Immunoglobulin G4-related disease (IgG4-RD) is a newly defined disease entity, while the exact pathogenesis is still not clear. Identifying the characters of IgG4-RD in proteomic and transcriptomic aspects will be critical to investigate the potential pathogenic mechanisms of IgG4-RD. We performed proteomic analysis realized with iTRAQ technique for serum samples from eight treatment-naive IgG4-RD patients and eight healthy volunteers, and tissue samples from two IgG4-RD patients and two non-IgG4-RD patients. Transcriptomic data (GSE40568 and GSE66465) was obtained from the GEO Dataset for validation. The weighted correlation network analysis (WGCNA) was applied to detect the gene modules correlated with IgG4-RD. KEGG pathway analysis was used to investigate pathways enriched in IgG4-RD samples. As a result, a total of 980 differentially expressed proteins (DEPs) in tissue and 94 DEPs in serum were identified between IgG4-RD and control groups. Three hundred fifty-four and two hundred forty-seven genes that most correlated with IgG4-RD were detected by WGCNA analysis in tissue and PBMC, respectively. We also found that DEPs in IgG4-RD samples were enriched in several immune-related activities including bacterial/viral infections and platelet activation as well as some immune related signaling pathways. In conclusion, we identified multiple processes/factors and several signaling pathways that may involve in the IgG4-RD pathogenesis, and found out some potential therapeutic targets for IgG4-RD. Immunoglobulin G4-related disease (IgG4-RD) is a newly defined immune-mediated disease with common clinical, serological, and pathological features . Common IgG4-RD is also characterized by alterations in acquired immune system, in which aberrant expansion of plasmablasts, CD4+ cytotoxic T cells, and follicular T helper cells have been observed –7. In adDue to the lack of ideal animal models, and limited sample origin, high throughput, and bioinformatics techniques may help understand the underlying pathogenesis of IgG4-RD more deeply. Transcriptome-wide profiling, as a downstream level of genome-scale mapping, can reveal a systemic dynamics of molecular interaction . RecentlProteins are effectors of biological function, and exert critical important roles in the pathogenesis of diseases. Investigation into proteins is crucial for the development of methods to realize early disease diagnosis, prognosis assessment and to monitor the disease development . ProteomIn conclusion, based on proteomic and transcriptomic analyses, we have not only identified several differently expressed proteins in serum and tissue samples from IgG4-RD patients compared with healthy people, but also illustrated some features of immuno-inflammatory reactions in IgG4-RD, which also helped provide information of its potential therapeutic targets. These results may provide clues to the elucidation of the pathogenesis of, and the development of therapeutic agents for IgG4-RD.We studied eight diagnostic serum samples from eight treatment-naive IgG4-RD patients (25–70 years old) at Department of Rheumatology and Immunology, Wuhan Tongji Hospital . The diaIn this study, the Isobaric tags for relative and absolute quantitation (iTRAQ) technology was applied to investigate the proteasome of serum and tissue samples. First, the ProteoMiner Protein Enrichment Kit  was applied to deplete the high abundance proteins. Then, the protein solution (100 ug) with 8 M urea was diluted 4 times with 100 mM TEAB buffer. Trypsin Gold  was used to digest the proteins with the ratio of protein: trypsin = 40: 1 at 37°C overnight. After trypsin digestion, peptides were desalted with Strata X C (Phenomenex), and vacuum-dried according to the manufacturer's protocol. Peptide labeling was performed by iTRAQ Reagent. Peptide Fractionation were realized with a HPLC Pump system (Shimadzu LC-20AB) coupled with a high pH RP column. Supernatants of fractions were loaded on UHPLC system (Thermo Scientific™ UltiMate™ 3000) equipped with a trap and an analytical column, and peptides separated from nanoHPLC were subjected into the tandem mass spectrometry QEXACTIVE HF X  for data-dependent acquisition (DDA) detection by nano-electrospray ionization. The parameters for MS analysis are listed as following: electrospray voltage: 2.0 kV; precursor scan range: 350–1,500 m/z at a resolution of 60,000 in Orbitrap; MS/MS fragment scan range: >100 m/z at a resolution of 15,000 in HCD mode; normalized collision energy setting: 30%; dynamic Exclusion time: 30 s; automatic gain control (AGC) for full MS target and MS2 target: 3e6 and 1e5, respectively; the number of MS/MS scans following one MS scan: 20 most abundant precursor ions above a threshold ion count of 10,000.Protein identification and quantification were realized by software IQuant . The proGSE40568 and GSE66465 from Gene Expression Omnibus (GEO). Specifically, LSG samples in GSE40568 were obtained from Japanese patients with IgG4-RD (n = 5) as well as from Japanese patients with SS (n = 5) and HCs (n = 3) who had been followed up at the University of Tsukuba Hospital , Tokyo Women's Medical University Hospital , and Kyushu University Hospital  (n = 2) and after steroid (n = 2) therapy who registered in the research project of the Research Program for Intractable Disease of the Ministry of Health, Labor, and Welfare (MHLW) of Japan and HCs (n = 4)  . PBMC sa (n = 4) .https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/). In this study, we construct a scale-free network (R2 = 0.9) based on the criteria that soft-thresholding power β were set as 20  of each gene were calculated after expression matrix were imported and normalized. Genes with CV >5% were log2 transformed, and the corresponding expression data was applied as input for WGCNA analysis. Then weighted co-expression networks were constructed by employing blockwiseModules function in the WGCNA package (t-test” function in R package (version 3.5.1). Expression matrix from GEO datasets were extracted and normalized by using R package “GEOquery”. WGCNA analysis was realized with R package “WGCNA” (version 1.68)  . KEGG/GOon 1.68) , 21. Infon 1.68) . Withoutp < 0.05). Among them, we found there were 12 DEPs  overlapped in the comparisons of tissue and serum and all those DEPs upregulated in IgG4-RD patients, suggesting their importance to IgG4-RD.A total of 980 (542 up-regulated and 438 down-regulated) differentially expressed proteins (DEPs) in tissue , while 9To understand the function of these DEPs which may involve in IgG4-RD, functional enrichment analysis of these DEPs identified in tissue/serum between IgG4-RD and control samples were carried out. Results indicated that most tissue upregulated DEPs were involved in terms including immune related cells activation  and cell adhesion , and infection related processes such as human immunodeficiency virus 1 infection, Epstein-Barr virus infection, and Salmonella infection etc. . Howevern = 5), pSS (n = 5), and HC samples (n = 3) (p = 7e-5)  >5% as input for the construction of WGCNA using GSE40568 dataset from LSC samples. Combined with the topological overlap matrix with the hierarchical average linkage clustering method, we detected the 13 modules in IgG4-RD ( (n = 3) . Among t = 7e-5) . Then, a = 7e-5) , S4.n = 2), IgG4-RD after treatment  and HC samples  based on the criteria referred in method section  >5% as input for WGCNA. In this study, 14 modules were detected by WGCNA in IgG4-RD before treatment , and can mediate the release of danger associated molecular patterns (DAMPs). Both PAMPs and DAMPs are ligands of pattern recognition receptors (PRRs), which can be found in many cellular components involved in immune reactions. Activation of PRRs can modulate the functional states of immune-related cells, which further influence the process of immune responses. Based on our proteomic data, many infection related terms such as “Human immunodeficiency virus 1 infection,” “Epstein-Barr virus infection” and “Bacterial invasion of epithelial cells” were enriched in IgG4-RD tissue , Table 2In proteomic data, up-regulated DEPs from both serum and tissue samples of IgG4-RD patients were enriched in “Platelet activation” pathway , 3. MeanUnderstanding the significantly enriched signaling pathways in IgG4-RD may help to search the potential therapeutic targets. In proteomic data, “Rap1 signaling pathway” and “NF-κB signaling pathway” were enriched in IgG4-RD tissue , while tIn order to find out the potential therapeutic targets for IgG4-RD, we extracted all proteins (not only DEPs) involved in top 30 KEGG/GO BP terms enriched from all DEPs in our tissue proteomic data . One thoTo our knowledge, the pathogenesis of IgG4-RD is still not clear and this is the first report providing new insights to help the illustration of potential pathogenic mechanisms underlying IgG4-RD based on proteomic and transcriptomic data.Existence of strong immune responses in IgG4-RD is well-known. Different T cell subsets may interact with B cell subsets in involved tissue, and further lead to the down-streaming fibrogenesis, which can also be partly reflected by our study  30–34).–34.30–34Our study detected significant enrichment of “Fc gamma R-mediated phagocytosis” process in IgG4-RD tissue , which mIn this study, besides immunoglobulins produced by plasmablasts/plama cells, we found level of several cytokines, including CXCL13, IL-27, and IL-18, elevated significantly in IgG4-RD tissue. Cytokines exert essential effects on the immunoinflammatory process. IL-27 can act as antagonists to suppress Th1, Th2, Th9, and Th17 responses, while promote the proliferation and the expression of T-bet, EOMES, and IL-12Rβ2 associated with increased production of IFN-γ and cytotoxic activity . IL-18 iin vivo.Infectious agents can modulate the status of immune responses, and even be the triggers of systemic lupus erythematosus (SLE) and Sjögren's syndrome . Our anaEnrichment of platelet activation related processes in IgG4-RD patients was also observed in our study , 3, 5. AGenes (or proteins) related to several signaling pathways were also detected in IgG4-RD samples (tissue or PBMC), including MAPK signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, TGF-β signaling pathway, NF-κB signaling pathway and Rap1 signaling pathway . All theHow to treat IgG4-RD is another task we are facing. Despite glucocorticoids and rituximab are effective in the induction therapy, and application of conventional steroid sparing medication  can help control the disease, the problem of relapse and side effects after long-term usage of these medications are still remarkable –50. BaseIn summary, we provided the first integrative analysis of IgG4-RD via both proteomic and transcriptomic data, and described a landscape of biological processes of this mysterious disease, which indicated some potential pathogenic molecules and immunoinflammatory responses, and provided several potential therapeutic targets for the treatment of IgG4-RD. There are also limitations in our study. Firstly, despite the aim of our study is to provide the landscape at the level of mRNA and protein of IgG4-RD, the sample size of our analysis was relatively small, and we didn't enroll more IgG4-RD-like samples , in our analyses; secondly, the transcriptomic data were originated from the published data of other centers, although analyses to them showed overlaps with our proteomic data, which may reflect the reliability of our analyses indirectly; thirdly, our analyses were based on the data of bulk samples, which cannot provide the information of specific cell types. Therefore, further studies with large sample size, and at single cell level are needed. Besides, other types of omics  and validation with laboratory experiments are also important to help us understand this mysterious disease more deeply.GSE40568 and GSE66465 can be downloaded from Gene Expression Omnibus Dataset (GEO Dataset: http://www.ncbi.nlm.nih.gov/geo/). The proteomic data is available from the corresponding author on reasonable request.Data of The studies involving human participants were reviewed and approved by Tongji Hospital, Tongji Medical College, Huazhong University of Science, and Technology Institutional Review Board Approval. The ethics IRB ID is: TJ-C20151109. The patients/participants provided their written informed consent to participate in this study.SC and YC made the sample collection, data analyses, and wrote this manuscript. SL and CY revised the manuscript and provided important advice. LD and FZ designed this program, and are the corresponding authors of this paper. All authors contributed to the article and approved the submitted version.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.


