Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients' sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 10 Yet IgM tissue deposits are a common finding in diverse inflammatory conditions –28 and e2 proteins or 104-105 random peptides. The IgM repertoire has been previously probed by protein arrays , whereas human monoclonal IgM paraprotein was isolated from an IgM myeloma patient's serum selected from the biobank at the Center of Excellence for Translational Research in Hematology at the National Hematology Hospital, Sofia (with the kind cooperation of Dr. Lidiya Gurcheva). In both cases, IgM was purified using affinity chromatography with polyclonal anti-μ antibody coupled to agarose . A 7-mer random peptide library  was panned overnight at 4°C on pooled human IgM adsorbed on polystyrene plates at a concentration of 0.1 mg/ml, washed, eluted with glycine buffer at pH 2.7, and immediately brought to pH 7. The eluate was transferred to a plate coated with monoclonal IgM and incubated according to the same protocol, but this time, the phage solution was collected after adsorption and amplified once, following the procedure described by Matochko et al. (Sera were obtained from randomly selected patients with glioblastoma multiforme (GBM), brain metastases of breast (MB) or lung (ML) cancers, and non-tumor-bearing patients (C)  of the Neurosurgery Clinic of St. Ivan Rilski University Hospital, Sofia. The samples were acquired according to the rules of the ethics committee of the Medical University in Sofia, after its approval and obtaining informed consent. The sera were aliquoted and stored at −20°C. Before staining, the sera were thawed; incubated for 30 min at 37°C for dissolution of IgM complexes; diluted 1:100 with phosphate-buffered saline (PBS), pH 7.4, and 0.05% Tween 20 with 0.1% bovine serum albumin (BSA); further incubated for 30 min at 37°C; and filtered through 0.22-μm filters before use. The serum IgM reactivity was analyzed on different sets of peptides defined in microarray format.in situ as 7-mer peptides attached to the surface through their C-terminus and a common spacer GGGS. The layout was in a format of a single field of up to 5,500 or five fields of up to 600 peptides in randomly positioned duplicates. The chips were blocked for 60 min using PBS, pH 7.4, and 0.05% Tween 20 with 1% BSA on a rocker; washed 3 × 1 min with PBS, pH 7.4, and 0.05% Tween 20; and incubated with sera in dilutions equivalent to 0.01 mg/ml IgM (~1:100 serum dilution) on a rocker overnight at 4°C. After 3 × 1-min washing, the chips were incubated with secondary antibodies at room temperature (RT), washed, rinsed with distilled water, and dried by spinning in a vertical position in empty 50-ml test tubes at 100 × g for 2 min.The customized microarray chips were produced by PEPperPRINT™  by synthesis https://github.com/ansts/IgMimoPap1 and https://github.com/ansts/IgMimoPap2). For algorithm details, see The microarray images were acquired using a GenePix 4000 Microarray Scanner . The densitometry was done using the GenePix® Pro v6.0 software. All further analysis was performed using publicly available packages of the R statistical environment for Windows (v3.4.1)  as well as in-house developed R scripts . At the same time, an almost complete diversity of sequences of that length could be interrogated. As a repertoire template, we used an experimental preparation of human immunoglobulins for intravenous use enriched in IgM. It represents a pool of the repertoire of ~10,000 healthy donors. Total IgM was isolated from it by affinity purification. The phage eluted from the IgM repertoire was adsorbed on a monoclonal IgM to filter out phage binding to the constant regions, thereby focusing only on the mimotopes  of diversity 10imotopes . The pepimotopes . Two sepimotopes . The lowThe overall amino acids residues frequencies (AAF) in the mimotopes selected from the phage library showed a skewing in favor of G, W, A, R, T, H, M, P, and Q and against C, F, N, Y, I, L, and S , when coTo gain insight into the mimotope sequence space, the set of 224,087 selected mimotope sequences was subjected to clustering using the GibbsCluster-2.0 method . This al5 peptides is impractical for routine diagnostic use. A way to scale down the mimotope probe array would be to include a representative sequence of each of the naturally existing 790 clusters. Only the sequence with the top score from each cluster was kept as a mimotope prototype for the cluster. These are expected to sample evenly  the mimotope sequence space, as ensured by the GibbsCluster algorithm.The mimotope library of more than 200,000 sequences is a rich source of potential mimotope candidates for vaccine or diagnostics. Yet the size of a probe containing an array of 10The mimotope sequence diversity in each cluster was significant. Thus, cutting down so much the list of representative sequences would seem counterintuitive with respect to our goals. Nevertheless, this approach was chosen because such a symmetric sequence set was hypothesized to address a much wider range of IgM clones than the mere number of mimotopes chosen because of the well-documented polyspecificity of the majority of the antibodies probed.The sequence clusters were found to vary with respect to the probability of random occurrence of such a group of sequences, which was used to rank them by significance . As the The relevance of this mimotope library to the complete repertoire of broadly expressed IgM reactivities and the scope of their diversity could be established by comparing several different peptide libraries with different properties.in silico to check the representativeness of the selected mimotope set and the relevance of the clustering found. To this end, 2.3 × 106 random 7-mer sequences were scored and ranked according to their similarity to each of the 790 clusters of mimotopes defined above. The random sequences that were the least related to any of the clusters in the selected library were used as a negative control  uncertainly clustered sequences as reflected in their KLD scores as shown by the GibbsCluster algorithm (NG1 and NG2); (2) two groups of five high scoring clusters as lower diversity libraries (C5_1 and C5_2); (3) random 7-mer sequences predicted to belong to some of the five highest scoring clusters based on PWM profile scores (C5P); and (4) random 7-mer sequences (RND) (see n = 3), MB (n = 3), and non-tumor-bearing neurosurgery patients  was analyzed using the sets of peptides described in p < 0.001) average reactivity than NG1, RND, or NGR. Interestingly, the library NGR, which was in silico purged of sequences scoring high with the 790 profiles of mimotope clusters defined, had indeed the lowest reactivity. It was significantly lower than that of both the weakly clustering peptides (NG1) and the random sequences (RND)  . This isNext, the capacity of the different libraries to sample symmetrically the space of 7-mer IgM mimotope reactivities in the IgM repertoire was tested. The mean nearest neighbor distance (MNND) was used for that purpose as a statistic indicating clustering of the data. Peptides that have similar reactivity profiles with different sera (thus carrying redundant information) would map to points in the reactivity space that lie close to each other. This clustering in some regions of the space would lead to a lower MNND. The library SYM ranked second only to NG2  by this z transformation of the correlation coefficients to allow for comparison by linear models, the means of those z values for each library were used to compare the libraries  was used to visualize the structure of the mimotope sequence space as represented by the general mimotope library produced by deep panning. To represent the sequences as vectors of real numbers, each amino acid residue was represented by five scores based on the z1–z5 scales published by Sandberg et al.  see Sup. Next, tAn important aspect of the usage of mimotopes as igome probes is their interpretability. Using large mimotope libraries provides an opportunity to generalize the type of structures targeted by the antibody repertoire studied. The numerous tests may allow for signal to emerge despite the noise due to poor representativeness of conformational epitopes, polyspecificity, mimotope/epitope sequence length disparity, etc.To explore the capacity of the library SYM to reveal general properties of the antigens targeted by the natural IgM repertoire, we used NCBI blastp program to find SYM homologous short sequences in the non-redundant (nr) database of human proteins , 37. The5 random peptide-based profiling of antibody repertoires can discriminate GBM from other tumors; (2) reports indicating the role of natural IgM antibodies in the immune surveillance against tumors  reports by Merbl et al.  that bott tumors , 41–43; t tumors –46. FurtFor this assay, we used sera from a set of 34 patients with brain tumors. The main goal was a “proof of principle” test demonstrating the capacity of the assay to provide mimotope reactivity profiles suitable for building predictors for randomly selected pathology. The distribution of patients by diagnosis  is shown in A two-dimensional projection of the cases on the 582 positive reactivities by multidimensional scaling (MDS) showed no separation (data not shown). This is expected because the peptide library is not targeted to any particular pathology. It represents rather a universal tool for IgM repertoire probing and mapping to a highly multidimensional feature space. The information in the reactivity profiles when all features are used is so rich that it makes practically each patient