means that the computation effort of each fault localization attempt should be proportional to the changes made from the previous attempt. In other words, we should avoid re-computation whenever possible, taking advantage of the fact that the program remains the same, or at least remains similar.It is often necessary to apply fault localization to several bugs in the same program, or even to several programs with different bugs. Therefore, it is desired that the fault localization algorithm be Algorithm FOSFL can be easily made incremental for the case of different bugs of the same program. In this case, several successive calls are made to the algorithm using the same program formula e with an expression e itselfNote that I-FOSFL is useful not only for fault localization of different bugs of the same program, but also whenever the SDG remains the same during successive fault localization calls. This is the case when considering different mutated programs AllRepair, was presented. This algorithm uses the mutation scheme in order to repair programs with respect to assertions in the code. Unlike fault localization, where the motivation is repairing a bug for a specific input, program repair aims at repairing the program for all inputs. To avoid confusion, we refer to a repair for all inputs as a full repair. In[full repair is bounded: loops are unwound fully repaired if no assertion is violated along executions with at most buggy. For the rest of this section, we refer to an In, a mutatpair. In, the notAllRepair is to obtain all minimal fully repaired mutated programs, where minimality refers to the patch used in the program. It goes through an iterative generate-validate process. The generate phase chooses a mutated program from the search space, and the validate phase checks whether this program is fully repaired, by solving its program formula. The mutated program is fully repaired iff the formula is unsatisfiable.As its name implies, the goal of M and line l, there is a boolean variable M is applied to line l. A boolean formula is constructed and sent to the SAT solver, where each satisfying assignment corresponds to a program in the search space. The SMT solver is used for the validate stage. The program formula of the mutated program is solved to check if it is buggy or not. To achieve minimality, when a mutated program created using a patch The generate-validate process is realized using an interplay between a SAT solver and an SMT solver. The SAT solver is used for the generate stage. For every mutation Let Blocking such programs prunes the search space, but only in a limited way. No pruning occurs when the mutated program is buggy.In this paper, we extend the algorithm of with a ffl-AllRepair, whenever a mutated program is found to be buggy during the validation step, its program formula is passed to the fault localization component along with the model obtained when solving the formula. The fault localization component returns a set of locations Figure Let fl-AllRepair is sound and complete.Algorithm AllRepair, available at https://github.com/batchenRothenberg/AllRepair. In this section, we present experiments evaluating the contribution of the new fault localization component to the program repair algorithm. We refer to the algorithm of[We have implemented our fault localization technique and its integration with mutated-based program repair in the tool rithm of, withoutThe difference between the algorithms lies in the repair loop. In case a mutated program is found to be buggy, the AllRepair algorithm will only block the one program, while the FL-AllRepair algorithm might block a set of programs. Therefore, the number of repair iterations required to cover the search space can only decrease using the FL-AllRepair algorithm. On the other hand, the cost of each iteration with fault localization is strictly higher than without it. Our goal in this evaluation is to check if the use of fault localization pays off. That is, to check if repairs are produced faster using FL-AllRepair than using AllRepair.Benchmarks. For our evaluation, we have used programs from two benchmarks: TCAS and Codeflaws. The TCAS benchmark is part of the Siemens suite[ns suite, and is ns suite, 34, 38.The Codeflaws benchmark is also Mutations. The mutations used in AllRepair (and accordingly in FL-AllRepair) is a subset of the mutations used in[mutation levels, where level 1 contains only a subset of the mutations available in level 2. Thus, level 1 involves easier computation but may fail more often in finding repairs. used in. We defiTable Setting. All of our experiments were run on a Linux 64-bit Ubuntu 16.0.4 virtual machine with 1 CPU, 4 GB of RAM and 40 GB of storage, provided using the Vmware vRA serviceinput. For each input, we run both the AllRepair and the FL-AllRepair algorithms with a timeout of 10 minutes and a mutation size limit of 2 .In total, 131 different repairs were found during our experiments, for 60 different inputs . In this count, we treat repairs fixing the same program in the same way as different, if they were produced using different mutation levels or unwinding bounds. This is because our evaluation is concerned with the time to find these repairs, and both the mutation level and the unwinding bound greatly influence this time.Because the time to produce a repair sometimes varied in several orders of magnitude depending on the input, we have chosen to split repairs into three categories: fast, intermediate, and slow, and examine the time difference separately for each category. Splitting repairs to categories was done according to the time it took to find them using the AllRepair algorithm. If that time was under 5 seconds, the repair was considered fast. If it was over 4 minutes, it was considered slow, and otherwise it was considered intermediate.Figure For the fast category Fig. a, there exclusively, while AllRepair reaches a time-out. Also, for the remaining 27 repairs, FL-AllRepair is faster in all cases but one. The time difference is now also very significant: FL-AllRepair is faster by 1512 seconds (around 25 minutes) on average.For the intermediate category Fig. b, the adTo sum up, the results show that in many cases our algorithm FL-AllRepair is able to save time in finding repairs. The savings are especially significant in cases where it takes a long time to produce the repair using the original AllRepair algorithm, and these are the cases where time savings are most needed.AllRepair’s performance was compared to that of four other automated repair tools: Angelix[GenProg[FoRenSiC[Maple[AllRepair was found to be faster by an order of magnitude than all of the compared tools, taking only 16.9  seconds to find a repair on average, where the other tools take 1540.7, 325.4, 360.1, and 155.3 seconds, respectively. Since in our experiments on TCAS fl-AllRepair was faster than AllRepair on average (and even when it was slower it was only by a few seconds), we conclude that fl-AllRepair also compares favorably to these other tools.The TCAS benchmark was recently used also in, where A Angelix, GenProg[GenProg, FoRenSiFoRenSiC and MaplC[Maple. AllRepaAllRepair (and fl-AllRepair) is limited compared to the other tools: AllRepair only uses mutations on expressions while Angelix, FoRenSiC and Maple allow replacing an expression with a template , which is then filled out to create a repair. GenProg allows modifying a statement as well as deleting it or adding a statement after it. Therefore, the other tools are inherently capable of producing repairs in more cases than AllRepair.In terms of repairability, the repair scheme used by AllRepair is able to find repairs for 18 versions (a result that we confirm in our experiments as well), while Angelix, GenProg, FoRenSiC and Maple found 32, 11, 23 and 26, respectively. But, what the study also showed, is that in repair methods that are based on tests, in many cases the repair found only adhered to the test-suite, but was not correct when inspected manually. When counting only correct repairs, AllRepair finds repairs for 18 versions , while Angelix, GenProg, FoRenSiC and Maple find 9, 0, 15 and 26, respectively. Since fl-AllRepair is able to find all repairs found by AllRepair, the same results also apply to fl-AllRepair.In the case of TCAS, the study showed that Dynamic slicing has been widely used for fault localization in the past, 45–47. are likely to be relevant to the failing execution, based on all executions. We see may fault localization techniques as orthogonal to ours , since in the trade-off between returning a small set of locations, and returning one that is guaranteed to contain all relevant statements, may techniques prefer the first, while must techniques prefer the second. In the context of repair, there are interesting applications for both.Other approaches for fault localization include spectrum-based (SBFL), 31, 44,FBFL techniques represent an error trace using an SMT formula and analyze it to find suspicious locations. These techniques include using error invariants, 17, 40,MUT-APR[FoREnSiC[Maple[SemGraft[In the literature there is also a wide range of techniques for automated program repair using formal methods, 33, 42.FoREnSiC and MaplC[Maple repair CSemGraft conductsmust fault localization, that carefully identifies program locations that are relevant for a bug, so that the set is sufficiently small but is guaranteed not to miss desired repairs. We also show that the notion of must fault localization should be defined with respect to the repair scheme in use. We show that our notion of must fault localization is particularly useful in pruning the search space of a specific mutation-based repair algorithm.In this work we define a novel notion of To the best of our knowledge, we are the first to investigate the widely-used notion of fault localization and to suggest criteria for evaluating its different implementation.