But are such fixed-dose regimens optimal or can alternative regimens be designed to increase efficacy? Yet, few mathematical models have aimed to identify optimal treatments based on biological data of infections inside a living host. In addition, assumptions to make the mathematical models analytically tractable limit the search space of possible treatment regimens (e.g. to fixed-dose treatments). Here, we aimed to address these limitations by using experiments in a Galleria mellonella (insect) model of bacterial infection to create a fully parametrised mathematical model of a systemic Vibrio infection. We successfully validated this model with biological experiments, including treatments unseen by the mathematical model. Then, by applying artificial intelligence, this model was used to determine optimal antibiotic dosage regimens to treat the host to maximise survival while minimising total antibiotic used. As expected, host survival increased as total quantity of antibiotic applied during the course of treatment increased. However, many of the optimal regimens tended to follow a large initial ‘loading’ dose followed by doses of incremental reductions in antibiotic quantity (dose ‘tapering’). Moreover, application of the entire antibiotic in a single dose at the start of treatment was never optimal, except when the total quantity of antibiotic was very low. Importantly, the range of optimal regimens identified was broad enough to allow the antibiotic prescriber to choose a regimen based on additional criteria or preferences. Our findings demonstrate the utility of an insect host to model antibiotic therapies in vivo and the approach lays a foundation for future regimen optimisation for patient and societal benefits.Mass production and use of antibiotics has led to the rise of resistant bacteria, a problem possibly exacerbated by inappropriate and non-optimal application. Antibiotic treatment often follows  in vivo insect model of systemic Vibrio infection was used for the first time to determine critical model parameters for optimal antibiotic treatment. By this approach, the optimal regimens tended to result from a large initial ‘loading’ dose followed by subsequent doses of incremental reductions in antibiotic quantity (dose ‘tapering’). The approach and findings of this study opens a new avenue towards optimal application of our precious antibiotic arsenal and may lead to more effective treatment regimens for patients, thus reducing the health and economic burdens associated with bacterial infections. Importantly, it can be argued that until we understand how to use a single antibiotic optimally, it is unlikely we will identify optimal ways to use multiple antibiotics simultaneously.Research into optimal antibiotic use to improve efficacy is far behind that of cancer care, where personalised treatment is common. The integration of mathematical models with biological observations offers hope to optimise antibiotic use, and in this present study an  B(t)>Bdead for any t∈. McMillan et al (2015) found in their experiments that the host (the larvae) died when the bacterial abundances reached approximately 109 [Bdead of 109. The success of an antibiotic treatment was then measured by the fraction of runs where the host survived to 192 h, denoted Nsurv.As this was a stochastic model, the behaviour of the antibiotic and bacteria population size changed with each run. For this reason, we carried out 5000 runs of the model, and counted the number of runs where treatment was a ‘success’: a model run was a ‘success’ if the host survived to 192 h, as measured by the bacterial population size staying below a threshold, i.e. tely 109 ; this prThe parameter definitions are given in Genetic Algorithms (GA) were proposed by John Holland in the early 1970s . They berμ, v, mμ, n, a, a1, a2 and k, represented by a vector of real numbers. For each set, the GA ran the mathematical model 5000 times, and computed how well the mathematical model fitted the data, as measured by a least squares approach calculating the difference between host survival at each 24-h interval from the mathematical model and the biological experiments; the fit is denoted by the function Fpar and is given in , where di denotes the quantity of antibiotic (measured in mg) to be given at time ti. The GA aimed to find the optimal dosage vector that minimised our objective function, which comprised of up to two components: first, to maximise the number of runs of the mathematical model where the host survived the infection, i.e. B(t)<Bdead for all t, denoted Nsurv; second, to minimise the total amount of antibiotic used, as measured by the sum of the entries in the dosage vector, ∑idi. In addition, we had constraints: ∑idi≤0.9 mg, where 0≤di≤0.9  Maximise survival vs. minimise total antibiotic’, where we increased the total quantity of antibiotic). For each regimen, the GA ran the mathematical model 5000 times, with a population size of 50 potential solutions (dosage vectors), for 80 generations and the whole process was repeated 50 times. At the end of the complete GA run, we produced 50 sets (one from each GA run), each containing 50 possible solutions (dosage vectors). The run lengths, generations and population sizes were arrived at from prior calibration of the configuration that confirmed that minimal further improvement in performance was gained by increasing these values.Antibiotic regimens are represented by dosage vectors To gain a more accurate estimate for the decay rate of the antibiotic (TET), the antibiotic activity in the haemolymph was measured over time ). Fittinrμ, v, mμ, n a, a1, a2 and k. The GA showed strong convergence and provided a set of parameter values that best fit the data. The model with these parameters showed a reasonable fit with the biological data , with dose d1 at 2 h and the second (remaining) dose at 24 h. This strategy used 0.9 mg of antibiotic in total. Our parameterised mathematical model was run for values of d1 from 0 to 0.9, in increments of 0.01, with survival rate recorded at 192 h .in vivo experiments  dose of 0.9-d1 is given at t2 hours. Given we only had two variables, d1 and t2, we carried out a brute force (exhaustive) search for d1 between 0 and 0.9 , and t2 at hourly intervals between 3 h and 24 h. In t2, and the size of the first dose d1. We found that the optimal strategy was to administer a first dose of 0.54 mg of TET at 2 h and a second dose of 0.36 mg at 11 h, giving a survival rate at 192 h of 0.795. This is significantly greater than the survival rate of 0.566 for administering all 0.9 mg of TET in a single dose at 2 h.iiiMultiple-doses, variable timings: We extended the search to find the optimal treatment regimen, using 0.9 mg of antibiotic, by allowing any number of doses. A GA was applied to this problem, allowing the individual dosage quantities and timings of these doses to vary, to find the regimen that maximised host survival at 192 h. We found the optimal regimen was a four-dose strategy, giving: 0.43 mg at 2 h, 0.22 mg at 7.1 h, 0.13 mg at 11.7 h, and 0.13 mg at 16.8 h, which gave a host survival rate of 0.803.  Given this is a stochastic model, it would be difficult to test experimentally in vivo whether the improvement over the two-dose treatment above, with survival of 0.795, is significant.ivMaximise survival vs. minimise total antibiotic: Taking a multi-objective approach, we aimed to maximise host survival at 192 h while minimising the total quantity of antibiotic used. Again, we allowed both the individual doses and the timing of these doses to vary. In r = 0.997), with a gradient of 2.12, i.e. every 0.1 mg increase in antibiotic gives an increase in host survival at 192 h of 0.212. In addition, we saw almost distinct boundaries between where the optimal regimen involved increasing the number of doses: a single  dose when using less than 0.49 mg, two-doses when using between 0.49 mg and 0.64 mg in total, three-doses when using between 0.64 mg and 0.9 mg in total, and four-doses when using more than 0.9 mg in total.di); (ii) the least number of doses (i.e. least number of non-zero entries in dosage vector); (iii) the lowest maximum dose (i.e. to minimise max(di)); (iv) the earliest final dose (i.e. to minimise max(ti)).To understand the form of the optimal dosage vectors in the Pareto front, we explored the treatment regimens that were found by the GA and extracted the best regimen for a particular objective. All the treatments in V. anguillarum) in an in vivo host (G. mellonella) that can be treated with different fixed-dose antibiotic regimens. The mathematical model of this system was then tested with additional experiments, including some new antibiotic regimens where daily doses were no longer fixed. The model was shown to perform well in these subsequent experiments, and hence provides a useful new approach for researchers investigating the optimisation of antibiotic therapy.The first aim of this study was to build and parameterise a mathematical model that accurately represents a systemic bacterial infection , to this model to find optimal antibiotic dosage regimens that maximised host survival, while also minimising total antibiotic usage. In searching for the optimal regimen, we relaxed the commonly made assumptions of fixed doses  at fixed intervals (e.g. every 24 h) – the widened search space of possible treatments makes the application of artificial intelligence algorithms essential. When using a fixed total antibiotic dose of 0.9 mg, the best two-dose regimen was to apply 0.54 mg at 2 h and 0.36 mg at 11 h, giving a host survival rate of 0.795. This only increased slightly to 0.803 with a four-dose regimen. However, both of these were predicted to be significantly greater survival than administering all 0.9 mg in a single dose at 2 h. When aiming to minimise the total quantity of antibiotic used while maximising host survival, all the treatments found  for daily doses –78. In tWith the increase in antibiotic resistant bacteria, research has begun to examine the effectiveness of using multiple antibiotics, either sequentially or together in combinations –82. StilS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(EPS)Click here for additional data file.