unique and a generalization impossible. In addition, the “curse of dimensionality” makes differentiating in 582 dimensional space hard. Therefore, a feature selection step would be necessary to construct a predictor for any diagnostic task.A combination of filtering and wrapping feature selection techniques was applied next. The filtering method used was a selection of individual features with highly significant expression in at least one patient. The wrapping techniques were recursive feature elimination followed by a forward selection algorithm. The feature to remove (respectively to add) at each step was selected so as to improve maximally the separation of the patient data clusters of interest when mapped on the remaining features. This iteration was repeated until no further improvement of the separation is possible see . It was Interestingly, this two-stage feature selection strategy (bootstrapping RFEDS variability and pooling recurring features) helped improve the generalization considerably. Testing of the model of 43 dimensional data with just a few cases is impractical. Therefore, the dimensionality of the IgM reactivity data was reduced from 43 to 2 using MDS. The SVM model, constructed on the basis of the two surrogate features obtained by MDS, successfully classified the GBM and non-GBM cases not only of the training but also of the testing set of sera .Thus, we were able to show that a rationally designed small library of 586 IgM mimotopes contains potentially a huge number of mimotope profiles that can differentiate randomly selected diagnoses after appropriate feature selection.High-throughput omics screening methods have extracted profiles from different dynamic diversities  and used them as biomarkers. The use of the antibody repertoire as a source of biomarkers has also been defined and approached in multiple ways. First came the technically minimalistic, but conceptually loaded, semiquantitative immunoblotting, developed 20 years ago –53. The The deep panning approach relies on next-generation sequencing (NGS) and thus requires balancing between sequence fidelity and diversity. Even with diversity affected by discarding sequences of one and two copies on the one hand, and overgrowth of phage clones on the other, our strategy still manages to find a general representation of the mimotope sequence space by identifying clusters of mimotopes. This relatively small set of sequence classes is hypothesized to be related to the modular organization of the repertoire defined previously . AlternaThe central role of prolines in the nAb mimotopes has been observed previously . Tcherny5 derived by deep panning reflects the recurrent IgM specificities found in the human population. A library of random peptides with sequences selected to be least related to the observed 790 cluster profiles reacted very weakly with IgM from patients' sera. This fact suggests that not only does the library of a little over 200,000 mimotopes represents the IgM mimotope space but also that the 790 cluster profile matrices are collectively a promising model of it. The good coverage of the IgM reactivity space by this mimotope library most probably is facilitated by the polyspecific binding of IgM and the small, flexible peptides.The mimotope library of diversity 10Although the large mimotope library can be used as is in peptide arrays when applicable, its size is not very practical for routine diagnostics. The classification in 790 clusters was used to produce a smaller and more applicable library, SYM, for clinical use. It contains basically representative sequences from the most significant clusters. SYM represents more efficiently the mimotopes' main reactivity patterns found in the phage selection experiment when compared with seven other libraries chosen to represent key alternative concepts. The precision of that representation can be adjusted by expanding the small library if necessary. Including more mimotopes from the set of 224,087 can be done in a similar fashion, sampling further the existing sequence clusters. Another improvement may be to include a couple of related sequences to each of the mimotopes, for example, those immediately adjacent in the same cluster, for a statistically robust signal.An interesting though not unexpected property of the public IgM igome found is its idiotypic connectivity. Overlap with immunoglobulin variable domain J regions proved the prominent feature of the human protein sequence fragments homologous to the peptides in SYM library. The actual epitopes of IgM should be mostly conformational. Nevertheless, both linear idiotypic epitopes (idiotopes) and fragments of them are probably represented in the CDR3 loops so as to produce statistically detectable signal in the BLAST results. It has long been known that linear epitope models yield clear structural idiotypic representation in CDR3 loops . InteresSYM could be used as a tool for the study of the IgM repertoire, as a source of mimotopes for design of immunotherapeutics –64, but 70 different subsets. This is an estimate of only the qualitative outcome—presence or absence of reactivity. Thus, the information provided by a typical IgM binding assay with the library is probably enough to describe any physiological or pathological state of clinical relevance reflected in the IgM repertoire. Of course, this is just an estimate of the resolution of the method. The number of naturally occurring profiles and their correlation with clinically relevant states will determine the actual capacity. Another important consideration is the significant probability to find profiles correlating to any state by chance. Therefore, extensive testing of the models to prove their ability to generalize is indispensable.The optimal feature set for GBM diagnosis we find has 43 mimotopes. If the library provides in the order of 500 significant reactivities and the profiles are typically of around 50 features, the theoretical capacity of this approach is >10The novelty of our approach is based on the combination of several previously existing concepts.First, early studies have argued that the physiologically autoreactive nAbs comprise a consistent, organized immunological compartment , 65–68. Second, germline variable regions are characterized by polyspecificity or cross-reactivity with protein and non-protein antigens . It seemhttp://www.iedb.org) collection of 4 × 104 linear B-cell epitopes close to 5,000 entries are <8 residues long. Thus, the peptide length of seven residues makes possible the interrogation of the repertoire with a library of 109k-mers, which is at the same time complete and highly diverse.Third, peptide arrays have been used for some time now as probes of the antibody repertoire , 76–79. 70 distinct profiles. The major tasks ahead are (1) exploring the concept of reproducibility for the sequences of IgM mimotopes by further deep panning experiments and (2) designing studies aimed at efficiently extracting specific diagnostic profiles and building appropriate predictors, for example, for predicting immunotherapy responders or side effects and predicting the risk of malignancy in chronic inflammation as well as other conditions involving immune activity.The phage display-generated library provides a rich source of mimotopes that can be screened for different theranostic tools focused on particular targets. On an omics scale, the smaller optimized mimotope library proposed here probes the repertoire of broadly expressed IgM reactivities efficiently, mapping its dynamic diversity to a space of potentially over 10https://github.com/ansts/IgMimoPap1 and https://github.com/ansts/IgMimoPap2).The datasets generated for this study can be found in the GITHUB (The studies involving human participants were reviewed and approved by Ethics Committee at the Medical University Sofia. The patients/participants provided their written informed consent to participate in this study.in silico work, supervised experiments except for the sequencing as well as the overall project execution, and prepared the manuscript. MH ran the phage display experiments. VK and MT ran the microarray experiments up to data processing and cataloged and maintained the seroteque. VS participated in the conceptualizing of the paper and the overall design of the experiments, supervised the phage display experiments, and together with K-MH and LM-Z carried out the DNA isolation, PCR, and sequencing. EH supervised the sequencing task and participated in conceptualizing the project and the preparation of the manuscript. SP and MT performed the data processing of microarray scans. TV and TK-E participated in conceptualizing the project, analysis of the results, and the preparation of the manuscript. DF was responsible for the patient selection, informed consent, ethics committee protocol preparation, blood collection, and serum preparation.AP conceptualized the project, analyzed the results performing all the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: Uganda remains seismically vulnerable to earthquakes, which constitute one of the most deadly naturally triggered disasters in the world. This is not surprising given the country’s location in the East African Rift Valley System.Method: This paper draws mainly on the authors’ live event experience and some media reports to narratively outline the nature of a sizable earthquake, which measured a magnitude of 5.7 on the Richter scale that struck Uganda and other countries within the Lake Victoria Basin region on 10th September 2016 in the afternoon.Results: Rakai - a district in central region was the worst affected in Uganda. It witnessed the death