Blossom-end rot in tomatoes is often used as a model system to study fruit calcium deficiency. The study of blossom-end rot development in tomatoes has been greatly impeded by the difficulty of directly studying and applying treatments to the affected cells. This manuscript presents a novel method for studying blossom-end rot development after harvest in immature whole fruit and in pericarp discs.Pericarp discs removed from the bottom pericarp of immature healthy fruit developed blossom-end rot like symptoms, corresponding to a decrease in L* value and an increase in a* value. Symptoms also developed in columella tissue, but not in stem-end pericarp tissue, similar to patterns observed during blossom-end rot development on the plant. Ascorbate oxidase and peroxidase activity, which are elevated in blossom-end rot affected fruit compared to healthy fruit, were both correlated with colorimetric measures of tissue darkening in discs. Respiration rate was higher in discs that later developed blossom-end rot symptoms, with increased respiration in asymptomatic discs on day 1 of storage being associated with symptom development on day 2. Calcium chloride and ascorbic acid treatments inhibited symptom development, demonstrating the potential of this method to provide causal evidence.Results indicate that symptom development in this system is consistent with blossom-end rot development with regards to location, color change, and the activity of key enzymes. This system has the potential to be used to elucidate the cause of fruit calcium deficiency and improve knowledge of the biological basis for calcium’s diverse effects on fruit.The online version contains supplementary material available at 10.1186/s13007-021-00728-3. Blossom-end rot (BER) in tomatoes and peppers causes significant losses in the vegetable industry each year. BER is characterized by water-soaking of the tissue and cell death in the blossom-end pericarp. This is followed by blackening and sometimes drying of the affected tissue. BER occurs during fruit development prior to physiological maturity . While cMany previous studies on the biological drivers of BER development in tomatoes and peppers have relied on comparing measurements from BER affected fruit to healthy fruit. This approach has identified several possible causes of cell death. Comparing BER affected tomatoes grown in a low calcium hydroponic solution to healthy fruit grown with a higher calcium hydroponic solution, Mestre et al.  found a Pericarp discs have been used previously to study ripening in tomatoes , 7. The Immature green tomatoes (Variety Harris Moran 4885 (HM)) showing no symptoms of BER were observed to develop BER symptoms during storage after harvest, if harvested just prior to the usual timing of BER development (approximately 8–12 days after pollination) on the plant  seeds were obtained from Agseeds Unlimited  and Solanum lycopersicum L. (var. Rutgers and Ailsa Craig) seeds were obtained from the Tomato Genetics Resource Center . This variety is recognized by the tomato industry as highly susceptible to BER. Seeds were sprouted in peat pellets using double deionized water (ddH2O). Approximately 2 weeks after germination, seedlings were moved to a greenhouse and transplanted into 7.6 L pots with a mixture of 1/3 peat, 1/3 sand, and 1/3 rosewood compost, augmented with 1.56 kg m−1 dolomite lime. Plants were irrigated daily until pots were dripping with a solution containing 150 ppm nitrogen, 50 ppm phosphorus, 200 ppm potassium, 175 ppm calcium, 55 ppm magnesium, 120 ppm sulfur, 2.5 ppm iron, 0.02 ppm copper, 0.5 ppm boron, 0.50 ppm manganese, 0.01 ppm molybdenum, 0.05 ppm zinc, and 0.02 ppm nickel. Flowers were manually pollinated and tagged. HM fruit used for whole fruit experiments were harvested 9 days after pollination. HM and Rutgers fruit used for pericarp disc experiments were harvested 21 days after pollination.Whole fruit were stored blossom-end up in a closed container under a constant flow of humidified air to reduce the accumulation of ethylene and carbon dioxide. Fruit were photographed daily.2O and blotted dry on sterilized filter paper. Discs were weighed and placed skin side down in 24 well tissue culture plates , noting the original fruit and tissue location.Discs were prepared from the stem-end pericarp, blossom-end pericarp, and columella tissue on the day of harvest of 21-day-old tomato fruit. Fruit were surface sterilized in approximately 150 mL of 1% sodium hypochlorite for 1 min, then thoroughly rinsed in double deionized water. A cork borer (approximately 13.5 mm) was used to excise cylindrical tissue samples. For discs made from pericarp tissue, locular and endocarp tissue was removed using a razor blade. Discs were rinsed in ddHFor discs made from columella tissue, a 13.5 mm diameter cylinder was cut from the stem-scar to the center of the blossom-end of the fruit. The blossom-end pericarp and stem-scar were removed, producing a cylinder containing columella tissue with small amounts of locular and seed tissue. This cylinder was cut into discs approximately 2 mm in height, and these were rinsed in ddH2O and blotted dry using sterile filter paper. The discs were placed in a 24 well tissue culture plate with the disc locations from the stem-end to the blossom-end preserved. Tissue culture plates were stored without lids in a closed container under a constant flow of humidified air to reduce the accumulation of ethylene and carbon dioxide.After approximately 3.5 days, discs were scored based on their symptom development on a 0 to 5 scale, with 0 being no symptom development and 5 being complete