Flexible electronic devices have gained significant interest due to their different potential applications. Herein, we report highly flexible, stretchable, and sensitive sensors made of sprayed CNT layer, sandwiched between two polymer layers. A facile fabrication process was employed in which the CNT solution was directly sprayed onto a patterned bottom polymer layer, above which a second polymer layer was casted to get a sandwiched composite structure. Varying amounts of CNT solution  were sprayed to get conductive CNT layers of different thicknesses/densities. The physical characteristics of the conductive CNT layers were studied through SEM and optical images. The starting electrical resistance values (without strain) as well as the changes in electrical resistance against human body motions were monitored. The synthesized samples exhibited good response against finger and wrist bending. The conductivity of the samples increased with increase of CNT solution volume while the sensitivity followed the inverse relation, suggesting that the sensors with controlled sensitivity could be fabricated for targeted strain ranges using the proposed method. Wearable electronic devices are getting increasing attention due to their several applications ,2,3. ForTM silicone polymer, which served as a dielectric layer.Recently, efforts were made to deposit conductive fillers onto polymer substrate using hot press , inkjet In this work, we report a facile fabrication process in which CNT solution was directly sprayed onto patterned polymer substrate, using a simple spray gun. Since quantity of CNTs is of vital importance in terms of electrical conductivity and sensitivity, varying amounts of CNT solution were sprayed to obtain conductive CNT layers with different thicknesses/densities. The deposited CNT layers were characterized by electrical resistance measurements and SEM/optical image analyses. Following this, the sensors were tested for detecting different human gestures.TM 00-30), multi-walled CNTs, and isopropyl alcohol (IPA) were procured from Smooth-on Inc. , Hyosung Inc. , and Samchun Chemicals , respectively. The tensile strength, specific gravity, and elongation at the break of polymer were 200 psi, 1.07, and 900%, respectively. The length, diameter, and specific gravity of CNTs were 10 μm, 12–40 nm, and 1.32, respectively.The silicone polymer  and was kept at 70 °C for 2 h. In parallel, the CNT solution was prepared by adding 0.05 g CNTs in 100 g IPA. The solution was sonicated for 1 h, followed by 1 h of stirring at 300 rpm. After 2 h curing of the bottom polymer layer, the spray areas were specified (3 × 50 mm2 for each CNT strip) by applying PI tape. An air sprayer, with an attached pressure gauge to control the spraying pressure (1 bar), was used. The glass plate was kept on a hot plate at 110 °C for rapid evaporation of IPA, which was essential for getting a uniform layer of CNTs. Five types of samples  were prepared by using 10, 25, 40, 70, or 100 mL of CNT solution, respectively. After spraying, the samples were kept at 80 °C for 30 min to ensure complete evaporation of IPA. Then, the electrodes were made using copper tape, followed by deposition of the top polymer layer to get a sandwiched structure. Lastly, individual samples were obtained by cutting with a paper cutter.The fabrication steps are shown in Multiple image analysis techniques  were employed to visually examine the CNT layers. The electrical resistance values (non-strained) of three replicas of each sample type were recorded through a digital multimeter  to investigate the connectivity of CNTs in the deposited layers. Following this, the efficiency of samples for detecting several human joint movements was explored to demonstrate the potential of as-synthesized samples to be used in wearable devices.The SEM micrographs and optical images are shown in The resistance values of the non-strained samples were recorded. Sample S10 showed a highly unstable resistance of about 21 MΩ. Moreover, it often went out of the range of the multimeter showing a complete loss of electrical path, resulting from the poor connectivity of CNTs. Hence, sample S10 was excluded from further analysis. As the spraying volume increased to 25 mL, a sudden decrease in resistance was observed. Samples S25, S40, S70, and S100 showed about 704, 41, 12, and 4 kΩ resistance values, respectively. This suggests that the CNTs were well-connected in the layer, ensuring a stable electrical path. These findings are compatible with the image analysis results. The thickness of the conductive layers and their voids can be associated with the observed resistance values. Based on these observations, it could be said that the connectivity of CNTs in the layer could be controlled by using a specific amount of spraying solution in this facile fabrication process, to achieve the targeted sensitivity and stability of the sensor.Lastly, the samples were tested for detecting human joint movements. Specifically, finger and wrist bending were monitored by attaching the samples on index finger and wrist, respectively. The response of the samples in form of changes in resistance against finger and wrist bending are shown in The sensors also showed a good response against wrist bending. Since the bend angle in wrist bending (~60°) was less than the finger bending (>90°), the change in resistance was normally lower than the change against the finger bending . SimilarIn summary, the efficiency of the facile fabrication process was demonstrated. The conductive CNT layers were directly deposited onto the polymer. Using the proposed method, increasing amounts of CNTs could be easily spray-deposited, while avoiding issues like the non-uniform dispersion of CNTs in polymers, stiffening of the polymer itself, etc. The synthesized samples showed good sensing capabilities against different human motions. Furthermore, sensitivity of the samples was found to be dependent on the amount of CNT solution used. Hence, a specific amount of CNT solution could be used to achieve the desired sensitivity to target specific human motions.