of four people; 20 people were admitted to the hospital with injuries; a total of 590 people were affected; and serious structural damages mainly in buildings were reported, leaving many either razed to the ground or left with cracks.Discussions: Although this earthquake was less devastating in terms of injuries and fatalities compared to two previous earthquakes in Uganda, based on the Modified Mercalli Intensity Scale it was still considered to be severe. Therefore, this paper identified some proactive lessons as far as earthquake risk reduction in Uganda is concerned, which among others include: encouraging earthquake-resistant buildings; the safety of essential infrastructure; earthquake early warning systems supported by free global technologies; and the safety of rescue workers along with prioritizing the psychosocial needs of rescue teams. With all this in mind, the September 2016 earthquake should serve as a timely reminder that there is a real need for the proactive ex-ante earthquake preparedness rather than risking an expensive post-ante approach to responding to any future devastating earthquakes in Uganda.Keywords: Earthquake, Uganda, disaster risk reduction, ex-ante approach, post-ante approach The countries situated in the seismically active areas, however, are far more prone to the earthquakes and their associated impacts.Earthquakes are spontaneous events that constitute one of the most deadly naturally triggered disasters, which can cause extensive fatalities, injuries, disabilities, displacement, physiological distress, extensive property damage and devastating economic losses1,3,4,6,10,11,Earthquakes are known to trigger geohazards such as landslides, mudslides, tsunamis, avalanches, floods, volcanic eruptions, fires, technological accidents, water contamination, epidemics and panic-driven stampedes in crowded areas7The Lake Victoria Basin, which includes Uganda, remains a seismically active area and it has experienced many earthquakes including the most recent one which occurred on 10th September 2016. Since Uganda is one of the countries located in the seismically active areas, what was the nature of the September earthquake? Also, what lessons are to be derived as far as earthquake risk reduction in Uganda is concerned?This paper draws mainly on the authors’ live event experience and reviews of some media reports to narratively outline the nature of a sizable earthquake which measured a magnitude of 5.7 on the Richter scale that struck Uganda and other nearby countries within the Lake Victoria Basin region on 10th September 2016 in the afternoon. After explaining its nature, the present paper identifies and divulges some proactive lessons as far as earthquake risk reduction in Uganda is concerned.Description of the September 10th 2016,5.7 Magnitude Earthquake in the East AfricaOn 10th September 2016 (Saturday) at 15:27:28 local time (12:27:28 GMT/UTC Time), the Lake Victoria Basin region experienced an earthquake measuring a magnitude of 5.7 on the Richter scaleIt should be noted that the four countries which experienced this earthquake are situated in the East African Rift Valley System (EARVS), which is known to have significant seismic fault lines running through it. The EARVS which extends from the Red Sea/Gulf of Aden to Malawi is formed by the western and eastern rift branches14,14.According to the United States Geological Survey (USGS), the origin of the earthquake was some 200 km or more from the east and west branches of the EARVS14Earthquakes and Seismological Monitoring System in Uganda. Over the years, earthquake shocks of varying magnitudes have been recorded in different parts of the country – especially in the Rwenzori Mountains which is one of Uganda’s most seismically active areas. The Fort-Portal area, which is proximate to the Rwenzori Mountains, recorded 418 shocks out of all the 588 shocks that were felt in Uganda as a whole between 1907 and 1942. Other places that have seen significant numbers of shocks between 1910 and 1960 include Mubende (55 shocks), Mbarara (46), Hoima (36), Entebbe (30), Kabale (23) and Kampala (6). These areas are known epicenters of tremors in Uganda8. Tremors vary in magnitude but the most destructive earthquakes so far in the history of Uganda, occurred on the 20th March 1966 in the Toro region and on the 5th February 1994 in the Kabarole district measuring 6.6 M and 6.2 M respectively. In the Toro region earthquake, 150 people were killed and 1,300 injured while in the Kabarole district earthquake 8 people were reported to have been killed together with property destruction estimated at US$ 61 million. The two events were linked to fault rifting in the EARVS8,19. Other areas of seismological importance include the Rwenzori mountains which recorded approximately 800 seismic events per month with local magnitude ranging from -0.5 to 5.1 between February 2006 and September 2007Records of Uganda’s vulnerability to earthquakes date back to 189718,20,21. ENTT, HOI and KIL use MEQ-800 analogue ORION Digital equipment while MBAR uses special equipment as part of the Global Seismological Station Network (IRIS-GSN). The equipment automatically collects, transmits, processes, analyzes and interprets real-time waveform data using Seisan Earthquake Analysis Software20,21. Ultimately, this should help to inform the timely detection, preparedness and response to earthquakes and their related hazards.In response to the geological risks, Uganda established a seismological monitoring system which is monitored through the National Seismological Network (NSN). Potential earthquakes and their related hazards in Uganda are monitored from four seismic stations which form the NSN, under the Department of Geological Survey and Mines. The stations are located in Entebbe, Hoima, Kilembe and Mbarara. They use the station codes of ENTT, HOI, KIL and MBAR respectively19,23. The NPDPM articulates the measures which are related to mapping earthquake prone areas; developing public awareness on earthquake preparedness; developing earthquake resistant building standards and ensuring adherence to building codes and regulations. Other measures specified within NPDPM include: conducting geological studies and research; promoting seismic safety activities and acquiring the relevant technology to monitor and detect the occurrence of earthquakes. The Ministry of Energy and Mineral Development is designated as the leading authority in the field of earthquakes and it collaborates with other stakeholders to implement these measuresThe seismological monitoring would, however, be much more effective if it was utilized to create a well-organized and comprehensive earthquake damage risk reduction policy. Unfortunately, at present Uganda does not such a policy. Instead, some few earthquake preparedness actions and codes are included in the National Policy for Disaster Preparedness and Management (NPDPM) and the Seismic Code of Practice for Structural Designs (SCPSD) respectively22Uganda’s Experience with the September 2016 Earthquake,21,22,13,This earthquake caught the vast majority of the people by surprise, despite the presence of seismological monitoring systems in Uganda as noted above, which should detect any potential earthquake occurrence in a timely manner20,(figure 1). This is because the district stretches to the Uganda-Tanzanian border, close to the Kagera region which was the epicenter of the earthquake. Only four people were reported dead, and a total of 590 people were affected mainly in Rakai district. Based on this, the fatality rate was low if compared with more or less similar earthquakes of the same magnitude in Uganda and elsewhere for example the 1994 Kabarole 6.2 earthquake and 2003 6M Bam in Iran. Apart from the 20 people who were admitted as patients to the Kakuuto hospital with serious injuries, it was further reported that a local medical nurse at Kateera Health Center II sustained head injuries.While Uganda felt the consequences of the earthquake, Tanzania, a neighbor of Uganda in the South was much more affected by it. Tanzania’s Kagera region was reported to have been the most devastated with ~11 deaths, ~440 injuries and the total estimated economic damage at $ 458 million13,4,10,11,Given that the need to make vital infrastructural buildings earthquake resistant cannot be over emphasized– especially key emergency medical and health buildings. A similar situation, though with far more devastating consequences to health facilities and workers, was witnessed in the major earthquakes of Bam, Gujarat, Haiti, Ica, Kashmir, Sichuan and Tohoku-Oki3,5,7,25. As a direct consequence, the families which were most affected became homeless since they had to abandon their houses to sleep in plantations. Others were left with little or no choice other than to continue sleeping in their quake-damaged houses. However, there has been no clear estimates to date of the real monetary value of the losses and damages that were calculated or projected to be associated with the earthquake unlike in Tanzania as noted above which recorded $ 458 millionEarthquakes are known to cause immense economic losses, which occur either immediately or after long-term periods4Shallow earthquakes generally tend to be more damaging than deeper quakes whose seismic waves have to travel farther to the surface. The farther they travel the more kinetic energy they lose. This is why the shaking caused by shallow quakes may cause large seismic slips, which can eventually lead to mega-thrust earthquakesHowever, emergency shelter (tents) and basic facilitates were much needed for the homeless or displaced victims whose homes