deterioration and blackening of the tissue. Discs were turned upside down and disc color was measured by inverting a Minolta colorimeter  and measuring the color of the pericarp through the bottom of the plate. Disc color was measured through the bottom of the plate rather than from the top of the well to decrease effects of ambient light. In respiration and treatment testing experiments, disc color was measured through the top to reduce the mechanical damage and potential contamination associated with flipping discs pericarp side down for color measurement from the bottom. Measuring from the top is recommended for future studies. Discs were then weighed, frozen in liquid nitrogen, and stored at −80 °C for subsequent enzyme analysis. To determine weight loss trends over time, 6 Rutgers discs from the stem-end pericarp, side pericarp, and blossom-end pericarp were weighed on Day 0, Day 2, and Day 3.5 in a separate experiment.To compare color changes in discs to the color change of fruit on the plant, 4 fruit exhibiting BER symptoms were harvested. A thin layer of skin was removed from one spot within the BER affected tissue and two spots on the stem-end to expose the pericarp tissue for comparable readings with the discs. Color was measured at these spots as stated above, and the two measurements from the stem-end were averaged for each fruit to create 4 stem-end measurements and 4 blossom-end measurements.g and 4 °C, and the supernatant was used as a crude enzyme extract. Enzyme extracts for ascorbate oxidase activity on whole fruit were prepared using the same method.After 4 days of storage, color was measured on 24 stem-end and 24 blossom-end discs from HM fruit. These discs were frozen and stored at − 80 °C until enzyme extraction. Discs were ground with a pestle as they thawed in a room temperature mortar with 100 mM phosphate buffer, pH 6. The resulting homogeneous mixture was then centrifuged for 20 min at 20,0002O2. H2O2 was added last to start the reaction. The increase in absorbance at 420 nm, associated with purpurogallin formation, was monitored over 3 min. The slope of the regression line for the plot of absorbance over time was used to calculate enzyme activity on a min−1 g−1 fresh weight basis. The extinction coefficient used for the calculations was 12.0 (mg/mL)−1 cm−1.Peroxidase activity was assayed spectrophotometrically using a BioTek H1 multimode plate reader . The total reaction volume of 100 μL included 70 μL of ultrapure water, 3.33 μL of 100 mM phosphate buffer, 10.66 μL of pyrogallol, 10.66 μL of enzyme extract, and 5.33 μL 0.5% H−1 g−1 fresh weight basis was calculated using 2.8 mM−1 cm−1 as the extinction coefficient.Ascorbate oxidase was assayed using a total reaction volume of 100μL including 75.33 μL of ultrapure water, 3.33 μL of 100 mM phosphate buffer, 10.66 μL of 250 µM ascorbic acid, and 10.66 μL of enzyme extract. The change in absorbance at 290 nm over 2 min was measured, and activity on a min2 production (μl CO2 min−1 g−1 fresh weight) was measured using an infrared CO2 analyzer .Respiration rate was measured in HM discs by sealing each of the wells in the 24 well plate with an adhesive PCR plate cover  and attaching an adhesive septum  to the top. Samples (1 mL) were collected by inserting a syringe into the airspace above the discs. Respiration rate was measured 1, 2, and 3 days after harvest. Wells were sealed for 5–11 min. CO2 or 500 mM ascorbic acid and shaken gently on a rotating platform for 15 min. Controls included 15 min treatments in ddH2O (pH 5.58) or ddH2O adjusted to pH 2.00 on 8 discs each from the same fruit. The discs were blotted dry on sterile filter paper and placed in a 24 well tissue culture plate. The plates were stored in a humidified container as stated above. After 3.5 days the discs were photographed and their color measured. For color measurement of the treated discs the discs were left with the pericarp tissue facing up and the colorimeter was placed directly above each well.Calcium chloride and ascorbic acid treatments were applied to discs as 15 min dips. Discs were prepared from the blossom-end pericarp tissue of 4 HM fruit as stated above. After rinsing and blotting, 2 discs from each fruit  were placed in 15 mL of either 10 g/L (90.1 mM) CaClStatistical analysis was carried out using SAS On Demand for Academics . Pearson’s correlation for enzyme analysis and color measurements was completed on a total of 48 discs, 24 from stem-end pericarp and 24 from blossom-end pericarp. A general linear model procedure was used to determine significant differences in weight loss and a Tukey’s honest means separation was completed. Pearson’s correlation comparing respiration on day 1 with color measurement on day 2 was completed on 24 discs, comprised of 12 stem-end discs and 12 blossom-end discs.Additional file 1: Figure S1. Sampling locations and visual symptom scale. Fruit tissues (a) used for discs included the stem-end, middle, and blossom-end pericarp, as well as columella tissue. Representative (b) discs are shown for each rating on the 0–4 visual symptom rating scale.Additional file 2: Video 1. Video depicting blossom-end rot symptoms spreading during storage of pericarp discs from immature green tomatoes. The first and third rows of discs were obtained from the stem-end of fruit and the second and forth rows of discs were made from the blossom-end of fruit.Additional file 3: Figure S2. Ascorbate oxidase activity in BER unaffected and BER affected whole fruit harvested 21 days after pollination. Each bar is the mean of 4 replicates, and bars with the same letter were not statistically different (p > 0.05).