Early prevention methods in massive concrete structures to control the heat of hydration and, consequently, the development of cracks due to thermal expansion are important subjects, since these cracks may compromise structural integrity. In the present study, the sugarcane residues in massive concrete were used in order to investigate the reduction in the heat of hydration, the thermal expansion resistance, and also the fresh and mechanical properties of the concrete. The results showed that, by adding 2.0% of bagasse fiber and 5.0% of pozzolanic material to the concrete, the heat of hydration was reduced, and the strain due to the thermal expansion was smaller than the control mixture. Moreover, the compressive, flexural, and split tensile strength increased in comparison to the control mixture. The reaction between water and cement in massive concrete structures like dams, pavements and piers produces heat and increases the temperature of the concrete ,2. DurinSeveral previous researchers have tried to reduce hydration heat by replacing a certain amount of cement with pozzolanic materials, such as silica fume, fly ash, and oil palm fuel ash. Their results show that, when a certain amount of these materials is used in place of cement, it is possible to decrease the temperature of the concrete ,7,8.Nili et al. investigated the effects of supplementary cementitious materials on the temperature rising profile, heat evolution and early-age strength development of medium- and high-strength concrete. A total of 13 different mixtures were prepared, with two water-cement ratios (0.3 and 0.46). Natural pozzolan, fly ash, and silica fume were included in the specimens. The results showed that natural pozzolan, particularly fly ash served to decrease the amplitude of peak temperature, delay the occurrence of the peak, and decrease the sharpness of the temperature rising profiles .Awal et al. investigated the performance behavior of palm oil fuel ash (POFA) in reducing the heat of hydration of concrete. Two concrete mixes, namely OPC (ordinary Portland cement) concrete, i.e., concrete with 100% OPC as control, and POFA concrete, i.e., concrete with 30% POFA and 70% OPC were prepared, and the temperature rise due to heat of hydration in both the mixes was recorded. It has been found that palm oil fuel ash not only reduced the total temperature rise, but also delayed the time at which the peak temperature occurred. The results obtained and the observations made clearly demonstrate that the partial replacement of cement by palm oil fuel ash is advantageous, particularly for mass concrete where thermal cracking due to excessive heat rise is of great concern [Apart from the use of pozzolanic materials, there are several countermeasures that can improve thermal crack resistance and decrease the effect of the strain shrinkage of the concrete . The utiSarabi et al. used waste turnery steel fibers in massive concrete in order to control the generated cement hydration heat and, consequently, the potential of cracking due to the thermal expansion. The amount of cement used was reduced without changing the compressive strength. By substituting a part of the cement with waste steel fibers, the costs and the generated hydration heat were reduced, and the tensile strength increased. The results showed that, by using 0.5% turnery waste steel fibers and, consequently, by reducing to 32% the cement content, the hydration heat was reduced to 23.4%, without changing the compressive strength. Moreover, the maximum heat gradient was reduced from 18.5% in the plain concrete sample to 12% in the fiber-reinforced concrete sample .Recently, the application of agro-residues in concrete has gained interest due to the high amount of these materials available throughout the world, and also due to environmental issues such as the incorrect disposal of these residues. Among several types of agro-residues, there is the bagasse, which is a fibrous residue of the sugarcane . NaturalThe sugarcane bagasse is mostly composed of cellulose, hemicellulose, and lignin. Cellulose, hemicelluloses, and lignin are in the family of polysaccharides, and these polysaccharides are composed of various types of sugars. Therefore, these sugars act as setting and hydration retarding agents . The com2 for 24 h [The sugarcane residues (raw bagasse and burned residues) were acquired from a sugar mill in Okinawa Prefecture, Japan. In the case of the raw bagasse, it was dipped in water at 30 °C for 30 min and then dried in the open air for 14 days. The intent of this process was to reduce the residual sugar content of the bagasse and eliminate impurities ,20,21. Afor 24 h ,24. In tThe specimens were made using ordinary Portland cement (C). The fine aggregates (S) and the coarse aggregates (G) were acquired from manufacturer, Nishijima, Hyogo, Japan. In order to comparisons, a commercially available fly ash (FA) grade II was used. The physical properties of these materials are given in For the concrete mixture, two kinds of admixtures were used in this study to satisfy the requirements of the fresh concrete. The properties of the water-reducing agent (WRA) and air-entraining agent (AEA) are shown in max) was 15 mm.The mix proportions of the concrete are shown in For each concrete mixture, specimens of Φ100 mm × 200 mm were cast in order to determine the compressive strength (JIS A 1108), elasticity modulus (JIS A 1149), split tensile strength (JIS A 1113), porosity rate and water retention rate of concrete. Furthermore, specimens of 100 mm × 100 mm × 400 mm were prepared to determine the flexural strength (JIS A 1106). Note that three specimens were used for each test.The porosity rate test was based on the mass of the specimen. First, the specimens were completely dried in a drying furnace at 105 °C for 24 h. Afterwards, the specimens were stored in a room at 20 °C and relative humidity of 60% until the temperature of the specimen decreased and became equal to the ambient temperature, then the mass of the specimen was weighed. Subsequently, the specimens were immersed in water at 20 °C for 48 h, then the surface-dried mass of the specimen was weighed. The porosity rate was determined by the following equation:3);3).After the porosity rate test, the moistened specimens were placed in a room at 20 °C and relative humidity of 60%, and the mass change of specimen was recorded every 24 h for 7 days. This mass change was determined as water retention rate.In addition, in order to simulate a massive concrete structure, 2 specimens of 300 mm × 300 mm × 300 mm of each concrete mixture were prepared. The temperature and the strain of the concrete were measured by two thermocouples (T-G-0.65) and two mold strain gauges (PMFL-60) embedded in concrete, respectively. The thermocouples and the gauges were settled at the surface (cover thickness of 20 ± 2 mm) and the center (cover thickness of 145 ± 2 mm), as shown in All the concrete mixtures were mixed using a laboratory pan-type mixer. First, the coarse aggregate, cement/ashes, and fine aggregate were put in the mixer and mixed for 30 s. Next, the water/fibers were put in the mixer and mixed for an additional 120 s. Before casting, the slump flow and the air content were conducted on the concrete mixture to determine its workability.After the casting, all specimens were cured in the laboratory at 20 °C for 24 h. The specimens were then de-molded and cured underwater, at 20 °C for 28 days. However, the massive concrete specimens were not de-molded and were instead cured in the air at a room temperature of 20 °C and 60% RH for 28 days. During the curing period, the temperature and the strain due to the thermal expansion of concrete were measured by using thermocouples and gauges, as shown in The slump test and air content test results are shown in The air content in the case of the concrete without fibers is about 4.7%. However, the amount of air increases as the amount of mixed fibers increases. For the cases of BF2, BF5, BA, and FA, the air content values obtained were 8.4, 15.6, 6.1, and 5.5%, respectively. The rise in the air content with the addition of bagasse fiber can be explained due to fine bubbles of air clumping together while the concrete mixes; consequently, the amount of empty space may increase.The plot of temperature versus time for the different mixtures and the ambient temperature can be seen in As In the case of BF2 and BF5, the difference in the peak temperature of the heat of hydration was around 0.3 °C. As the amount of fibers increases in the case of BF5, a high number of empty spaces between the fiber and the matrix are created, reducing the thermal conductivity. Therefore, the absorption of the hydration heat by the surrounded fiber components becomes difficult, resulting in the same temperature as BF2.In the cases where the bagasse ash and the fly ash were added into the mixture to replace the sand, the temperature was approximately 1 °C lower compared to the case of C; however, the temperature was about 3 °C higher than for those mixtures where bagasse fibers were added. The reason for this increase in temperature in the cases of BA and FA, compared to BF2 and BF5, is due to the difference in the water to binder ratio. As shown in 2O was added, subsequently, it was allowed to stand for about 2 h. Later, this mixture was centrifuged at 8000 rpm at 20 °C for 30 min. Then, 10 mL supernatant was lyophilized and dissolved in 1 mL of acetonitrile/water (CH3CN/H2O = 75/25). This solution was used as a sample to analyze the reducing sugar. Afterward, 0.2 mL of sample, 0.4 mL of reagent A (NaCO3 (40 g/L), Glycine (16 g/L), CuSO4·5 H2O (0.45 g/L)) and 0.4 mL of reagent B were mixed. The mixture was heated at 100 °C for 12 min and rapidly cooled. Then, 1 mL of H2O was added, and the absorbance at 450 nm was measured. From the calibration curve prepared with glucose, the reducing sugar content of the sample was determined as a glucose equivalent. This result indicates that the reducing sugar content was 1.05 mg/g, confirming the presence of water-soluble sugars that affected the hardening and hydration of cement. These soluble sugars may form a protective layer around the hydrating cement [Furthermore, from g cement , preventg cement . As a coMoreover, The relationship between the heat of hydration and the strain on each mixture is shown in The strain due to the thermal expansion of each mixture is shown in Furthermore, As shown in However, in the cases of BA and FA, where the ash was added, the porosity rate of the concrete decreases in comparison to the case of BF2, which contains the same volume of fiber. Although the solid content per cubic meter of BA and FA is higher than the solid content of BF2, the reason for this may also be related to the filler effect of the ash. When adding ashes to concrete mixtures, the ashes act to filling the voids and reduce the number of pores and probable microcracks of the concrete during pozzolanic reactions.The water retention rate of each concrete mixture is shown in As shown in However, after 120 h, the water retention rate of BF5 decreases to nearly the same values as BF2. The natural fibers are very hydrophilic ,26. For In cases when 5% of ashes were added, the water retention ratio is smaller than the BF2 case, even though these cases contain 2% of fibers added in the mixture. In particular, in the case of FA, the water