were made unsafe to inhabit and accommodate them. This paper argues that the affected people would have been unnecessarily exposed to disease outbreak such as malaria, diarrhea and acute respiratory infections. Many were left in situations that could have exacerbated their vulnerability to psychosocial distresses like posttraumatic stress disorders. Additional injuries from the falling materials inside the cracked houses were inevitable to those who continued to reside in the quake damaged homes which were then further damaged by aftershocks. More so, the problems for those most affected by the earthquake did not end there. Some victims might have been expected to pay for the costs of the transportation and the treatment of the injured as well as for the repair of damaged structures. This situation not only exposed victims to health-related risks but it also exposed them to further impoverishment at a time when their already meager incomes were needed for providing for their family’s most basic needs.Is Uganda Prepared to Respond to Potential Earthquakes and Related Hazards Based on the Nature of the September 2016 Earthquake?,15,17,21. This situation is also heavily compounded by the ongoing political and socioeconomic dynamics in the country. One of the noteworthy challenges is the inappropriate construction methods and materials often used for different types of building structures in Uganda. They fail to comply with the existing building engineering codes to make them structurally more stable and resistant to earthquakes. Buildings which fail to comply with required construction codes can easily collapse on their occupants during earthquakes. It is important to note that, the number of buildings being built in many urban areas of Uganda is skyrocketing but unfortunately many of them are non-compliant with the essential building codes. This problem has been of particular concern in Kampala, the capital city, which lies in a seismically active zone next to Lake Victoria and hosts a population estimated at 4.5 million. Only recently has more stringent construction measures started to be enforced.There is no escaping the fact that Uganda will continue to be vulnerable to future earthquakes given its location in the EARVS8,Uganda as a whole is vulnerable to incurring heavy fatalities from earthquakes in places of excessive population growth. Mostly the riskiest places are those which are characterized by dense and informal settlements. Kampala is a case example with many earthquake susceptible housing structures which are compounded by the unregulated development of 62 informal settlements (slums)29There is also a great need to enforce building codes and to ensure where possible that quality earthquake-resistant building designs are implemented. Needless to say, that is absolutely essential for vital infrastructural buildings including those that are essential for the delivery of vital services such as the police stations (like the one that was destroyed by the earthquake in Rakai district). The buildings of that nature need to comply with the minimum standards of the existing SCPSD regulations. Also, where possible the SCPSD should be regularly revised to address the changing construction and engineering dynamics whenever any important lessons are learned from both naturally triggered and human-induced disasters around the world. It’s quite evident that new technology and improved construction methods and materials continue to be developed. So it’s important that the SCPSD keeps pace with these changes so that any of the important opportunities they advance to make cost-effective improvements are leveraged.,6,7,12. This paper notes that the September earthquake in Uganda should be an opportunity for enhancing the resilience and safety of essential infrastructure, along with the need for preplanning emergency responses.Besides the case with protecting hospitals and police stations as noted above, it’s also evident that earthquakes often damage and destroy essential infrastructure such as roads, airports, telecommunication facilities and networks, electricity and water infrastructure5Earthquake early warning systems (EEWS) can help to greatly minimize the consequences of earthquakes by providing better understanding of their precursory activities,Aside from caring for traumatized victims, the psychosocial needs of the rescue teams are an important driver to be prioritized to enable the success of the response activities to disasters. At times, some response measures undertaken by the rescue workers during disasters like earthquakes can be complex and expose them to life changing and potentially deadly occupational hazards. For instance, responding effectively to the needs of victims after the occurrence of earthquakes by providing the emergency medical care to any severely injured people; searching for and rescuing the victims entrapped in the wreckage. Similar attention needs to be prioritized for the firefighting and rescue workers who at times find themselves at high risk of losing their own lives or developing physiological distress33This paper aimed at outlining the nature of the 2016 September earthquake as well as identifying some lessons for earthquake risk reduction in Uganda. Besides, some of the lessons could also be applied in responding to other disasters and emergency situations in Uganda in general. With the above review in mind, the 2016 September earthquake should serve as a timely reminder that there is a real need for the proactive ex-ante earthquake preparedness rather than an expensive post-ante approach to responding to future earthquakes. This applies to not only the Rakai district and other known earthquake prone areas, but to Uganda as a whole as far as earthquake risk reduction in Uganda is concerned. However, that needs to be aligned to the SCPSD and some policy actions enshrined in the NPDPM, and above all the Sendai Framework for Disaster Risk Reduction 2015 – 2030 with special attention given to its priority number four: enhancing preparedness for effective response and to “Build Back Better” in recovery, rehabilitation and reconstruction.All relevant data are indicated within this paper, and it does not report new data apart from that herein referenced.This paper received no funding.The authors have declared that no competing interests exist.Joseph Kimuli Balikuddembe, Email: jbalikuddembe.k@scu.edu.cn


A13 exhibited excellent protective activity for tobacco to against Tobacco mosaic virus (TMV) at 500 μg/mL, with a value of 70.4 ± 2.6% compared with control treatments, which was better than that of the plant immune activator chitosan oligosaccharide (49.0 ± 5.9%). The protective activity is due to compound A13 inducing tobacco resistance to TMV, which was related to defense-related enzymes, defense-related genes, and photosynthesis. This was confirmed by the up-regulated expression of proteins that mediate stress responses and oxidative phosphorylation.Plant immune activators can protect crops from plant virus pathogens by activating intrinsic immune mechanisms in plants and are widely used in agricultural production. In our previous work, we found that curcumin analogs exhibit excellent biological activity against plant viruses, especially protective activity. Inspired by these results, the active substructure of pentadienone and quinazoline were spliced to obtain curcumin analogs as potential exogenously induced resistant molecule. Bioassay results showed that compound  Tobacco mosaic virus (TMV) is a well-studied plant virus that causes massive crop loss. The prevention and treatment of TMV remain a problem worldwide . PreviouA number of different types of compounds with stable and effective activities as plant immune activators have been explored so far. As one of the important plant immune activators, chitosan oligosaccharide (COS) can induce disease resistance in plants and enhances a plant’s defense against pathogens ,14,15. Ctrans-ferulic acid derivatives, which are α,β-unsaturated carbonyl compounds, display a favorable inhibitory activity against TMV, and they can enhance the disease resistance of tobacco by increasing the activity of defense enzymes and promoting photosynthesis [A13 induces resistance was analyzed, including the defense-related enzyme activity, chlorophyll content, defense genes, and differentially expressed proteins (DEPs). The results show that compound A13 can effectively enhance the resistance of tobacco, and it can be used as a plant immune activator.It is well known that most of plant immune activators are metabolic products of organisms or derive from the metabolic products. So, finding a plant immune activator that is based on a natural product is an effective way to develop new drugs. In our previous study, we found that ynthesis . Curcumiynthesis ,21,22,23ynthesis . SearchiA1–A20 were synthesized with the intermediates 4-chloroquinazoline and -1-(substituted-phenyl)-5-(4-hydroxyphenyl)-penta-1,4-dien-3-one. The intermediate 4-chloroquinazoline was synthesized as described in a previous report [E, 4E)-1-(substituted-phenyl)-5-(4-hydroxyphenyl)-penta-1,4-dien-3-one was synthesized by using a previously described method [The target compounds s report . The intd method . The tarA1–A20 against TMV were tested by using the half-leaf method in vivo [A3, A8, A9, A15, and A20 against TMV was comparable with that of COS (49.0 ± 5.9%) at 500 μg/mL, with values of 50.6 ± 2.8%, 47.3 ± 9.3%, 48.8 ± 3.3%, 47.6 ± 9.8%, and 53.8 ± 3.0%, respectively. Interestingly, compounds A12 and A13 exhibited superior protective activity against TMV over the control agent COS, with values