In view of limited resources during the COVID-19 pandemic, there is an urgent need to create novel programs to meet the changing demands of trainees towards developing and strengthening their skills in healthcare research. During the COVID-19 pandemic, digital learning has become an invaluable tool by providing more learning opportunities. Through the use of platforms available for distant learning, we made our pre-existing online research program more interactive. Through collaboration in small groups, the trainees developed their research and mentorship skills and were able to meet the goal of submitting their research projects as abstracts. All of the abstracts were accepted for publication. Because of the COVID-19 crisis, many trainees have paused their research and are seeking opportunities to restart their research efforts and strengthen their skills while others have had to modify their projects. There is a major concern that an increasing number of trainees who are seeking competitive post-graduate training or fellowships are pressured to take an extra year of research to make up for the lost opportunities [We describe an innovative Online Research Program developed at the University of California, Irvine that trains premedical and medical students about the basics of healthcare research with an emphasis on literature review and evidence-based medicine. The cascading nature of our program has been described previously . Under tI am really glad that I was able to participate in this program and develop my research skills and my mentoring capabilities.The program was very beneficial for both the coaches and students! The faculty mentor is very responsive to any questions/concerns throughout the program. I also appreciated the flexibility with a schedule that this program allowed. Overall, a wonderful experience that I would love to participate in again.Overall, I think the second time around I felt more confident in organizing my time and communicating with the rest of the students. I also feel like I gleaned a lot of information from the faculty mentor about residency and fellowship programs and the importance of work–life balance.I completed this program with the same faculty mentor last year and I really enjoyed it. I like that there were zoom meetings and more interaction this year in comparison to last year-a great improvement.In this study, we only focus on the feedback from the coach mentors. All of the coaches responded to our survey. Based on a 4-point Likert Scale ranging from 1 = decreased and 4 = significantly increased, the average rating of the coaches on the impact of the program in promoting their interest in research was 3.6, their leadership skill was 3.9, their mentorship skills were 3.9, and their ability to do a literature review was 3.7. All of the coaches rated their satisfaction with the structure of the program and their research topic at the highest rating of 4. The qualitative feedback survey included the following comments:Our goal was met as all of the 10 abstracts submitted by the coaches as the first author were accepted for publication in the Journal of Investigative Medicine. Of the 10 abstracts, five were related to health inequalities and the other 5 were related to cutting-edge COVID-19 research topics.We believe that there were several reasons for the effectiveness of our program during the summer of 2020. In their feedback, the returning coaches stated that they preferred the 2020 program compared to the previous years because the interactive and collaborative nature of live video meetings in real time helped them to be innovative and develop their mentorship and leadership skills. As Palloff describes, collaborative learning that allows sharing of ideas among the students is very important in forming the foundation of a learning community . The coaFinally, faculty mentor’s commitment to mentorship contributed to the success of the program. She encouraged a comfortable but goal-oriented environment where the coaches provided guidance to students through near-peer mentorship. Having known a few of the coaches over the years, the faculty mentor was able to assess each of their capabilities and develop an individualized plan to make sure the coach would succeed in mentoring high school students and completing their research projects with the goal of publication. The goal of publishing all projects as abstracts as well as the positive responses from the coaches suggest that our virtual-based research program has been successful and can be implemented at other institutions.


The idea is to take as input a specification, a program, and a verification witness, and produce a new specification and a transformed version of the original program such that the transformed program satisfies the new specification if the witness is useful to confirm the result of the verification. Then, an ‘off-the-shelf’ verifier can be used to validate the previously computed result (as witnessed by the verification witness) via an ordinary verification task. We have implemented our approach in the validator, and it was successfully used in SV-COMP 2020 and confirmed 3 653 violation witnesses and 16 376 correctness witnesses. The results show thatimproves the effectiveness (167 uniquely confirmed violation witnesses and 833 uniquely confirmed correctness witnesses) of the overall validation process, on a large benchmark set. All components and experimental data are publicly available.Witness validation is an important technique to increase trust in verification results, by making descriptions of error paths (violation witnesses) and important parts of the correctness proof (correctness witnesses) available in an exchangeable format. This way, the verification result can be validated independently from the verification in a second step. The problem is that there are unfortunately not many tools available for witness-based validation of verification results. We contribute to closing this gap with the approach of  Formal software verification becomes more and more important in the development process for software systems of all types. There are many verification tools available to perform verification . One of The idea to witness the correctness of a program by annotating it with assertions is as old as programming , and fro, which we describe here. Witness validation is an interesting problem to work on, and there is a large, yet unexplored field of opportunities. It involves many different techniques from program analysis and model checking. However, it seems that this also requires a lot of engineering effort.Yet, although considered very useful, there are not many witness validators available. For example, the most recent competition on software verification SV-COMP 2020)0202 showvalidation via verification is a construction that takes as input an off-the-shelf software verifier and a new program transformer, and composes a witness validator in the following way  can lead to strong validators.All data and software that we describe are publicly available .The conversion from the source code into a CFA and vice versa is straight forward, provided that the CFA is deterministic. A CFA is called Since our goal is to validate  the statement that a program fulfills a certain specification, we need to additionally model the property to be verified. For properties that can be translated into non-reachability, this can be done by defining a set entclass1pt{minimaconcrete data state is a mapping from the set V of program variables to their domain concrete state is a pair of control location and concrete data state. A concrete program path is then defined as a sequence concrete executionIn order to reason about the soundness of our approach, we need to also formalize the program semantics. This is done using the concept of concrete data states. A given in, where cprotocol automatonQ of states, a set of transition labels V to represent the state invariant.V to represent the guard condition for the transition to be taken. An observer automaton [true. Correctness witnesses are represented by observer automata in which the set of final states is empty.Witnesses are formalized using the concept of protocol automata . A prototomaton , 34, 36 C = T L, and a witness automaton W = g in the CFA only those transitions When given a CFA op is a pure assumption of the form f(V) then op is an assignment of the form C by projecting their pairs  on their first element. Let C, i.e., It is evident that the automaton  reducer, and thethat was used in SV-COMP 2020 did not yet support strict witness validation.For explaining the validation of results based on a violation witness, we consider the witness in Fig. C in Fig. Correctness witnesses are represented by observer automata. Figure entclass1pt{minimacheck the invariants in the witness anduse the invariants to establish that the original specification holds.The idea of a result validator for correctness witnesses is to q and the invariant of q does not hold, and we add self-loops such that the