retention was smaller than the control mixture. This reduction may be related to the pozzolanic reaction of the ashes, in which the water has been consumed by the fibers.2 for 24 h, the internal alkali solution of the fibers may have been activated on the surface of the ash particles at an early age, accelerating the pozzolanic reaction during the water immersion curing period.On the other hand, 2, the elastic modulus is in the range of 31–33 GPa. Although the modulus of elasticity in the case of C and FA is slightly higher than this range, in the case of BF5, the elastic modulus decreases drastically to around 28 GPa. This is due to the high amount of fiber added to the mixture, which increased the porosity and reduced the modulus of elasticity.According to the Japan Society of Civil Engineers (JSCE) , in the 2 for C, BF2, BF5, BA, and FA, respectively. From these results, it can be concluded that the higher the amount of fiber added, the higher the acquired flexural strength is. However, in the case of BF5, the flexural strength was not proportional, resulting in a flexural strength near to that of BF2. This tendency indicates that, when a high amount of fibers are added into concrete, these fibers may not effectively transfer the stress to the other cross-sections. This may be due to the fact that, with the addition of a high amount of fibers, the concrete density tends to decrease and, consequently, the porosity tends to increase. Therefore, the high number of interfacial voids between the fiber and the matrix may increase, affecting the transmission of stress.In addition, 2, respectively.From 2, approximately 7% lower in comparison to the control mixture. Due to the fact that the tensile failure section is limited to the one in the tensile splitting test, the effect of fiber bridging to redistribute the stress to another cross-section is limited, a phenomenon that is even more pronounced due to the high amount of voids in the fiber/matrix interface.However, when 5% of fiber was added, the split tensile strength decreased to around 2.87 N/mmThe use of sugarcane residues to replace sand may be a way to make low-cost and environmentally friendly materials. In addition, their use in concrete could reduce the heat of hydration and the strain due to the thermal expansion, which may be a countermeasure against the generation of cracks in massive concrete.The slump decreased with the addition of the fiber amount compared to the control mixture. However, the amount of air increased as the amount of mixed fibers increased.With the addition of the bagasse fiber, the heat of hydration of all mixtures was reduced. In the case when 5% of the bagasse fiber was added, the peak temperature neared 48 °C, approximately 4.5 °C lower than the control mixture.In the cases in which the bagasse fiber was added, the peak temperature was reached later than the control mixture. In the case when 2% of the bagasse fiber was added, the temperature peak was achieved 26 h after the concrete placement, while the control mixture temperature peak was achieved 4 h earlier.In the case of the control mixture, the strain rose to a value of approximately 55 μ, while, in the case of the mixture in which 5% of bagasse fiber was added, the strain value was 30 μ, a difference of about 25 μ.In the case where the bagasse fiber was added, the compressive strength decreased. However, the compressive strength increased when the ashes were added to the mixtures, thereby exceeding the control mixture.The flexural strength of all concrete specimens with added fiber exceeded the value of the control specimen.The split tensile strength increased when 2.0% of the fiber content was added to the mixture. On the other hand, with 5.0% of the fiber, the split tensile strength decreased.The results obtained in this study can be summarized as follows:


Immunoglobulin G4-related disease (IgG4-RD) is a chronic multisystem immune-mediated disease. Histologically, it presents as infiltration with IgG4-secreting plasma cells affecting many organs such as the pancreas, lacrimal glands, salivary glands, kidneys, and arteries, resulting in chronic fibrosis and scarring within the tissue which over time forms into a mass. It is a slow-growing process that usually remains indolent until it causes a mass effect or tissue infiltration.Our patient was found to have a pancreatic mass on imaging concerning pancreatic neoplastic lesions. He subsequently underwent biopsy for histology/pathology and IgG4 levels, which led to our diagnosis of IgG4-RD. Imaging, blood tests, and, most importantly, histopathological features of the involved organ are important in determining the diagnosis. This is important as a treatment plan will be based on the given diagnosis, and patients with IgG4-RD respond very well to systemic steroid therapy. Immunoglobulin G4-related disease (IgG4-RD) is a multisystem chronic mass-forming inflammatory condition characterized by tissue infiltration with lymphocytes and IgG4-secreting plasma cells, storiform fibrosis, and obliterative phlebitis histologically . DiagnosA 60-year-old male with a history of cryptogenic liver cirrhosis and newly diagnosed diabetes presented with abdominal discomfort and unintentional weight loss for a few months.The patient’s physical examination showed mild diffuse abdominal pain, otherwise unremarkable. His labs showed a slight elevation in alkaline phosphatase, mild elevation in globulin level at 4.3 g/dL, normal C-reactive protein, and mild elevation in erythrocyte sedimentation rate of 28 mm/hr. Alpha-fetoprotein, carcinoembryonic antigen, and CA 19-9 levels were normal. PPD and QuantiFERON gold were negative; antinuclear antibody, anti-smooth muscle antibody, and antimitochondrial antibody levels were also normal; hepatitis panel was negative. He underwent further imaging, including computed tomography (CT) with contrast scan of the abdomen, which showed the following: a poorly defined 4.5 cm mass in the pancreatic body encasing the splenic artery, indicative of pancreatic adenocarcinoma; mild intrahepatic bile duct dilatation, primarily on the left liver lobe, with circumferentially thickened gallbladder wall; a 2.6 cm mass at the deep margin of the umbilicus possible rectal cyst vs. metastasis; or postsurgical changes. It showed soft tissue thickening anterior to the distal abdominal aorta and encasing of inferior mesenteric artery suspicious for possible metastatic disease or chronic post-inflammatory changes. The patient was furthered referred to oncology for evaluation.Our patient then underwent esophagogastroduodenoscopy, which showed two columns of trace distal esophageal varices with no stigmata for bleeding; no bending was performed. It also demonstrated antral gastritis, mild diffuse portal hypertensive gastropathy, and normal appearance of the duodenum. A random biopsy of the second portion of the duodenum was performed to rule out IgG4 deposition. Duodenal biopsy results showed IgG4-positive plasma cells, with no evidence of dysplasia or malignancy  demonstrated an irregular mass identified in the pancreatic body Figure  and pancPathology from the pancreatic head and tail mass showed fibrosis and inflammation suggestive of chronic pancreatitis. Given these findings, there was a concern for IgG4-RD, which was confirmed as IgG levels returned back elevated as follows: subclass 1 at 1,469 mg/dL subclass 2 at 1,027 mg/dL subclass 3 was normal, subclass 4 >300 mg/dL, and total serum globulin 3,127 mg/dL.The patient subsequently was started on prednisone and had received two months of systemic steroid therapy.Follow-up CT abdomen with contrast in two months showed improvement in the ill-defined area of decreased attenuation in the tail of pancreas; the overall appearance is most consistent with resolving pancreatitis, now measuring 1.9 x 1.5 cm. IgG levels decreased to the following levels: subclass 1 at 531 mg/dL , subclass 2 at 220 mg/dL, subclass 3 at 25 mg/dL , subclass 4 still elevated at 413.5 mg/dL , and IgG serum 1,155 mg/dL .Although there was an improvement in imaging with steroid therapy, IgG4 levels were still elevated; therefore, our patient was then started on rituximab therapy and received four doses of rituximab. Follow-up IgG4 panel showed significant improvement along with imaging evidence. IgG subclass 4 levels showed improvement to 164.4 mg/dL.Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States . SurgicaIgG4-RD is a chronic multisystem immune-mediated disease that leads to tissue infiltration with IgG4-secreting plasma cells resulting in chronic fibrosis and scarring within the tissue which over time forms into a mass. It is a slow-growing process that usually remains indolent until it causes a mass effect or tissue infiltration. It can affect any tissue/organ within the body, and thus can have a variable presentation. The most commonly affected organs are the pancreas, lacrimal glands, salivary glands, kidneys, and arteries, and based on the organ affected, it can be known as many different names ,3-6. ForPatients generally present late as initially, they feel well and unlikely present as critically ill. However, due to the chronic inflammatory process, it can cause fatigue, weight loss, and obstructive jaundice, as well as vague abdominal pain if affecting the pancreas, as seen in our patient .The diagnosis of IgG4-RD relies on clinical evaluation, laboratory testing, imaging, and histopathological findings. Serum IgG4 levels may or may not be present and are not required for the diagnosis of IgG4-RD, as can also be found to be normal in patients with IgG4-RD . Thus, hHowever, if IgG4 levels are detected in patients with IgG4-related pancreatitis, they are usually significantly elevated in comparison with pancreatic cancer. Although mild elevation of IgG4 serum levels can be seen in pancreatic cancer , patientRadiological studies such as CT/PET scans or MRI are essential in evaluating these mass-forming lesions based on the symptoms, although findings may not be as useful or help distinguish between pancreatic cancer and IgG4-related pancreatitis. The definitive diagnosis is still based on the formal biopsy with its characteristic histopathological findings that are identical in all of the involved organs. These include dense lymphoplasmacytic infiltrate with high levels of IgG4-producing plasma cells, storiform fibrosis, and obliterative phlebitis ,9.Open surgical biopsy is very invasive in obtaining a confirmative pathological specimen. The use of EUS-guided fine needle aspiration (FNA) can help evaluate a solid pancreatic mass, is less invasive, and can be useful in obtaining biopsy specimens for histopathological analysis . As seenIt is important to distinguish benign pancreatic masses from malignant cancer in the early phase as effective treatment can be administered as soon as possible because prognosis and treatment for both are quite different. Treatment for IgG4-RDs depends on the severity of symptom onset. The first line of treatment for IgG4-RD involves systemic glucocorticoids . SubsequPhysicians should be familiar with the clinical presentations and diagnostic criteria of IgG4-related autoimmune pancreatitis resulting in a mass-like lesion vs. pancreatic cancer. An awareness of the differences between these two diseases may avoid misdiagnosis, prevent any unnecessary operative interventions, and result in prompt treatment.