of 66.5 ± 3.0%, and 70.4 ± 2.6%, respectively. Meanwhile, the curative activity of compounds A1, A3, A11, A12, A13, and A14 against TMV was comparable to that of COS (34.1 ± 8.9%) at 500 μg/mL, with values of 55.8 ± 3.7%, 58.1 ± 10.0%, 49.9 ± 9.6%, 42.0 ± 5.5%, 42.9 ± 2.2%, and 50.8 ± 7.7%, respectively.The antiviral activities of the target compounds  in vivo . The plaN. tabacum cv. K326 with the same growth at the six-leaf stage was selected, and compound A13 was smeared over whole leaves at 500 μg/mL. Solvents (1% tween) and COS were used as the negative (CK) and positive controls (COS), respectively. Tobacco leaves were inoculated with TMV after 24 h, and they were placed, and cultured, in a greenhouse. Four treatments were prepared: CK, CK + TMV, COS + TMV, and A13 + TMV. Tissues were collected on 0, 1, 2, and 3 day after the tobacco leaves were inoculated with TMV for assays on defensive enzyme activities and chlorophyll content. The tissue samples were obtained in triplicate.A13 were investigated and presented in A13 + TMV treatment group decrease from day 0 to day 3, reaching its minimum on day 3. On day 3, the CAT activity was higher in the A13 + TMV treatment group than in the other treatment groups. The results showed that compound A13 can effectively increase the CAT activity in plants. The superoxide dismutase (SOD)   B activitse (POD) C activitA13 on chlorophyll contents, we studied the changes in chlorophyll content , enhanced disease susceptibility 1 (EDS1), superoxide dismutase (SOD), catalase 1 (CAT1), phenylalanine ammonia lyase 4 (PAL4), pathogenesis-related genes 1 (PR1), and nonexpressor of pathogenesis-related genes 1 (NPR1), in the A13 + TMV treatment group were remarkably up-regulated on day 3. Specifically, the expression levels of ICS1, CAT1, and PR1 in the A13 + TMV treatment group were 1034.70, 876.13, and 876.13 times higher than those in the CK treatment group, respectively. The results show that compound A13 can effectively enhance the activity of the defense enzymes in plants. Notably, the expression levels of PR1 and CAT1 in the A13 + TMV treatment group were 17.44 and 4.15 times higher than those in the COS + TMV treatment group, respectively. Furthermore, the expression levels of SOD in the A13 + TMV treatment group was lower than that in the COS + TMV treatment group. The results showed that the compound A13 can induce the expression of defense-related genes in tobacco, thus achieving antiviral effect.As shown in A13 treatment on the proteins in the TMV-inoculated tobacco, we detected and identified total protein samples by using label-free LC−MS/MS on day 3. We used the iBAQ algorithm of the label-free protein spectrum technique to quantify the protein in tobacco leaves from the A13 + TMV treatment and control groups. As shown in A13 + TMV and CK + TMV treatment groups, respectively .To study the effect of the ectively . The volectively . Among tA13-induced proteins (p ≤ 0.05). The GO-annotated map of A13 + TMV versus CK + TMV shows that the DEPs involved in cellular components . Different proteins were mapped using the KEGG database (p < 0.05). Oxidative phosphorylation  was found to be the main enrichment pathway, which includes 11 specific proteins, including the TMV capsid proteins. As shown in A13 enhances tobacco’s resistance to viral invasion by altering the up-regulation and down-regulation of differentially expressed proteins in the oxidative phosphorylation pathway.To study the potential link between DEPs and biological functions, we used the KEGG to identify potential pathways for different proteins in the treatment groups , soluble inorganic pyrophosphatase (Ppa1), ATP synthase gamma chain (ATPeF1G), ATP synthase delta chain (ATPeF1D), V-type proton ATPase catalytic subunit A (ATPeV1A), and V-type proton ATPase subunit H-like (ATPeV1H) were up-regulated (A13 + TMV versus CK + TMV). Of which, Ppa catalyzes the decomposition of pyrophosphate into phosphoric acid, which is important for maintaining the reduction of cellular levels [Ppa1 controls the cellular level of inorganic pyrophosphate [ATPaF1G, ATPeF1D, ATPeV1A, and ATPeV1G are ATP-related enzymes encoding genes that promote ADP to ATP conversion in plants to provide energy for a plant’s metabolism. It can be seen from the down-regulation of the ATP synthase subunit b protein that compound A13 can accelerate the decomposition of ATP to provide energy, thereby preventing plants from resisting pathogen invasion. In summary, A13 can enhance a plant’s immune response and resistance to TMV.Oxidative phosphorylation is an important biochemical process in plants that provides energy for their life activities. This process is divided into substrate-level phosphorylation and oxidative phosphorylation. Oxidative phosphorylation is accompanied by ATP formation during biological oxidation and is regulated by key proteins, such as ATP synthase, cytochrome, and NADH dehydrogenase. The differential proteins inorganic pyrophosphatase /E0.1%1cm260nm, where E represents the extinction coefficient for TMV; and E0.1%1cm260nm is 3.1Virus concentration (mg/mL) = . The sample was homogenized and incubated at 35 °C for 0.5 h and then centrifuged at 6500 rpm for 15 min. The absorption spectra of Ca and Cb at the wavelengths of 663 and 645 nm, respectively, were recorded in a reference solvent. The measurements were tested in triplicate. Ca, Cb, and the total chlorophyll content (Ct) were calculated using the following formula:The chlorophyll content of each test sample was measured four times every day in accordance with a previously described method . Each tra (mg/L) = 9.784OD663 − 0.990OD645Cb (mg/L) = 21.426OD645 − 4.650OD663Ct (mg/L) = Ca + Cb = 5.134OD663 + 20.643OD645C−1 fresh weight) was calculated as follows:Quantification  according to the manufacturer’s instructions. RNA was reverse-transcribed using a cDNA kit  in accordance with manufacturer’s instructions. The experiments were performed in 10 μL of reaction volume, which was analyzed using SYBR Premix Ex TaqII (Sangon) and an iCycleriQ multi-color real-time PCR Detection System . 2O, containing 0.1% formic acid (FA) for liquid chromatography–tandem mass spectrometry (LC-MS/MS). The measurements were tested in triplicate.All of the tobacco protein was extracted in accordance with a previously described method . A tobacWe analyzed peptides by using a triple time-of-flight (TOF) 5600 mass spectrometer . A peptide solution (8 μL) was injected in full-loop mode, and 1% acetonitrile (ACN) and 0.1% FA in water were equilibrated on a Chrom XP Trap column . The sample was washed for 10 minutes at a flow rate of 300 nL/min on reversed-phase chromatography  with a linear gradient formed by mobile phase A  and mobile phase B . The sample was washed again at a flow rate of 300 nL/min for 20 min. Peptides were eluted by the Triple TOF 5600 MS, which uses a data-dependent model with an automatic switch between TOF−MS and product ion acquisition by Analyst (R) Software (TF1.6) . β-Galactosidase was used to calibrate each pair of samples for 10 min of elution and 30 min of identification.t-test of the iBAQ data of two-samples was used to identify DEPs between the treatment and control groups.We used MaxQuant  version http://www.uniprot.org/) was used to annotate the classification of DEPs by GO on the KEGG. GO items lacking the corresponding annotations were removed from the protein list, and the IDs listed for the proteins were mapped at the biological process, cellular component, and molecular function levels [http://www.geneontology.org/), and the protein amount for each GO item was calculated. The label-free proteomics was used as the target list. The background list was generated by downloading the GO database.The Uniprot software (n levels . DEPs (ep < 0.05).To produce true and reliable data, we repeated each measurement three times thrice and completely arranged the results in a random design. SPSS version 16.0  was used to analyze the experimental data. ANOVA and Tukey’s multiple comparison were used to determine significant difference . This protective activity of compound A13 is due to its ability to induce disease resistance in plants by influencing defense-related enzymes, defense-related genes, and photosynthesis. In addition, compound A13 can induce stress responses and oxidative phosphorylation. So, compound A13 can induce plant disease resistance in plants and may be considered to be a new plant immune activator for controlling viruses in plants. Our data provide scientific evidence to support the further investigation of plant immune activators.Taken together, this study shows that novel curcumin analogs bearing the quinazoline moiety have favorable inhibitory activity against TMV in vivo at 500 μg/mL. In particular, compound


This paper describes the piloting of a culturally modified version of the FDIC Money Smart Peer-to-Peer Program. The pilot study examined the use of Mentorship Teams comprised of two persons one (60 years and over) and another (30 - 59 years) who collectively provided mentorship and financial education to four working-age adults. The pilot was designed with four (4) phases: Phase 1 included recruitment and training of Mentors, Phase 2 the assignment of Mentors and Mentees, Phase 3 application of the intervention, and Phase 4 the evaluation of outcomes. This paper will review the design and recruitment processes, modifications made to the existing Money Smart Peer-to-Peer Program for cultural sensitivity and financial system factors, implementation challenges (including barriers and facilitators) and lessons derived from initiation and program implementation.