automaton stays in the new accepting state forever. In sum, for each invariant, there are two transitions, one with the invariant as guard (to assume that the invariant holds) and one with the negation of the invariant as guard  state if it does not hold). This transformation ensures that the resulting automaton after the transformation is still a proper observer automaton.We can achieve the second goal by extracting the invariants from each state in the product automaton participated as validator. We did not perform a separate evaluation because the results of SV-COMP are complete, accurate, and reproducible; all data and tools are publicly available for inspection and replication studies (see data availability in Sect. This section describes the results that were obtained in the 9th Competition on Software Verification (SV-COMP 2020), in whichExecution Environment. In SV-COMP 2020, the validators were executed in a benchmark environment that makes use of a cluster with 168 machines, each of them having an Intel Xeon E3-1230 v5 CPU with 8 processing units, 33 GB of RAM, and the GNU/Linux operating system Ubuntu 18.04. Each validation run was limited to 2 processing units and 7 GB of RAM, in order to allow up to 4 validation runs to be executed on the same machine at the same time. The time limit for a validation run was set to 15 min for correctness witnesses and to 90 s for violation witnesses. The benchmarking framework2.5.1 was used to ensure that the different runs do not influence each other and that the resource limits are measured and enforced reliably [Benchmark Tasks. The verification tasksValidator Configuration. Since our architecture  framework[ramework, 41. BotAutomata-Based Software Model Checking. Our approach is also similar to that of the validator [constructs the product of CFA and witness, and applies a similar instrumentation. In both cases,’s transformer produces a C program, which can be passed to an independent verifier.dator . For viodator , Sect. 4Reducer-Based Conditional Model Checking. The concept of generating programs from an ARG has also been used to successfully construct conditional verifiers [acts as initial verifier that does not try to reduce the search space and instead just instruments the invariants from the correctness witness as additional specification into the program.rifiers . Our appVerification Artifacts and Interfacing. The problem that verification results are not treated well enough by the developers of verification tools is known [s known  and thers known  or on cos known .Test-Case Generation. The idea to generate test cases from verification counterexamples is more than ten years old [ars old , 39, hasars old , 33, andars old , 29, 35.Execution. Other approaches [roaches , 22, 28 Debugging and Visualization. Besides executing a test, it is important to understand the cause of the error path [or path , and theor path , 9, 26.validation via verification, which is a two-step approach that first applies a program transformation and then applies an off-the-shelf verification tool, without development effort.We address the problem of constructing a tool for witness validation in a systematic and generic way: We developed the concept of , which has already been successfully used in SV-COMP 2020. The validation results are impressive: the new validator enriches the competition’s validation capabilities by 164 uniquely confirmed violation results and 834 uniquely confirmed correctness results, based on the witnesses provided by the verifiers. This paper does not contain an own evaluation, but refers to results from the recent competition in the field.The concept is implemented in the witness validatorThe major benefit of our concept is that it is now possible to configure a spectrum of validators with different strengths, based on different verification engines. The ‘time to market’ of new verification technology into validators is negligibly small because there is no development effort necessary to construct new validators from new verifiers. A potential technology bias is also reduced.Data Availability Statement. All data from SV-COMP 2020 are publicly available: witnesses [ 1.0 is available on GitLabtnesses , verifictnesses , and bentnesses 7. The vatnesses  and on t


Metagenomic next-generation sequencing (mNGS) is a new technology that allows for unbiased detection of pathogens. However, there are few reports on mNGS of lung biopsy tissues for pulmonary infection diagnosis. In addition, radial endobronchial ultrasound (R-EBUS) is widely used to detect peripheral pulmonary lesions (PPLs), but it is rarely used in the diagnosis of peripheral lung infection.  The present study aims to evaluate the combined application of R-EBUS-guided transbronchial lung biopsy (TBLB) and mNGS for the diagnosis of peripheral pulmonary infectious lesions.  From July 2018 to April 2019, 121 patients from Tianjin Medical University General Hospital diagnosed with PPLs and lung infection were enrolled in this prospective randomized study . Once the lesion was located, either TBLB or R-EBUS-guided-TBLB was performed in randomly selected patients, and mNGS was applied for pathogen detection in lung biopsy tissues. The results of mNGS were compared between the TBLB group and R-EBUS-guided TBLB group. In addition, the clinical characteristics and EBUS images from 61 patients receiving bronchoscopy for peripheral lung infectious detection were analyzed and compared with the results of mNGS. P=0.030, odds ratio 17.742; 95% confidence interval, from 1.325 to 237.645). Anechoic areas and luminant areas of ultrasonic image characteristics were correlated with lower positivity rate of mNGS .  The positivity rate of mNGS in R-EBUS-guided TBLB was  that was significantly higher than  in the TBLB group. Difference in the position of R-EBUS probe and image characteristics of peripheral lung infectious lesions affected the positivity rate of mNGS. Tissue collected by R-EBUS within the lesion produced higher positivity rate than samples collected adjacent to the lesion ( R-EBUS-guided TBLB is a safe and effective technique in the diagnosis of peripheral lung infectious lesions. R-EBUS significantly facilitates the accurate insertion of bronchoscope into the lesions, which improves positivity rate of mNGS analysis in pathogen detection. The R-EBUS probe position within lesion produced a higher positivity rate of mNGS analysis. Nevertheless, the presence of anechoic and luminant areas on ultrasonic image was correlated with poor mNGS positivity rate. Pulmonary infection is a leading cause of death and morbidity worldwide . HoweverPathogen identification by mNGS is based on unbiased detection of nucleic acids isolated from clinical samples, including but not limited to tissue, body fluids, swabs, and bronchoalveolar lavage, stool or polymicrobial abscesses . ApplicaEtiology inspection in PPLs is challenging compared to central pulmonary lesions due to the limitation in obtaining histopathological specimens. Conventional bronchoscopy has difficulty in reaching the exact location of the lesions in the peripheral lung. CT-guided percutaneous biopsies are associated with a higher risk of complications including pneumothorax or hemoptysis , 12. OncA prospective randomized trial of bronchoscopic procedures was performed in Tianjin Medical University General Hospital between July 2018 and April 2019. During this period, bronchoscopy was performed in 151 patients who were suspected to have peripheral lung infection by chest spiral CT. Among these patients, 30 patients were excluded from the study due to histopathological diagnosis of lung cancer and 121 patients were eventually diagnosed as pulmonary infections. Among these patients, 61 patients were diagnosed by R-EBUS-guided TBLB and the other 60 patients by TBLB. Pathogen identification in lung biopsy tissues was performed by mNGS. Lung lesion was considered as PPL when its location was beyond the segmental bronchus [Electronic video bronchoscope , ultrasonic host , R-EBUS with a 1.4 mm diameter , and Biopsy forceps  were used in performing R-EBUS-guided TBLB or TBLB.TBLB was performed according to diagnostic flexible bronchoscopic application guide (2008 Edition) published by Chinese Medical Association Respiratory Diseases Society . BronchoLesions were located by studying the chest CT. The biopsy procedure was repeated until lung tissue spilled over the surface of the biopsy forceps. In the TBLB group, the operator maneuvered the bronchoscope to the suspected bronchi as far as possible until resistance was met and then conducted forceps biopsy. In the R-EBUS-guided TBLB group, the EBUS probe was inserted into the suspected bronchi through the working channel of the bronchoscope until resistance was met to detect the PPLs. Once the lesion was located, R-EBUS was performed through the working channel of the bronchoscope until resistance was met. Then, the EBUS probe was inserted into the suspected bronchi to detect the PPLs before R-EBUS-guided TBLB was performed. All pathological