The core part of a hybrid truss bridge is the connection joint which combines the concrete chord and steel truss-web members. To study the mechanical behavior and failure mode of steel–concrete connection joints in a hybrid truss bridge, static model tests were carried out on two connection joints with the scale of 1:3 under the horizontal load which was provided by a loading jack mounted on the vertical reaction wall. The specimen design, experimental setup and testing procedure were introduced. In the experiment, the displacement, strain level, concrete crack and experimental phenomena were factually recorded. Compared with the previous study results, the experimental results in this study demonstrated that the connection joints had the excellent bearing capacity and deformability. The minimum ultimate load and displacement of the two connection joints were 5200 kN and 59.01 mm, respectively. Moreover, the connection joints exhibited multiple failure modes, including the fracture of gusset plates, the slippage of high-strength bolts, the local buckling of compressive splice plates, the fracture of tensile splice plates and concrete cracking. Additionally, the strain distribution of the steel–concrete connection joints followed certain rules. It is expected that the findings from this paper may provide a reference for the design and construction of steel–concrete connection joints in hybrid truss bridges. Steel–concrete composite bridges are widely used because they combine the advantages of prestressed concrete box girder bridges and steel truss bridges. A composite bridge with steel truss members instead of traditional concrete webs attracted the attention of researchers, which was also known as a hybrid truss bridge (HTB). HTBs have a lighter weight and smaller beam height than prestressed concrete box girder bridges, enhancing the bridge span. The Arbois Bridge , which wThe steel–concrete connection joint is considered the most important part of hybrid truss bridges, transferring the load between the concrete chord and steel truss web ,22,23,24In this paper, we sought to investigate the mechanical behavior and failure mode of steel–concrete connection joints. More specifically, this study aimed to determine the following specific research directions: (1) the ultimate bearing capacity and corresponding displacement of the proposed joints, (2) the typical failure mode of such joints, (3) the strain distribution rule of the main components, and (4) the key component of the steel–concrete connection joint to carry the external load. Therefore, we conducted static loading tests on two joint specimens with the scale of 1:3. Such experimental investigations of the proposed joint form may enrich the experimental data and provide an experimental reference for the design and construction of such joints in hybrid truss bridges.As shown in The static tests of the steel–concrete connection joints were carried out in the National Engineering Laboratory for Technology of Geological Disaster Prevention in Land Transportation, and the experimental setup is shown in Before the formal multi-step loading, pre-loading was performed to avoid assembly clearance. The loading step of 400 kN was adopted within 0–2000 kN. Then, when the horizontal load was in the range of 2000–3000 kN, the loading step was reduced to 200 kN. Finally, the loading step of 100 kN was applied until the end of the loading procedure. The laser displacement sensor (D1) was used for measuring the horizontal displacement in the loading direction. Furthermore, the strain gauges were installed to monitor the strain of the main components, including the concrete chord, gusset plates, PBL shear connectors, and the steel truss-web members, as shown in To precisely investigate the failure modes of the proposed connection joints, the experimental phenomena and data were recorded in detail. For Specimen 2, the first crack of the concrete chord emerged near the loading end at the load of 3400 kN, but this crack did not spread in the latter loading stages. Furthermore, the relative location malposition between the gusset plates and the splice plates with hand holes was also observed. Then, the local buckling of the compressive splice plates and the slippage of high-strength bolts were observed, as shown in Based on the experimental observation of two specimens, the order of the destruction course of the proposed connection joints was the local buckling of the compressive splice plates, the slippage of high-strength bolts, the cracking of the concrete chord, the fracture of the gusset plates and the tensile splice plates. Clearly, two specimens showed similar test phenomena and failure modes. Nonetheless, the damage degree of Specimen 2 was greater than that of Specimen 1, for example, the gusset plates of Specimen 2 were pulled to the point of total fracture. The difference in the experimental results between the two specimens is attributable to the fact that the first fracture of Specimen 1 dissipated part of the energy, making Specimen 1 unable to continue to bear a greater load, and also causing the damage degree to be less than that of Specimen 2. Compared with previous tests ,30,31,32Specimen 1 was selected as the narrative object to avoid redundancy in the following sections. The strain results of the concrete chord are set out in The strain results of the compressive truss web are displayed in Compared with the previous experimental results in In the experiments, Specimen 1 and Specimen 2 shared similar failure modes, such as the fracture of gusset plates, the slippage of high-strength bolts, the local buckling of compressive splice plates, the fracture of tensile splice plates and concrete cracking. In particular, the fracture of gusset plates, the slippage of high-strength bolts and concrete cracking were the most prominent failure modes. Moreover, it can be seen from the comparison of the failure modes between the proposed joints and the previous test specimens that the fracture of the gusset plates and the slippage of the high-strength bolts seemed to be two typical failure modes of such connection joints with high-strength bolts.The strain distribution of the steel–concrete connection joint followed certain rules. For the concrete chord, the strains decreased quickly in the loading direction. On the contrary, the strains increased in the vertical direction. For the gusset plates, in the loading direction, the strains reduced gradually from the middle to the edge. In the vertical direction, strains of the embedded gusset plate enhanced from the top to the bottom, but the strain variation tendency of the exposed gusset plate was not obvious. For the steel truss-web members, the areas near the inside of the angle between the tensile truss web and the compressive truss web had greater strain values than in other areas. For PBL shear connectors, the strain distribution patterns were in keeping with the patterns of the embedded gusset plates. The third-row PBL shear connectors resisted most of the load rather than the other two-row connectors.The exposed gusset plate was the key component of the steel–concrete connection joint to resist the load. The exposed gusset plate was at a higher strain level than the other members at the same loading step. Under the extreme condition, the exposed gusset plates of both specimens experienced a severe tensile fracture and were therefore unable to continue to bear the load. Furthermore, in the ultimate state, the fracture of the gusset plates prevented the specimen from continuing to bear the external load.In this investigation, the purpose was to fully study the mechanical behavior and failure mode of steel–concrete connection joints by static model tests, especially to determine the strain distribution rule of the main components and the typical failure mode of such joints. The following conclusions can be drawn based on the present study.In this study, we focused on the experimental investigation of steel–concrete connection joints. However, the numerical analysis is also important for a full understanding of mechanical behavior and failure mode of such joints. We will perform the numerical analysis in another article in detail.