Simulation-based health professional education (HPE) is widely practiced in resource-rich regions, yet it is underutilized or ineffectively delivered in resource-poor ones, particularly when we fail to consider local contexts such as infrastructure, literacy, and culture when developing educational programs. Such an approach would be an example of failure of delivery, or the inability to bring services to people whose diseases have proven therapies. It is the biggest obstacle facing global health. This paper is a review of the literature and the authors’ experience in developing, delivering, and evaluating sustainable HPE programs in resource-poor regions, wherever in the world they may be. Failure of delivery, or the inability to bring services to people whose diseases have proven therapies, is the biggest obstacle facing global health . AlthougIt is appealing to combine humanitarianism with hands-on patient care, and opportunities are increasing for health professionals to embark on such missions . HoweverSimulation is a globally applicable HPE intervention, used to teach a variety of medical skills and procedures. It is risk-free to patients, and as such is a moral health imperative . If emplThis paper is a review of the literature and the authors’ experience in developing, delivering, and evaluating sustainable HPE programs in resource-poor regions. We present 12 key steps that may help achieve this goal.First, know the local context. Brazilian philosopher and educator Paulo Freire said, “transformation is valid only if it is carried out with the people, not for them” . Follow The problem applies equally to educational programs. During a recent visit to Haiti, completing a simulation-based professional development exercise, we were shown an example of what could be called simulation-related systemic harm. While touring the hospital grounds, we learned that a shipping container full of unused equipment was generously donated by an NGO, meaning to form a modern emergency department, but was unusable because of electrical incompatibilities and the lack of available personnel with the necessary time or expertise to incorporate new system. Expensive high-fidelity simulators should not be sent to resource-poor regions without extensive local consultation. Thoroughly assess the equipment’s utility: Are the plugs and voltage compatible? How does the equipment react to environmental conditions? Can minor repairs be managed locally? Growing evidence suggests that many clinical tasks and skills are learned just as well from simple trainers as from complex human mannequins -20. UndeSecond, conduct a needs assessment. Collaborate with local colleagues and carefully consider who the students are, what skills they need to learn, and what local teaching resources are available. Sophisticated simulation equipment is a highly valued investment that should come with a warning label: do not overestimate how well this tool applies to a resource-poor setting. Heed this warning and use a needs assessment tool to ask local educators what works for them . Our HaiWe used two simple needs assessment tools to develop our collaborative program: SWOT   and CIPPFinding a local champion is our third step. The educational program is more likely to succeed if visitors collaborate with a local project champion who leads it from start to finish . He or sA culturally relevant reward may be an effective incentive for your project champion when, as is often the case, no salary is available. For example, one of the authors identified potential titles for two project champions working at Addis Ababa’s Black Lion Hospital Simulation Centre in Ethiopia. These unpaid educators’ leadership and commitment were remunerated with the positions of Director and Patron; both individuals became instrumental in rapidly expanding the hospital’s simulation program.Fourth, align educational goals with your partners’, and understand that the program belongs to them. Clearly establish each collaborator’s contribution to the program, and iteratively review, reinforce, and modify the plan when necessary. Careful examination and reflection by all parties is essential, both before the project starts and as it unfolds. Be clear that everyone understands the program’s objectives, and agrees that local ownership is the goal.Strengthen this approach with a memorandum of understanding, or an agreement among collaborating parties. Although the document may hold little or no legal value, it states common goals and charts a course for achieving them. Understand that novel ideas must eventually become organized , and thaFifth, garner support from all angles: administrative, collaborative, and financial. Administrative leaders can lend support if they want you to invest in training local health professionals . Two sucTop-down initiatives also need bottom-up collaborations to provide soil for young roots . A top-dSeek financial support for the program. Recently, public and private sectors have collaborated to achieve a common objective: benefit that does not compromise public interest . Thus, sFinancial support signals that the project is highly regarded. For example, a peer-reviewed grant presents the project to a broad academic community. Funding also establishes accountability, sets timelines, and supports travel, enabling participants from resource-rich and -poor regions to experience one another’s contexts. It also supports salaries and essential equipment. Based on our collective experience, even projects with small awards are more successful than those with no funding at all.Sixth, realize that a fast pace may slow you down in resource-poor regions. Culturally diverse team members may speak different languages. They may also have different perceptions of the local hierarchy, and choose to navigate it in unfamiliar ways. Although visitors may be comfortable questioning leaders, doing so is considered disrespectful, for instance, in some Asian cultures . ApproacWhile in Haiti, for example, we delivered a simulation workshop with the local educators. While we delivered our lectures, we were unprepared for our audiences’ constant influx and egress through a squeaky swinging door and for their continuous cell phone use. Our participants clearly had other work to do, now. This behaviour may have slowed us down, but it sped them up.Seventh, use materials local to where you are teaching. For example, we taught chest tube placement in a remote part of our own Canadian backyard. We built our task trainer with hard foam from a construction site and pork ribs from a local grocer. We mounted it on a banker’s box and draped it all in surgical cloth. The local educators taught the procedure with their own thoracentesis kits. Further afield in Ethiopia, participants in one study used shoelaces to practice their surgical throws . ShoelacLocal markets are also a vibrant living display of the social determinants of health. Send local and visiting learners there together, and see if they bring back a better mutual cultural understanding along with their raw material. Use simulators to teach locally relevant practices, and remember that inappropriate teaching tools are harmful when used in conditions of privation. They divert busy educators from delivering more sustainable education.Eighth, use local knowledge. Knowledge Translation (KT) is a scholarly approach that can translate the program’s findings into practical local knowledge that feeds back into and improves the program . TranslaLocal knowledge is the program’s bedrock; publish and disseminate it. Co-authorships will help illuminate your partners’ scholarly work, as will publishing in the local language. Some international academies feature local work and raise its visibility by linking up with smaller journals . VisibilNinth, train more trainers. Simulation-based teaching takes time and effort ; this isTenth, anticipate consequences. Projects are unlikely to evolve predictably. Be attuned to this and remain flexible . IdentifThe eleventh step is to fade away. Aggressively and persistently plan for independence. Enable local program control once the learning environment is strong enough to support the new knowledge and skills. Although visits between regions are desirable, avoid contributing to the "brain drain" that may come with internationally recognized professional credentials. Teach locally relevant skill sets; unused ones are likely to wither. Ideally, the collaborations built among administrative leaders, the local champion, and both teams, working from the aforementioned solid structural foundations, will leave a sustainable and well-integrated project. Fading away does not mean disappearing altogether; withdrawal plans should include long-distance support and periodic reassessments .Finally, evaluate the program. William Boyd (1990) wrote: “The last thing we learn about ourselves is our effect” . So it iEvaluation models have evolved over time; they once simply judged whether a program fulfilled its designer’s objectives. Roadmaps now take a more nuanced look at how a syllabus operates within its unique environment . They aiHealth inequities and limited access to health education exist in resource-poor areas. Simulation-augmented HPE is a moral health and educational imperative, one that can help us achieve the 2015 Millennium Goals and the 2030 Sustainable Development Goals if we collaboratively spread it to the resource-poor regions both in developing countries and our own backyards. We describe a way to develop and deliver a sustainable simulation program to resource-challenged regions, based on both a review of the literature and the authors’ experience.