results were inspected by two experienced pathological doctors. To determine if iatrogenic pneumothorax had developed, initial chest radiographs were obtained 4 hrs after the procedure and follow-up chest X-rays the following day.The lung biopsies were separately sent to clinical microbiology and histopathology laboratories within 2 hrs for analyses. The remaining or leftover tissue homogenates were stored at −70°C for mNGS.The lung biopsies were cut into small pieces according to standard procedures. The 1.5 mL microcentrifuge tube with 0.7 mL lysis buffer and pieces of tissue sample and 1°g 0.5 mm glass bead were attached to a horizontal platform on a vortex mixer and agitated vigorously at 2800–3200RPM for 30 min. 0.3 mL sample was separated into a new 1.5 mL microcentrifuge tube, and DNA was extracted using the TIANamp Micro DNA Kit  according to the manufacturer's recommendation. RNA extraction shared same tissue collection procedures as mentioned above, the extraction kit QIAamp Viral RNA Mini Kit  was used for extraction of RNA, and then complementary DNA (cDNA) was generated from an RNA template by reverse transcription.ftp://ftp.ncbi.nlm.nih.gov/genomes/). RefSeq contains 4,061 whole genome sequences of viral taxa, 2,473 bacterial genomes or scaffolds, 199 fungi related to human infection, and 135 parasites associated with human diseases. Combining the results of controls and calibrators, data-analytical algorithms were used to exclude microorganisms that were not significantly related to infection. Microorganisms with clinical significance were reported with the sequencing reads of the micro-organisms detected at the genus/species levels.DNA libraries were constructed through DNA-fragmentation, end-repair, adapter-ligation, and PCR amplification. Agilent 2100 Bioanalyzer  was used for quality control of DNA libraries. Quality qualified libraries were sequenced by BGISEQ-50 platform . At leasThe results of the mNGS-based approach were estimated by 2 independent clinical specialists not associated with the study.t-tests were used to compare the means of the independent variables. Pearson's chi-square test or Fisher's exact test was used for categorical variable. Multivariate logistic regression tests were used to further confirm the results of independent variable analysis. P value <0.05 was considered as statistically significant.SPSS  was used for statistical analyses. The data are expressed as mean (standard deviation). Paired P > 0.05.A total of 121 patients with peripheral lung infectious lesions were enrolled in a prospective randomized study, 60 patients in the TBLB group and 61 patients in the R-EBUS-guided TBLB group. Their baseline characteristics are shown in P=0.026).Final diagnosis of targeted lesions is shown in More than half of the 121 enrolled patients had hematological malignancies, including 70 immunocompromised and 51 immunocompetent individuals. Immunocompromised individuals were more susceptible to lung infections than the immunocompetent ones. The most common pathogens detected by mNGS are shown in P=0.013, odds ratio 6.080; 95% confidence interval (CI), from 1.630 to 22.685). However, the significance was lost after multivariate analysis combined with the R-EBUS probe position and EBUS images characteristics , from 0.858 to 89.417). Distance from the chest wall of the lesion was not associated with the diagnostic yield of mNGS (P=0.057). The presence of probe within the lesion were associated with significantly better diagnostic yield with radial EBUS than when the probe was found adjacent to the lesion , from 1.325 to 237.645).In the R-EBUS group, the diameter of lesion region >3 cm was associated with significantly better diagnostic yield by univariate analysis , 1.588 to 23.107). However, the significance was lost after multivariate analysis combined with the R-EBUS probe position and EBUS images characteristics , from 0.903 to 81.885). Anechoic areas and luminant areas of ultrasonic image characteristics were correlated with lower diagnostic yield of mNGS , from 1.595 to 200.399; P=0.042, odds ratio 16.745; 95% confidence interval (CI), from 1.106 to 253.479).In our study, pathogens were successfully detected by mNGS mostly in the lesion with homogeneous internal echoes , and 4 lesions  with homogeneous internal echoes were negative in mNGS analysis. More than half of cases exhibited the characteristics of anechoic area in mNGS negative cases , while only 5 cases had positive mNGS results with anechoic areas, included 1 case of pulmonary tuberculosis and 1 lung abscess. There were 8 cases involving luminant areas with positive mNGS results , and 8 cases with negative mNGS . Homogenous internal echoes were correlated with better diagnostic yield of mNGS from lung biopsy tissues by univariate analysis (P > 0.05).Over all, complications related to TBLB group occurred in 4 patients (6.6%): mild bleeding was developed in 2 patients (3.3%) and pneumothorax was developed in 2 patients (3.3%). One patient (1.6%) developed mild bleeding, and one patient (1.6%) developed pneumothorax in the R-EBUS-guided TBLB group. The bleeding was improved after local application of norepinephrine and intravenous drip of vasopressin. The pneumothorax was resolved spontaneously without chest tube drainage. No patient suffered from severe hemorrhage, air embolism, respiratory failure, or pulmonary infection after the procedure. There were no premature terminations of the procedure, and none of the patients died due to application of the procedure. No significant differences in the occurrence of complications were detected between the TBLB group and R-EBUS-guided TBLB group (P < 0.05). Previous studies reported that the diagnostic yields of utilize EBUS with a guide sheath or EBUS-guided biopsy in PPL were 53–75.9% [Lack of accurate etiology diagnosis remains the major cause of high morbidity and mortality in pulmonary infections, which increases healthcare expense. mNGS is an unbiased and rapid technique capable of detecting a broad range of pathogenic bacterial, viral, fungal, and parasite simultaneously in clinical samples. R-EBUS is a safe and valuable technique, which has been demonstrated to increase diagnostic yield for PPLs . Current53–75.9% –18, 37. This study showed that the diagnostic yield of pathogens detection by mNGS with TBLB was not related to the size of lesion and its distance to the chest wall of peripheral infection lesions; this is not consistent with previous studies , 33, 36.Our study indicated that ultrasound image characteristics of peripheral lung infectious lesions were correlated with the diagnostic yield of mNGS. The image of anechoic areas and luminant areas of ultrasonic image characteristics were correlated with worse diagnostic yield of mNGS from lung biopsy tissues. Lesions with anechoic areas and luminant areas might result from necrosis, where airway inflammation is light, pathogen load is low, and lesions tend to be chronic. Therefore, the diagnostic yield of mNGS approaches after TBLB is low.There were several limitations to our study. First, it was conducted at a single institute owing to the difficulties in recruiting subjects, so there was not sufficient sample size. Second, the majority of patients had hematological malignancy, and potential selection bias might influence the results. Third, we did not use fluoroscopy, electromagnetic navigation bronchoscopy, or virtual bronchoscopy with R-EBUS, all of which could have further improved the diagnostic accuracy of radial EBUS-guided biopsy. Considering the medical expenses, the enrolled patients did not receive mNGS analysis of bronchoalveolar lavage fluid or serology at the same time. Whether lung tissue mNGS is superior to bronchoalveolar lavage fluid or serology in patients with pulmonary infection required further investigation. Furthermore, studies designed as multicenter trials are needed to validate the result from this study.Pneumonia is a common infection that often lacks pathogen diagnosis. Rapid and accurate pathogen detection and identification of pathogen is critical for the precise antibiotic therapy. Many patients are on antibiotic therapy, which limits the yield of culture-based testing. Thus, we hope the early use of mNGS testing to identify the pathogen in the everyday clinical practice. For PPLs, we can combine with R-EBUS. Considering the high cost of mNGS test, we recommend the early use of mNGS testing in failure cases of empirical treatment and severe cases.Although there are some limitations, our study firstly demonstrated that pathogens diagnosis of peripheral lung infectious lesions by mNGS in TBLB was meaningful and that R-EBUS-guided TBLB is useful in confirming the accurate insertion of the bronchoscope into PPLs, which improved diagnostic yield of mNGS. The R-EBUS probe position within the lesion was correlated with higher diagnostic yield of mNGS from lung biopsy tissues. EBUS patterns of peripheral pulmonary lesions with anechoic areas and luminant areas were correlated with the diagnostic yield of mNGS.