Hypertensive disorders of pregnancy (HDP) are a group of morbid pregnancy complications, with preeclampsia (PE) being the most common subclassification among them. PE affects 2%–8% of pregnancies globally and threatens maternal and fetal health seriously. However, the only effective treatment of PE to date is the timely termination of pregnancy, albeit with increased perinatal risks. Hence, more emerging therapies for PE management are in urgent need. Originally introduced as the first-line therapy for type 2 diabetes mellitus, metformin (MET) has now been found in clinical trials to significantly reduce the incidence of gestational hypertension and PE in pregnant women with PE-related risks, including but not limited to pregestational diabetes mellitus, gestational diabetes mellitus, polycystic ovary syndrome, or obesity. Additionally, existing clinical data have preliminarily ensured the safety of taking MET during human pregnancies. Relevant lab studies have indicated that the underlying mechanism includes angiogenesis promotion, endothelial protection, anti-inflammatory effects, and particularly protective effects on trophoblast cells against the risk factors, which are beneficial to placental development. Together with its global availability, easy administration, and low cost, MET is expected to be a promising option for the prevention and treatment of PE. Nevertheless, there are still some limitations in current studies, and the design of the relevant research scheme is supposed to be further improved in the future. Herein, we summarize the relevant clinical and experimental researches to discuss the rationale, safety, and feasibility of MET for the management of HDP. At the end of the article, gaps in current researches are proposed. Concretely, experimental MET concentration and PE models should be chosen cautiously. Besides, the clinical trial protocol should be further optimized to evaluate the reduction in the prevalence of PE as a primary endpoint. All of those evidence gaps may be of guiding significance to improve the design of relevant experiments and clinical trials in the future. Hypertensive disorders of pregnancy (HDP), a group of morbid pregnancy complications, include gestational hypertension, preeclampsia (PE), eclampsia, and chronic hypertension in pregnancy . Among tEmerging progress in understanding the preeclamptic pathogenesis leads to an interest in more novel agents to prevent and treat PE, among which metformin (MET) has preliminarily proved to be safe during human pregnancies and effective in the prevention or treatment of PE. Well known as the first-line therapy for type 2 diabetes mellitus , MET, inPreviously, the classic two-stage theory of PE proposed by Redman has been widely accepted; that is, the first stage mainly comprises poor placentation, resulting in the excessive release of antiangiogenic and proinflammatory factors into the maternal circulation, which is the reason for multiple clinical manifestations of the disease in the second stage . With a p = 0.029) and maternal weight gain (p < 0.001) in the MET-treated group compared to the insulin-treated group (p = 0.02). Likewise, average weight gains after enrollment were much lower in the MET group  with a daily dose of 1.0 g and the control group (n = 145). All pregnant women entered the study with a gestational age of less than 20 weeks and were followed throughout the prenatal period. In the results, the incidence of PE was lower in the MET group compared to the control . Similarly, in a double-blind, placebo-controlled trial, the researchers randomly assigned nondiabetic women who had a BMI of more than 35 kg/m2 to receive MET or placebo (225 women in each group) from 12 to 18 weeks of gestation until delivery. MET was initiated at a daily dose of 1.0 g, and the dose was increased by 0.5 g per week to the maximum daily dose of 3.0 g. The incidence of PE was lower in the MET group , as was the median maternal gestational weight gain (2 and examined the effect of MET at a dose of 0.5 g daily (increasing to a maximum of 2.5 g) or matched placebo per day from between 12 and 16 weeks of gestation until delivery. Although the study showed no significant differences between the MET and placebo groups in the rate of PE, the inflammatory markers IL-6 and CRP were both significantly lower in women given MET (Increased prepregnancy body mass index (BMI) is associated with increased risks of PE, particularly those women with prepregnancy BMI >30 kg/m2 . In a ra2 , obese pght gain . Anotheriven MET . The METiven MET . Differep = 0.005), GDM, and miscarriage and a nonsignificant decrease in PE , compared to the control group. Similarly, another randomized, placebo-controlled, double-blind, multicentre clinical trial also showed that MET induced a nonsignificant decrease in the incidence of PE  in pregnant women with PCOS, compared to the placebo-treated group , biceps skinfolds , and subscapular skinfolds  at age 2, than those in the insulin group. However, the total fat mass and percentage of body fat assessed by bioimpedance showed no difference (p = 0.049), waist  and arm  circumference, and the ratio of waist to height   . Those r = 0.02) . It is s = 0.02) . Notably = 0.02) . HoweverCurrently, most studies suggest that metformin has no negative effects on perinatal outcomes and newborns, and the American College of Obstetricians and Gynecologists (ACOG) indicated that MET could be reasonably used in pregnancy . HoweverAccording to the above-mentioned Redman’s theories, defective placentation plays a crucial role in the pathogenesis of PE, with trophoblast dysfunction regarded as the major trigger . Due to As mentioned in the preeclamptic pathophysiology, imbalanced secretion of angiogenic and antiangiogenic factors from the ischemic placenta results in dysplasia of the placental vessel network, systematic endothelial dysfunction, disturbance of vasomotion, and impaired ability to endothelial repair . AccordiVEGF, a familiar angiogenic factor in endothelial cells, not only plays an essential role in placental angiogenesis and vascular recasting but also participates in infiltration, proliferation, and differentiation of trophoblast cells . VEGF issFlt-1, a representative type of antiangiogenic factor secreted primarily by syncytiotrophoblasts into maternal circulation , is typiThe healthy endothelium plays a vital role in maintaining vascular integrity, coagulation and fibrinolysis function, leukocyte and platelet adherence, and inflammatory responses . Endothel-arginine to l-citrulline by endothelial nitric oxide synthase (eNOS) on villous endothelial cells. NO is frequently involved in angiogenesis and spiral artery remodeling, achieving the local drop of vascular resistance to preferentially shunt blood to the uteroplacental unit (2S)  treatment reduced symptoms of PE and returned sFlt-1 levels to normal  is followed by the increase of H2S tissue concentrations in the mouse brain, heart, kidney, and liver  , the pro(2S) (2S  and decr(2S)  are endothelial cell precursors  that conPE has been described as a highly inflammatory state, with low levels of anti-inflammatory molecules and high levels of proinflammatory factors and the activation of various inflammation-related signaling pathways in both the local fetoplacental unit and maternal plasma . FurtherEven though the preeclamptic pathophysiology has been studied for almost 200 years, it remains poorly understood, thus limiting the development of preventive and therapeutic interventions of PE. Due to the complexity and diversity of the etiopathogenesis of PE, more emerging therapies should be proposed to prevent and ameliorate this health hazard. Dubbed as the “wonder drug” of the current time, MET is reported to reduce the incidence of gestational hypertension and PE according to the clinical trial results available. In terms of the underlying mechanisms, MET itself has significant effects on the improvements of hyperglycemia, dyslipidemia, obesity, and insulin resistance, which are all verified as PE-related high risks. Furthermore, there are other plausible explanations of the preventive and therapeutic effects of MET on PE, including the promotion of angiogenesis, endothelial protection, anti-inflammatory effects, and particularly the protective effects on trophoblasts against risk factors, which are beneficial to placental development . While tConcretely, one problem is that the dosages of MET differ from each other in several reported experiments, leading to contradictory effects on the cellular bioactivities, such as the protective benefits on trophoblast with low-dose MET and the impairment with high dose. Thus the dose of MET in different experiments is supposed to be limited within a certain range. To tailor the dose of MET in future experiments, a proper understanding of the pharmacokinetics of MET during pregnancy, especially the placental MET concentration, is essential. In nonpregnant patients, the plasma concentration range of MET after liver metabolism is usually 10–40 μM with the oral administration of 0.5–2.5 g/d . HoweverAnother unsolved problem is whether the MET is effective for every subtype of PE. At present, scholars have reached a consensus that PE is more of a syndrome with various subtypes rather than a single disease. Consequently, the possible prevention and treatment exerted by MET on PE need to be further verified in a variety of preeclamptic animal models; this could be helpful in further defining the subclassifications and subtypes of PE for which MET may be applicable . Similar+0 to 31+6 weeks. Participants will randomly receive either 3 g of MET or placebo daily; the time from randomization until delivery and the maternal and neonatal outcomes will be exploratory. The PI2 clinical trial can fill the current research gap and have a guiding significance in the design of more and larger prospective clinical studies to verify the efficacy of MET in the treatment of PE in the future. The changes of key molecular indicators in serum, placenta, umbilical cord, and other clinical specimens should be simultaneously detected. Moreover, clarifying the relationship between MET and HDP with the combination of the basic experiments and clinical data will be very helpful.Last but not least, there is an urgent need for more clinical trials related to the preventive and therapeutic effects of MET on PE, particularly the clinical trials designed to evaluate the reduction in the prevalence of PE as a primary endpoint. Currently, PE is a secondary outcome in most existing studies, suggesting that MET is temporarily not recommended for the clinical prevention of PE, referring to the 2019 ACOG notice . BesidesIn summary, MET may be a reasonable alternative approach to prevent and treat HDP, but it cannot be ignored that there are still unsettled questions. We are looking forward to more related studies to be reported in the future, which can bring MET from experimental research to clinical use.YZ and XL drafted the manuscript by reviewing the literature. LY participated in the discussion and prepared the chart. The corresponding author LZ guided the formation of the entire manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Nature Science Foundation of China (No. 81741002 to Li Zou and No. 81703242 to Qingqing Luo).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.