Lymphovascular invasion (LVI) is associated with the development of metastasis in invasive breast cancer (BC). However, the complex molecular mechanisms of LVI, which overlap with other oncogenic pathways, remain unclear. This study, using available large transcriptomic datasets, aims to identify genes associated with LVI in early-stage BC patients.n = 1565) was used as a discovery dataset, and The Cancer Genome Atlas  cohort was used as a validation dataset. Key genes were identified on the basis of differential mRNA expression with respect to LVI status as characterised by histological review. The relationships among LVI-associated genomic subtype, clinicopathological features and patient outcomes were explored.Gene expression data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort , which were significantly associated with pathology-determined LVI status in both cohorts. The 10-year overall survival of subtype 2 was significantly worse than that of subtype 1.This study demonstrates that LVI in BC is associated with a specific transcriptomic profile with potential prognostic value. Lymphovascular invasion (LVI) is an early event in the development of metastasis and is a potent prognostic factor.6 Although the molecular profiles associated with tumour differentiation in terms of histological type and grade and development of lymph node metastasis have been well characterised,9 the molecular mechanisms of LVI and associated genes that may represent therapeutic targets or biomarkers remain to be identified. The main challenge in determining the molecular profiles associated with LVI status in BC stems from the lack of LVI status in the available large-scale molecular studies in addition to the inherent subjectivity of morphological assessment of LVI status.Outcomes for early-stage breast cancer (BC) patients have improved over recent decades as a result of better diagnostic accuracy, targeted drug therapies, in addition to improvements in early diagnosis.10 and The Cancer Genome Atlas (TCGA)11 cohorts are currently the largest genomic and transcriptomic datasets of early-stage BC patients with clinical follow-up. In this study, using these large transcriptomic datasets combined with thorough histological assessment of LVI, we applied bioinformatic analysis to evaluate the genes associated with LVI and assessed the prognostic value of genomic subtype based on LVI status.The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC)10 mRNA was extracted from primary tumours of female patients, and mRNA expression was evaluated using the Illumina TotalPrep RNA Amplification Kit and Illumina Human HT-12 v3 Expression BeadChips . LVI status of 1565 patients within the METABRIC cohort, which were histologically assessed using haematoxylin and eosin (H&E) stained slides. For the Nottingham subset included in METABRIC (n = 285/1565), LVI status was additionally assessed by immunohistochemistry (IHC) utilising CD31, CD34 and D2-40,12 and the final LVI status was confirmed using a combination of multiple H&E tumour sections and IHC. Considering the different methods of LVI assessment, cases were divided into two groups: (1) the Nottingham cases and (2) the remaining METABRIC cases (n = 1280). Gene transcript expression levels between LVI-positive and LVI-negative cases were compared for each group, as described in the ‘Bioinformatics analysis’ section.In the METABRIC study,11 cohort of female BC patients (n = 854) was extracted from the Genomic Data Commons Data Portal and cBioPortal website.14 Briefly, the datasets of mRNA expression from RNASeqV2 were accessed along with de-identified clinical information for several clinicopathological factors and outcomes. Digital H&E-stained slides from the TCGA_BRCA cohort were accessed via the cBioPortal website, and LVI status was quantified by an expert breast pathologist (LD).The data from the TCGA10 Differentially expressed genes (DEGs) between LVI-positive and LVI-negative cases were identified using the weighted average difference (WAD) method, and the DEGs were selected according to the WAD ranking.16 Lists of the top 350 genes associated with LVI for the WAD assay in both (1) the Nottingham cases in the METABRIC cohort (n = 285) and (2) other METABRIC cases (n = 1280) are shown in Supplementary Tables Analysis of mRNA expression data from METABRIC has been previously described.hort n = 85 and  was used to calculate significantly enriched gene ontologies and pathways associated with these genes.Statistical analyses were conducted using IBM SPSS Statistics for Windows, version 24.0 . The chi-squared test was used to assess differences among several clinicopathological factors, including LVI status, tumour size, lymph node status, histological grade, oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 (HER2) and molecular subtypes, as stratified by the LVI-associated genomic subtype.Kaplan–Meier survival curves of 10-year overall survival (OS) were plotted for the METABRIC and TCGA cohorts. The 10-year OS in this study was defined as the day of death within 10 years or the day of completing follow-up from the day of surgery. In univariate and multivariate analyses, 95% confidence intervals (CIs) were assessed using the Cox proportional hazards regression model to determine the associations between clinicopathological factors , including the LVI-associated genomic subtype and prognosis.p < 0.0001; TCGA: p = 0.00055), positive nodal status (METABRIC and TCGA: both p < 0.0001) and high histological grade  were LVI-positive and 930 (59%) were LVI-negative. The LVI-positivity rate was 41.1% (117/285) in the Nottingham cases and 40.5%  in the remaining METABRIC cases. In the TCGA cohort, 295/854 (35%) patients were LVI-positive and 559 (65%) were LVI-negative. In both cohorts, LVI positivity was significantly associated with large tumour size , positive nodal status , negative ER status  and negative PR status  as poor prognostic factors in both cohorts. In addition, significant prognostic factors included high histological grade  and positive HER2 status  in the METABRIC cohort. LVI positivity was an independent poor prognostic factor in multivariate analysis  and (2) remaining METABRIC cases (n = 1280) included 42 significantly overexpressed and 57 downregulated genes (Table The overlapping DEGs between 1) the Nottingham cases in the METABRIC cohort  and 2   Fig. . The denTo validate these results, hierarchical clustering was conducted on the TCGA cohort using the same 99 genes. The dendrogram classifying these 854 cases is shown in Supplementary Fig. p < 0.0001; Table In both cohorts, LVI positivity was significantly more prevalent in subtype 2 tumours than those of subtype 1 (METABRIC and TCGA: p < 0.0001), high histological grade (both p < 0.0001), ER negativity (both p < 0.0001), PR negativity (both p < 0.0001) and HER2 positivity  and serpin family E member (SERPINE) were present in our 99-gene set.In this study, we identified a 99-gene set significantly associated with LVI status in the METABRIC dataset. We validated this finding using the TCGA dataset. LVI is a biomarker for aggressive BC and is considered predictive for metastasis.24 suggested their gene set associated with LVI was related to extracellular matrix components using microarray data from 108 BC patients. Epithelial–mesenchymal transition (EMT)-implicated genes in prostate cancer have also been associated with pathways relating to the extracellular space.25 The extracellular matrix comprises a network of structural proteins, and reorganisation of this matrix is required for cancer to progress.26 The EMT is thought to play an important role in the process of metastasis to distant sites, and certain EMT markers are related to LVI status in BC.12 In the 99 gene LVI signature set, there are several genes associated with extracellular pathways that are implicated in BC prognosis. For example, heat shock protein 27 (HSPB1), is associated with BC aggressiveness and metastasis.27 HSPB1 expression is upregulated in the early phase of cell differentiation, which implies that HSPB1 may play an important role in controlling the growth and migration of cancer stem-like cells.28 Another example is apolipoprotein C1 (APOC1), which is considered as a prognostic biomarker for triple-negative BC.29 APOC1 is thought to regulate the inflammatory response in cancer tissues,30 which may be closely related to the elimination of proliferating cancer cells.31 Upregulation of MMPs is also related to cancer cell proliferation, invasion and epithelial-to-mesenchymal transformation and is indicative of a poor prognosis for BC patients.32 As an example, MMP-11, which belongs to the MMP family, promotes BC development by inhibiting apoptosis as well as enhancing the migration and invasion of BC cells.33 Additional functional studies of these genes are necessary to explore the association of aberrant gene function and proteins related to LVI in BC.The underlying molecular mechanisms driving LVI in BC, which are potential therapeutic targets, have yet to be identified. The 99 genes in the LVI-related gene signature from this study are significantly associated with extracellular pathways. In previous work, Klahan et al.12 In the Nottingham cases, we evaluated LVI status using strict criteria based on both morphology and immunohistochemistry. However, for the TCGA BRCA cohort, we evaluated LVI status using H&E-stained slides alone from the cBioPortal database. Although LVI evaluation using only one H&E slide is feasible, it may be difficult to clearly identify LVI negativity.34 In present study, the LVI-positivity rates were closely similar between the Nottingham cases, the remaining METABRIC cases and TCGA_BRCA cases using the different LVI-evaluations. Although our results might suggest the adequacy of LVI evaluation with only one H&E-stained slide, further analysis with