This article focuses on the process of designing the vital, participatory school-based intervention program aiming to increase the physical activity in schools. The program analyzed is Estonian nationwide comprehensive physical activity program Schools in Motion (SiM) that recently received European Commission’s #BeActive Education Award. The program has a good performance in terms of willingness of schools to participate in co-creation of program development, the high interest to join the program and zero dropouts, and strong partnership with ministries which enables to actively participate in policy making. Authors analyze the key elements of the planning, piloting, implementation, and scaling phases of the SiM program and share their lessons learnt in co-working with schools. The difficulties faced during the development process, the strengths and challenges associated with an interdisciplinary approach, and involvement of schools as experts have been addressed. Physical activity (PA) is an important lifestyle factor associated with a wide range of benefits in children’s health and development, including the prevention of overweight, obesity, and cardiovascular diseases as well as supporting academic achievement and mental health –3. It haKohl et al.  have staSchools are potentially powerful agents for changes to support PA levels  but therCurrent article is focusing on the design of the socially viable school-based PA intervention programs with the aim to inspire and offer tips for designing and implementing PA-enhancing interventions within school systems.The SiM program has been successfully scaled up, starting from 10 pilot schools in 2016 and reaching 110 schools in 2020. The SiM program is currently targeted at basic school . The group of schools who participate in the program is diverse in terms of size and location, involving rural schools with 15 students up to urban schools with more than 1300 students. The Estonian SiM program has received a great deal of attention in both Estonia and abroad. For example, SiM won the European Commission’s #BeActive Education Award in 2019, which recognizes activities in the field of education to encourage young people to be more physically active [The Estonian SiM program aims to achieve sustainable change towards a PA-friendly mode of everyday operation in schools, similarly to “Whole School” approaches to PA-intervention that have arisen from Toronto Charter , and ComFigure Moreover, the renewing of the physical education curriculum, where the sport-centered approach interchanged with the lifestyle-centered approach, is included. The renewing of physical education is an ongoing process but is not discussed in the current analysis. Finally, the schema highlights the role of different stakeholders and interest groups who should participate in the process to achieve appropriate, meaningful and sustainable results.During the implementation of the program, this model has maintained both a structuring and presentation function, although the elements depicted in the model are not presented equally in the program. Throughout the process it has been important to support the autonomy of the schools, e.g. every school creates their own action plan and schedule, schools are free to decide their aims and actions while the SiM model and program activities provide them with certain tools and general suggestions. All schools entering into SiM program participate in one-day program training “Start-up seminar” with school team , after that several training seminars and workshops are made available for teams, teachers, and students. Schools are free to choose seminar or workshop to participate in depending on their own action plan, needs and main focuses. To support the autonomy of the schools and prevent overload associated with project activities, there is no mandatory seminar schedule for schools. Generally, most schools have been highly motivated to participate in provided trainings and seminars.The development process in 2014–2019 can be divided into four main phases which main focuses and activities are presented in Fig. From 2014, we started to focus on the problem of children’s physical inactivity as a serious risk for public health and wellbeing given that, according to a national survey, only 16% of 11- to 15-year-old students met the PA recommendations every day in Estonia . We statBased on the scientific literature, we concluded that it is important to start the intervention from the youngest age groups, as the period between 6 and 12 years is critical in forming healthy lifestyle behavior and PA habits . The anaSpecial focus was given to theoretical frameworks that could have been used in the development process. A socio-ecological model  and compAs there was a lack of objectively measured PA data in school settings, we carried out a nationwide objective PA measurement among 7- to 13-year-old students, where 819 students from 13 schools wore the accelerometer for seven consecutive days , while iAdditionally, we carried out focus group interviews in three Estonian schools with varied sizes, locations, and opportunities for PA. We conducted 17 focus group interviews with 92 children aged 8–15 and 9 focus group interviews with teachers, school principals and parents. The aim of our qualitative research was to understand the perceptions of different stakeholders about PA in school settings and to identify how they evaluate the possibilities for PA, what the existing practices are, and whether they feel a necessity for change considering PA opportunities in school , 36. IntThe interviews indicated that overall attitude for PA is positive in schools. However, there is a need to initiate a change in school culture through: 1) creating new practices, e.g. possibilities for PA during recess and lessons by introducing new activities; 2) changing the meaning of PA, e.g. movement during the school day is normal and PA can support mental effort and academic achievement, PA is not only sport; 3) developing the skills of teachers and students, e.g. applicable techniques and methods to integrate PA to academic lesson and recess; and 4) providing supportive tools, e.g. materials/ideas for changing the physical environment . AccordiIn parallel with the creation of the program model, negotiation with the Ministry of Social Affairs and the Ministry of Education and Science started to inform them about the serious situation concerning the low levels of PA in children and the framework of evidence-based solutions was provided. Positive feedback from the ministries for initiating piloting was received. For more wide partnership a nationwide forum “Children’s physical activity levels are decreasing – how can we stop that?” was organized with the purpose of calling society, different activity groups and politicians to action.In 2016, the piloting of the SiM program received encouragement from the Ministry of Social Affairs and funding from the Council of Gambling Tax. The RDT sent out the call to participate in the SiM pilot program. In order to participate, schools had to send a motivation letter. Based on 18 received motivation letters, 10 schools of different size (per student), location , existence of outdoor area (yes/no) and number of students  were selected.Although the plan was to run the pilot phase with 10 schools from 2016 to 2019, more schools joined in during this period due to the strong interest from schools and additional funding, culminating in 78 network schools by 2019.As the problem targeted by SiM is multifaceted and complex, an interdisciplinary team was formed from experts from sport and health sciences, education, social sciences, psychology and communication. The leading institution was the Institute of Sport Sciences and Physiotherapy, with experts who had previous experience with applied projects. Two non-academic consultants were invited as consulting members of the team as they had previously led a smaller-scale project called Safe and Active School Day. Their existing contacts as well as expertise in educational settings proved