V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of  Vibrio parahaemolyticus, we resolved that distinct subpopulations of bacteria exist in the VBNC state and these have different resuscitation potentials and distinct proteomic profiles. We also discovered that deletion of the gene encoding the enzyme lactate dehydrogenase (lldD) triggered the cells to enter the VBNC state, and adding lactate to VBNC cells extended their resuscitation potential window. The ability for bacteria to survive in the VBNC state might be linked to their ability to overcome oxidative stress.Members of the Proteobacteria are reported to adopt a survival strategy and enter a viable but non culturable (VBNC) state, when exposed to stressful or non-permissible growth conditions. This is a characteristic employed widely in the natural environment in order for bacteria to survive harsh environmental conditions over a long period. In spite of the importance of the VBNC state in microbiology, we know little about the molecular makeup of VBNC cells. In this study, using the model organism  Vibrio species and V. parahaemolyticus is a good model organism for studying VBNC cells for a number of reasons  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Andreas J BaumlerAssociate EditorPLOS PathogensKarla SatchellSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The paper submitted by Wagley et al. present interesting findings on VBNC in Vibrio vilnificus (Vv). The authors shows that 2 subpopulation of VBNC cells exits: P1, composed of composed of small coccid cells, and P2, composed of larger, elongated or coccoid cells. The authors further characterize these 2 subpopulation by testing infectivity in Galleria and through proteomic analysis. The identification of these subpopulation is novel and interesting. The proteomic study is very useful and novel. The identification of lldD as a key protein for resuscitation is very interesting and will lead to new studies on the energetics of resuscitation.Reviewer #2: In the manuscript titled “Bacterial dormancy: a subpopulation of viable but non-culturablecells demonstrates better fitness for revival.”, Wagley et al., investigated the populationheterogeneity of the viable but non culturable (VBNC) state of V. parahaemolyticus. Using FACS and IFC, they thoroughly and convincingly showed that distinct populations exist with VBNC population. The authors went on to explore the differences between these populations and the starting microcosm using proteomic analysis, showing that lldD was one of the proteins that were differentially regulated. They then showed that lldD played a role in regulating the VBNC state and the metabolic product of lldD was able to rescue the loss of lldD. Overall, the study is carefully designed, compelling and identified new components in regulating bacterial dormancy.Reviewer #3: In this study, Wagley et al., investigated the mechanism by which different populations of V. parahaemolyticus enter the VBNC state using imaging flow cytometry (IFC), fluorescence activated cell sorting (FACS), and proteomics. The authors identify two distinct bacterial VBNC subpopulations (“P1” and “P2”) based on size and florescence intensity and find that these different abilities for revival. The authors also report altered virulence properties of these subpopulations in a larval model of infection. Proteomic analysis of these subpopulations identified lactate dehydrogenase as a bacterial factor that contributes to the VBNC state and addition of exogenous lactate was able to promote VBNC resuscitation.Overall, the experiments seem generally well performed and the data are interesting. However, further genetic analysis to investigate the mechanism by which lactate dehydrogenase contributes to VBNC state formation and improved experimental design with their infection model would solidify these findings.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: 1. For the flow cytometry please present the live and dead (boiled) controls. This would help the reader understand what was done, and how the gates were set. In addition, the result section should be revised. My understanding is that PI was used to test for viability, which was detected by Y610/20. P2 is therefore less viable than P1, but the author do not consider P2 as dead. Again, the controls (live and dead) would help the reader interpret this important dataset. Why not used Syto 9 signal instead of SSC? Was the flow cytometry done on 14 days stored culture or 5 day old culture .2. I am not convinced that the seafood samples contained Vv. I presume that the shrimps harbour a variety of microorganisms that could results in similar images. How does the author confirm that the cells the see are Vv? Microscopic morphology cannot be used to identify microorganisms, unless some kind of specific labeling was used (such as antibodies but this does not seem to be the case).3. In general, I find that the figures are of poor quality  and the legends could be significantly improved. I cannot read protein label on the proteome figures. The authors needs to describe what the experiment is and provide sufficient details for the reader to understand the figure without having to constantly refer to different part of the text.4. Several section of the results needs to be rewritten. The text is disjointed, the panels are not presented in the same order in the text and in the Figure and some panels are not presented at all  and . It ws very difficult for me to understand what was done. I suggest that the author present the experiments, and the important details, before presenting the results of it. In particular, the microcosm should be presented  as well as the general method to test resuscitation (PBS). I find the use of T12 and T50 confusing (as it seems to relate to time after the population is composed of VBNC cells only) rather than days. Also Fig 1B present data as VBNC+number of days. I presume VBNC is a specific time point but this is unclear.5. What is Fig. 4B?Reviewer #2: 1. Can the author provide possible explanation to why P2 has high PI signal but intact membrane?2. It is unclear whether the presence of distinct VNBC populations is specific to the condition tested in the manuscript. The authors should test whether distinct VNBC populations are also present when VNBC state is induced by a different stimulus, in order to support their claim (line 484-486).3. The authors suggested that VBNC subpopulations cannot be resuscitated inside G. mellonella, and that the larvae death was caused by the virulent compounds associated with whole microcosm. A direct way to test this hypothesis will be to infect the larvae with FACS-sorted P1 or P2 population without resuscitation.4. While it is definitely interesting that the addition of pyruvate can resuscitate VBNC, the underlying mechanisms remains elusive. It will be interesting to test the proposed antioxidant mechanism by assaying whether other antioxidant compounds can rescue the ldhA mutant.Reviewer #3: -In Figure 4, the authors state that they infected larvae with approximately 10^7 CFU of T0 cells and approximately 10^5 VBNC cells. Later they state that they infected with approximately 10^4 CFU of P1 or P2 sorted cells for infection. It’s difficult to make any conclusions on VBNC virulence given that the authors are using 2-3 logs less VBNC bacteria for infection. The authors should repeat the experiments by normalizing the VBNC cells up to 10^7 or reduce the inoculum of T0 to match the dose being used for VBNC/P1/P2 cells in this figure.-In Figure 4, the authors state that, “V. parahaemolyticus VBNC cells in subpopulation P2, when resuscitated, revert back to virulence capabilities of the log phase bacteria.” However, the authors do not demonstrate directly that virulence is responsible for this observation and it’s not clear whether increased virulence or if growth alone is responsible for reduced larval survival. The authors should address this by repeating the post-resuscitation P2 infection experiment using a non-virulent bacterial mutant and showing that larvae survival is rescued.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: Line 27: "displayed different abilities", please be more explicit. Which one is better?Author summary should be revised. It reads like it was written at the last minute, without revision.Line 53-65: the first paragraph of the intro needs reference for each statement.L230: different number of "event" were sorted from P1 and P2, could that affect the results of the infection assay?L242 and 249: I guess the author means Fig 4A, not Fig 4?L 259-263: this was hard to understand, the text should be revised. I don't see the link with the other part of this paragraph.L337: what does "more protein made redundant" means?Fig 6C is unclear. Are does proteins shared between P1 and P2?Fig 8B: the authors always show the curve until the population becomes all VBNC. Why not do the same for this?L424-425: "For the present studies we have consistently used fresh cultured bacteria." is in contradiction with the methods for the microcosm that mentioned 5 day old culture and 14 days stored culture.L468: Fig 4B?Reviewer #2: 1. In the intro, the transition between using transcriptomic and proteomic is not well supported. If transcriptomics cannot identify unannotated genes that are important for VBNC, it is unlikely that proteomics can.2. The low image quality of multiple figures significantly impacts the readability of the manuscript. Some figures were unreadable.3. The authors need to provide experimental details as what were considered as “stained as live and metabolically active” in the main text.4. Can the author provide possible explanation why P2 has high PI signal but intact membrane?5. How did the author determine the bacteria in the seafood were V. parahaemolyticus?6. What percentage of V. parahaemolyticus population in the seafood samples were VBNC? If all detected V. parahaemolyticus were VBNC cells, and these cells cannot be resuscitated in the host, does entering VBNC state mean a pathogenesis dead-end for V. parahaemolyticus? The authors should expand a bit on this in the discussion.Reviewer #3: -In Figure 1, I’m surprised by the low amount of variation  and apparent high level of reproducibility given the complexity of these experiments. Given the number of unknown factors that may contribute to the findings reported in Fig. 1, the authors should state explicitly how many biological replicates were performed to produce these data (this is not mentioned in the Results or Methods). If it wasn't already done, these experiments should be repeated independently at least 3 times to solidify the conclusions made in Figure 1, which sets the groundwork for the rest of the manuscript.-In Figure 3, the images should be labeled within the figure so that the reader doesn’t have to rely exclusively on the figure legend.-It would be interesting to analyze the cell morphology of the ΔlldD mutant-In Figure 8C, the authors should repeat this experiment (addition of sodium lactate) using the ΔlldD mutant.-Does inactivation of TCA cycle metabolism gene(s) also lead to cells entering VBNC state significantly earlier (like ΔlldD mutant)? Can you resuscitate VBNC stage of cells with other metabolites such as glycerol?-Does a ΔlldD mutant have altered virulence in the larval infection model? The authors should address this question experimentally.**********what does this mean?). 