the larger cohorts to assess the LVI status using both H&E and IHC slides is necessary to report accurately on LVI status.Comparison of the METABRIC and TCGA cohorts was a limiting factor in this study, in terms of the different methods used to quantify and statistically analyse gene expression and in the approaches to LVI evaluation. We previously developed a method for the accurate detection of LVI using immunostaining for CD34 or D2-40.36 For example, the different approaches can have different lower limits of detection or may encompass different genomic regions. Thus, we cannot assume that the methods are interchangeable, and doing so would require rigorous cross-assay comparisons.37 Although there is statistical agreement across the different cohorts in the present study, further analysis using identical technologies (microarray and/or NGS assays) may provide clearer validation of the LVI gene signature.Microarrays were used to evaluate mRNA expression in the METABRIC analysis. In contrast, RNA-seq using NGS was used in the TCGA analysis. Microarray platforms have been used and validated for nearly two decades, and this approach has been widely used for evaluating multi-gene expression. Conversely, the unbiased genome-wide RNA-seq method allows for the analysis of all annotated transcripts in addition to the identification of novel transcripts, splice junctions and noncoding RNAs. These technological and methodological differences may underpin the known challenges of relating microarray and RNA sequencing data between studies.In conclusion, we have confirmed the suitability and prognostic significance of our LVI-evaluation approach using the METABRIC and TCGA cohorts. We have determined genomic subtype associated with LVI status and patient outcome in BC, therefore, providing an experimental tool which may serve to unravel the complex gene networks associated with LVI with potential clinical relevance. Consistency between clinical cohorts stratified by LVI-gene signature may be further improved by using the same definitions and evaluation methods for LVI status.List of top 350 genes significantly associated with lymphovascular invasion in the Nottingham cohortList of top 350 genes significantly associated with lymphovascular invasion in the remaining METABRIC casesCorrelation between lymphovascular invasion and clinicopathological characteristicsSurvival analysis based on clinicopathological characteristics including lymphovascular invasionFull gene name list of the 99 genes significantly associated with lymphovascular invasionMean value, standard error of the mean (SEM), subtraction and weighted average difference (WAD) ranking in the 99 genes significantly associated with lymphovascular invasionSurvival analysis based on clinicopathological characteristics including LVI-related genomic subtypeSignificant pathways associated with LVI-related gene setThe dendrogram of METABRIC cases for hierarchical clustering analysisThe dendrogram of TCGA cases for hierarchical clustering analysis


TORCH including the pathogens of Toxoplasma gondii (TOX), rubella virus (RV), cytomegalovirus (CMV), and herpes simplex virus (HSV) causes intrauterine infections and poses a worldwide threat to women especially in pregnancy. In this study, we described the seasonal difference in TORCH infection and analyzed the anti‐TORCH IgM multipositive serum samples by the indirect immunofluorescence assays (IFA).To observe the seasonal influence of the anti‐TORCH IgG and IgM antibodies, a retrospective study was conducted with 10 669 women (20–40 y old) before pregnancy from August 2016 to July 2017. Totally 199 ELISA anti‐TORCH IgM multipositive serum samples were further tested by IFAs for false‐positive analysis.The prevalence of positive HSV1‐IgM, RV‐IgM, HSV2‐IgM, CMV‐IgM, and TOX‐IgM in the present population was 6.30%, 2.55%, 1.94%, 1.24%, and 0.67%, respectively. Additionally, the prevalence of positive RV‐IgM, CMV‐IgM, and HSV1‐IgM was statistically different among four seasons, with the highest positive rates of RV‐IgM (4.12%) in autumn, CMV‐IgM (1.75%) in summer, and HSV1‐IgM (7.53%) in winter. The confirmatory IFAs showed that the positive rates of RUV‐IgM, CMV‐IgM, and HSV2‐IgM were significantly different from those in ELISA screening experiments. Interestingly, only 32.7% (65/199) of the TORCH IgM multipositive results were consistent with those by the IFA, indicating that cross‐reaction caused false positives were common in ELISA IgM antibody screening.The TORCH infection displayed different prevalence among four seasons in our 12‐month retrospective study. The IgM multipositives by ELISA screening may need further confirmation analysis due to its relatively high cross‐reaction rate. In addition, two serum samples that were tested TORCH IgM multipositive by enzyme‐linked immunosorbent assay (ELISA) were confirmed and analyzed by the “gold‐standard” indirect immunofluorescence assay (IFA).22.1The TORCH screening results of 10 669 women before pregnancy in Beijing Obstetrics and Gynecology Hospital from August 2016 to July 2017 were used in seasonal analysis. In the period of January 2016 to July 2017 during which 19 652 women were screened for before‐pregnancy TORCH infection screening, a total of 199 serum samples that were tested IgM multipositive from the TORCH assays were collected and saved at −80°C.2.2TM TOX IgM/IgG; CaptiaTM Rubella IgM/IgG; CaptiaTM CMV IgM/IgG; CaptiaTM HSV 1 IgM/IgG; CaptiaTM HSV 2 IgM/IgG).The TORCH ELISA screening assays were performed on the TECAN Freedom EVOlyzer® (Switzerland) platform with the TORCH IgM/IgG detection kits obtained from Trinity, TX, USA . The TORCH IFA reagents used were listed as follows: IgM Indirect Fluorescence Kits  from SCIMEDX CORPORATION ; IgM Indirect Fluorescent Kits (RV Cat. 902701 and CMV Cat. 902123) from Hemagen .The ELISA experiments were performed according to the manufacturer’s instructions. Briefly, after the sample incubation step, the anti‐TORCH IgG/IgM antibodies in serum samples were bound to the solid phase with the TORCH‐specific antigens already attached to the surface of each reaction well. After excess antibody was removed by the washing buffer, the goat anti‐human IgG/IgM conjugated with horseradish peroxidase was added to the antibody‐antigen complexes, followed by the addition of the chromogen (tetramethylbenzidine). The relative color intensity indicating the concentration of the antibody in a patient serum was measured on an ELISA plate reader at the wavelength of 450 nm. The ELISA results were interpreted according to the manufacturer’s instructions, with signal/cutoff ratio greater than 0.9 as positive readings.The experimental protocol for the IFA assays was described briefly as follows. Cover each reaction well with PBS‐diluted samples or controls (10–20 μl per well). After incubation in a humidified chamber at 20‐25°C, rinse the slides thoroughly in a staining dish of PBS and change the buffer and wash for an additional 8 minutes. Cover each well with fluorescein‐conjugated goat anti‐human IgM. After incubation and washing steps as described above, place a small drop of buffered glycerol in each well and cover with a coverslip, then the slides were read immediately at a magnification of 500‐fold, and a positive reaction for TORCH antibodies was indicated by the presence of diffuse nuclear inclusion bodies that emit green fluorescence . TORCH IgM infection differences were calculated by chi‐square test; two‐sided 33.1With the ELISA screening assays described above, the positive rates of serum IgM antibodies to TORCH are listed in the order of decreasing prevalence: HSV1 (6.30%), RV (2.55%), HSV2 (1.94%), CMV (1.24%), and TOX (0.67%). As the IgM positivity is a strong indicator for primary infection, the TORCH IgG prevalence reflecting past exposure or vaccination was much higher than IgM, with CMV (92.05%), RV (89.74%), and HSV1 (81.56%) as the top prevalence IgG antibodies in the study population. See Table 3.2P < .05) was applied to the following study , suggesting the false‐positive of these antibodies with ELISA should not be neglected. Based on the IFA experiments  had consistent results with ELISA experiments. In other words, as much as 67.3% of the specimens had one or more false‐positive IgM reactions by ELISA. No false negatives were identified in the TORCH IFAs when compared with the corresponding ELISA results Table . To makets Table .4As seen in Table The CMV‐IgG had the highest prevalence in this study with 92.05%, which was consistent with the data reported previously, 60% in developed countries and almost 100% in developing countries.,vs rural areas) and methodologies. The risk of vertical transmission of HSV from mothers with a primary HSV infection to fetuses is about 25–50% and decreases to less than 3% in women with a recurrent HSV infection due to placental protecting IgG antibody.The HSV1‐IgM prevalence was the highest in TORCH in this study (6.3%), consistent with Feng’s and Su’s findings about the women in BeijingAs seen in Table The false‐positive ELISA results were confirmed by the “gold‐standard” TORCH‐specific IFA experiments. Three pathogen‐specific IgM antibodies  were observed to have statistically significant cross‐reactions in the ELISA screening assays, according to Table In our study, the most common TORCH co‐infection patterns were HSV1 + HSV2, TOX + HSV1 + HSV2 and CMV + HSV1 + HSV2. The anti‐HSV1 and anti‐HSV2‐IgM antibodies tended to cross‐react with each other relatively easily due to their similar antigenic determinants.As an essential part of decreasing the congenital diseases, TORCH‐specific antibody screening results should be evaluated with extra care due to the existence of high rates of false‐positive IgM reactions, to decrease the unnecessary psychological and economic burdens of patients. Click here for additional data file.