highly valuable in, for example, deciding how to communicate with or to motivate schools , which has to a large extent succeeded in Finland. Their program’s approach differs from many others as it is a so-called “bottom-up” approach, which supports schools’ own autonomy . We can During the planning phase of the SiM program, there was intensive debate regarding whether or not to include school visits to the program as they were considered too time-consuming and intrusive. However, a decision was made to test it and, during the first months of the pilot program, all schools were visited by the RDT. This was an informal visit which enabled us to gain a greater understanding of the schools and their peculiarities. In terms of the program development, it turned out that such school visits were invaluable as they helped us to understand that each school is unique and that it is impossible to develop a one-size-fits-all model. It became more evident that we can support the schools by developing materials and tools, carrying out training, and supporting the exchange of good practices and ideas for different program elements but the schools must have autonomy to choose their own focus and set of tools and methods . The aim was to deter enthusiastic schools from over planning only to realize during the process that they do not have resources to carry out all the started activities (see Tips 4 and 9 in Table Networking and exchange of practices (both good practices as well as failures) were encouraged and supported throughout the pilot program through different school visits and seminars for school teams on a regular basis. Each year there were at least two seminars for school teams whose aim was to support networking, obtain feedback about project activities and input into further development needs. Throughout the development and piloting process, a bottom-up approach and top-down approach have been used simultaneously. This means that the schools are considered as partners and experts whose feedback is thoroughly considered. At the same time different solutions and elements are developed and disseminated centrally by the program’s RDT. It can be said that schools in the SiM program are not intervention venues but active creators. The schools are also involved in the dissemination process as, in some cases, the experience and advice from other schools is more valued compared to the advice of university researchers  increase the awareness of the positive influence of PA on mental health, cognitive functions and learning; 2) provide new skill and exchange experiences; and 3) provide supportive and ready-to-use materials to the participants. The teacher training for active lessons consisted of two training days in order to help to anchor the skills and ideas learnt during the training and for them to gain confidence in using them. On the first training day the importance of PA on learning and academic achievement was highlighted. Throughout the day the techniques for reducing sedentary time and integrating PA into learning were modelled and the teachers had the possibility to play through various activities and discuss with colleagues where and how they could be used with their students. The teachers also received supportive materials and were requested to keep a diary of their practice for two weeks. After a month of individual practice at school, the teachers received the second training day, where the focus was more on the exchanging of experiences as well as getting new ideas to support the teachers’ motivation to carry on with the activities  for the students to use. As outdoor recess is not common in Estonia, schools were encouraged to try all-year outdoor recess as previous research had indicated the positive effect of outdoor recess on PA –45. HoweThe regulations concerning the outdoor recess are also crucial as, in Estonia, the weather conditions are cool and wet for most of the academic year and require appropriate clothing. This means that the students have to go to their wardrobe to get outdoor clothes, which is time-consuming and might require additional cleaning services as the students carry in dirt. Thus, the outdoor recess can bring additional costs that need to be acknowledged and pre-planned  research material consisting of examples or stories acts as a strong motivating factor for the RDT. Although this might only seem to be a positive side-effect, this could be a key driver for the RDT as the efficient intervention planning undoubtedly requires a great deal of engagement. Having occasional reminders in the form of comments, stories, and personal experience from school visits and observations is a highly needed impetus for this type of program. The collected qualitative data complements and helps to interpret quantitative data as the complexity of social situations and the multiplicity of factors makes it very difficult to ascertain causal relationships with only objective physical activity measurements or surveys  and social health are constantly presented. In addition, the necessity for a more active class environment which is based on the arguments related to health effects and supported by medical experiments that observed the “acute effects of a simulated school day with reduced sitting or usual sitting on adolescents’ cognitive function and cardiometabolic biomarkers”  is highlwww.liikumakutsuvkool.ee) was launched to communicate SiM activities and principles to a broader audience, such as schools outside the network, different stakeholders and interest groups. The webpage includes an overview of all SiM elements, tools and materials, coming events and a “Bank of Ideas” which is a database of methods, ideas and games on how to add PA to lessons, recess and environment.In 2018, a SiM webpage  implementers. Consequently, not only the effectiveness promised by the trials but also the prospective vitality of the suggested intervention in the “natural” settings have to be addressed in the design of the program, as Reis et al.  have strDespite the positive acceptance of SiM by schools, there are still many challenges ahead. The RDT has analyzed shortages in the program and continues to work out solutions. The further development has to be more age specific. Outdoor recess and playing during recess are attractive for younger children but not appealing for the older students aged 13–16, who are the most physically inactive group in basic schools. The training of new skills among teachers has to involve not only the development of personal skills, but also knowledge transfer to the other teachers and staff members at the in-school training sessions, inspiration seminars, regional training networks or teacher training institutions. The PA-friendly school concept also considers teachers’ and principals’ health and PA, and students’ families’ PA improvement. The social norms that discourage active transport from home to school as a sign of parental negligence are also a great challenge to meet via public communication during the coming years. The further development of SiM and its scaling-up will proceed in parallel, accompanied by action research. In addition, the rapid growth of participants requires creating a sustainable model for managing the network, providing enough support for participating schools and, concurrently, to keep the main principle for co-creative and collaborative approach throughout scaling-up process.The development and implementation of a comprehensive school-based PA intervention is a great challenge as the school is a complex social network comprising different interests, stakeholders and aims. We have managed to develop a flexible and comprehensive school-based PA program which has been well received by both schools and overall society. However, many challenges still lie ahead. We believe that sharing our experience – both our successes as well as the difficulties faced, and lessons learnt – will help to promote the design and implementation PA-enhancing interventions within school system and fight the overwhelming physical inactivity epidemic worldwide.