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This paper proposes a channel state information (CSI)-based prediction method of a human flow that includes activity. The objective of the paper is to predict a human flow in an outdoor road. This human flow prediction is useful for the prediction of the number of passing people and their activity without privacy issues as a result of the absence of any camera systems. In this paper, we assume seven types of activities: one, two, and three people walking; one, two, and three people running; and one person cycling. Since the CSI can effectively express the effect of multipath fading in wireless signals, we expected the CSI to predict the various activities. In our proposed method, the amplitude and phase components are extracted from the measured CSI. The feature values for machine learning are the mean and variance of the maximum eigenvalue derived from the auto-correlation matrix and variance–covariance matrix composed of the amplitude or phase components and the passing time of flow. Using these feature values, we evaluated the prediction accuracy by leave-one-out cross-validation with a linear support vector machine (SVM). As a result, the proposed method achieved the maximum prediction accuracy of 100% for each direction and 99.5% for two directions. Recently, expectations for sensing methods without any cameras have increased. In September 2019, the IEEE802.11 Committee established the project of channel state information (CSI)-based sensing. As of March 2020, the project name is SENS TIG [There are alternative methods of sensing, such as the use of a passive infrared ray (PIR) sensor or light detection and ranging (LiDAR) sensor. PIR sensors cover a low range, require multiple sensors to achieve reliable detection, and the detection accuracy is low for static persons . For exaThe IEEE802.11n/ac/ax standard applies the orthogonal frequency division multiplex (OFDM) and multiple input and multiple output (MIMO) technologies. The OFDM is a multi-carrier transmission using multiple subcarriers concerning the physical layer information. Since the subcarrier characteristic is different because of the effect of multipath propagation, the characteristic varies with the location and shape of objects. Thus, the information about the frequency domain is obtained by OFDM transmission. MIMO uses multiple antennas in the transmitter and receiver. Since the antenna location is different for each antenna, the effect of multipath propagation is different for each transmitting and receiving antenna pair. Thus, the information about the space domain is obtained by MIMO transmission. The CSI consists of frequency and space information, expresses the effect of multipath propagation, and corresponds to the space state between transmitting and receiving antennas. To date, CSI has been used for various studies such as human movement detection, human behavior classification, location estimation, object identification, human counting in a room, and human counting in a passageway ,17,18,19The objective of the paper is a prediction of human flow including various activities: one, two, and three people walking; one, two, and three people running; and one person cycling. In the example shown in In this study, a human flow that includes activity was classified by a support vector machine (SVM), and we clarify the feature values to achieve the best accuracy for the classification.This paper is the extended version of the conference paper . We condCSI can effectively express the effect of multipath propagation and multipath fading in wireless signals; thus, CSI-based estimation is suitable for environments where diffraction and scattering occur. Such situations are indoor, and many studies have been conducted indoors rather than outdoors. To date, CSI has been used for various studies such as human movement detection, human behavior classification, location estimation, object identification, human counting in a room, and human counting in a passageway ,17,18,19The related studies can be classified into non-moving and moving objects. In the case of non-moving objects, the multipath propagation is different depending on the location or the shape of objects where they exist between antennas. This characteristic is used for location estimation and object identification ,7,8,9,10Since the objective of the paper is a prediction of human flow, our proposed system was categorized into moving objects. Qian, K. et al. proposed the human detection method concerning human movement in an indoor passageway . This meTx and Rx be transmitting and receiving antenna numbers, respectively, and let TxN and RxN be the number of transmitting and receiving antennas, respectively. The CSI matrix can be expressed as follows:k denotes the received frame number. Linux 802.11n CSI Tool can obtain CSI on 30 subcarriers. Thus, let n be a subcarrier number, and each element of the matrix is expressed asn contains the amplitude and phase components and can be expressed as follows:Current Wi-Fi systems, such as IEEE802.11n/ac/ax, have functions of the OFDM and MIMO. Since the OFDM transmission uses multiple subcarriers, the effects of diffraction and scattering in the wireless signal are reflected in each subcarrier fluctuation. MIMO transmission makes multiple transmission paths composed of transmission and reception antenna pairs, and the CSI is different for each path. In Linux 802.11n CSI Tool, the CSI is obtained whenever the receiver receives the frame . Let Tx The phase in each subcarrier isn contains the effect of the timing and phase offset at the receiver. To eliminate the effect, this paper applies the following sanitized phase [The measured phase ed phase ,12 insteThe purpose of the proposal is to predict a human flow, including activities, such as one person or two people walking or running, between transmitting and receiving antennas. The proposed system is composed of mainly three parts, as shown in For the passage detection of human flow, the CSI amplitude is used, but the amplitude includes high-frequency noise ,15. The When a human moves across transmitting and receiving antennas, multipath fading occurs and the amplitude fluctuates between received frames. The correlation of amplitude among the frames decreases with the increase of fluctuation. In short, the amplitude characteristics in each subcarrier vary frame-by-frame. Focusing on the relative amplitude between CSI in frames, the amplitude is normalized as follows:window size using Equation (7). The window size corresponds to the number of frames. In the proposed system, the window size was set to 10. Let Next, we calculated the maximum eigenvalue of the auto-correlation matrix with window order whose elements are all one. In this case, the maximum eigenvalue in Equation (7) is one.If a human does not move across transmitting and receiving antennas, the correlation is one. The matrix Tx1–Rx1 path in Tx2–Rx3 path is very narrow and cannot be appropriately detected. To address this problem, we have to apply the final passing section instead of the primary passing section.In the passage detection, we assume that only one human flow is passed during observation. We describe the explanation of the detection method using In this system, the feature values are created from the amplitude and phase components of CSI. The lengths of the final passing section in each transmission path are also used as the feature values. As with the CSI amplitude, the maximum eigenvalue of phase, In addition to the two types of eigenvalues, (10) and  are usedk is in the final passing section in each transmission path. The lengths of the final passing section in each transmission path are also used as feature values. Specifically, since 3 × 3 MIMO has nine transmission paths, the maximum number of feature values is 4 eigenvalues  function to avoid frame collisions. That is, in the environment where the effect of interference is significant, the transmitter cannot send frames using this function. Since the CSI is obtained whenever the receiver receives frames in Linux 802.11n CSI Tool, there is no problem in the environment where the receiver can receive the frames.The human activity of flow comprises seven types: one, two, and three people walking; one, two, and, three people running; and one person cycling. People walked or ran, keeping a certain distance from the person in front. As shown in Prediction performance was evaluated by leave-one-out cross-validation. We evaluated the prediction performance for each direction and two directions, TR and RT directions, as shown in At first, we analyzed the performance when only the lengths of the final passing section were used. The prediction accuracies for TR and RT directions are 0.733 and 0.771, respectively. The definition of prediction accuracy is the ratio of the number of correct predictions and the total number of predictions. In the case of TR direction, the number of correct predictions was 77, and the total number of predictions was 15 × 7 = 105. Thus, the prediction accuracy is 77/105 = 0.733. In the case of RT direction, the number of correct predictions was 81, and the total number of predictions was 15 × 7 = 105. Thus, the prediction accuracy is 81/105 = 0.771. The confusion matrix for TR and RT directions is shown in Next, we analyzed the performance when only the CSI was used. The prediction accuracy is shown in We analyze the performance when using the length of the final passing section and CSI. The prediction accuracy is shown in Finally, we analyzed the performance for two directions , when using the length of the final passing section and CSI. The prediction accuracy is shown in Summarizing the above, we clarified that the length of the final passing section and the CSI have to be used as feature values to achieve good accuracy. Considering the computational complexity, we also clarified that the feature values to achieve the best accuracy are mean of amplitude, We compare our system with the conventional method in terms of accuracy and the number of feature values. Reference  conducteWe applied these feature values to our system, and the prediction accuracy is shown in This paper proposes the human flow prediction method. To predict the activity of human flow, we used nine types of feature values derived from the CSI amplitude, the CSI phase, and the passing time across transmitting and receiving antennas. The CSI characteristics can express the effects of diffraction and scattering in the wireless signals, and the passing time depends on the length of human flow. At first, the proposed system detects that the human flow moves across transmitting and receiving antennas. From the passage detection, the passing time between antennas is obtained as the feature values. While the human flow is moving across between antennas, the CSI amplitude and phase fluctuate. In this passing section, the feature values are obtained from the mean and variance of the maximum eigenvalue in the auto-correlation matrix and variance–covariance matrix composed of the CSI amplitude and phase. In the experiment, we set seven types of activities: one, two, and three people walking; one, two, and three people with running; and one person cycling. The maximum prediction accuracy was 100% for each direction and 0.995 for two directions. The number of feature values to achieve the best accuracy in our system is decidedly smaller than that of the conventional method.The contribution points of this paper are as follows. First, the antenna spacing in the transmitter and receiver is physically increased to extend the scanning area of CSI. The effect of the wide antenna spacing makes it easier to acquire exactly the activity and the passing time of the human flow. Second, the feature values to achieve the best accuracy are clarified. Considering the computational complexity, it is clarified that the feature values to achieve the best accuracy are the combination of the passing time and the mean of amplitude derived from the maximum eigenvalue of the auto-correlation matrix and the variance–covariance matrix composed of the CSI amplitude.A challenging issue for future research is the examination of antenna setting methods. In this paper, the antenna spacing was set to 1.5 m, and the scanning area for CSI was extended. We are planning to evaluate the prediction accuracy using different antenna spacing. The optimum sampling frequency will also be discussed in our future research.