Type 2 diabetes mellitus is one of the most common chronic diseases, which endangers peoples health and life qualities. Sodium-Glucose Transporter 2 (SGLT2) inhibitors have been widely recognized since their clinical application in blood glucose control. While, dyslipidemia caused by SGLT2 inhibitors has been identified that affected the prognosis of this disease.I2 test will be used to assess heterogeneity between studies. The robustness of meta-analysis results will be determined by sensitivity analysis. It will be assessed that evidence quality of the outcomes on the GRADE.We will retrieve 8 databases including English and Chinese. After multiple screening, all randomized controlled trials (RCTs) related to SGLT2 inhibitors will be included by the 2 authors and data will be extracted. After completion of the risk of bias assessment, we will use these effect values including risk ratio (RR), weighted mean difference (WMD) and 95% confidence interval (CI) to conduct data analysis. Chi-Squared test and The results of our research will be published in a peer-reviewed journal.The purpose of this systematic review and meta-analysis is to evaluate the association and degree of association between different doses of SGLT2 inhibitors and changes on blood lipid levels in patients with type 2 diabetes mellitus, in order to provide a reliable basis for clinical medication.INPLASY202040201. People with type 2 diabetes mellitus often suffer from dyslipidemia, characterized by elevated triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) and decreased high-density lipoprotein cholesterol (HDL-C), which are risk factors for atherosclerotic cardiovascular disease (CVD).,7 However, one of the adverse reactions of SGLT2 inhibitors is an increase in LDL-C and HDL-C levels, which may increase the risk of cardiovascular disease. EMPA-REG OUTCOME ClinicalTrial,9 has shown that empagliflozin, used to treat type 2 diabetes mellitus, was associated with a mild increase in LDL-C and HDL-C in patients. However, phase 3 trials showed that HDL-C concentrations were significantly higher than at baseline after 3 months of treatment with empagliflozin, but there was no significant difference in total cholesterol, LDL-C, and triglyceride concentrations. The outcome analysis of another study was similar. In the CANVAS Program,,13 similar to previous results, higher levels of HDL-C and LDL-C were observed in the canagliflozin group compared to placebo. A study of dapagliflozin found that it increased HDL cholesterol without significantly affecting LDL cholesterol. Results of the meta-analysis,16 of SGLT2 inhibitors treatment for type 2 diabetes mellitus confirmed that SGLT2 inhibitors, including empagliflozin and dapagliflozin, could increase the levels of LDL and HDL cholesterol.SGLT-2 inhibitors, which are widely applied in the management of type 2 diabetes mellitus, have shown favorable effects on glycemic control. SGLT2 inhibitors reduce blood glucose via inhibiting the reabsorption of glucose by the proximal renal tubules, resulting in osmotic diuresis and glycosuria, and its mechanism of action is insulin-independent.These findings are not entirely consistent, although some meta-analyses have shown that SGLT2 inhibitors are associated with an increase in HDL and LDL cholesterol, the latest studies are not adopted and the evidence of differences in SGLT2 inhibitor dosages is lacking. Therefore, it is worth exploring whether the effect of SGLT2 inhibitors on blood lipid levels in type 2 diabetes mellitus patients is related to the dosages of SGLT2 inhibitors and the degree of correlation.22.1https://inplasy.com/, with a registration number INPLASY202040201. Under the statement guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P), the current meta-analysis and systematic review will be performed rigorously. We will modify and update the details, which of adjustments made during the course of study, in the final report of INPLASY.According to the registration items prompt of International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY), we registered a protocol for systematic review and meta-analysis at 2.22.2.1All clinical randomized controlled trials on humans of SGLT2 inhibitors for the treatment of type 2 diabetes mellitus, both published and unpublished, will be collected in our study.2.2.2Participants diagnosed as type 2 diabetes mellitus aged ≥18 years will be included in the study, irrespective of race and sex, severity of illness, and other factors.2.2.3Trails where therapeutic agents are SGLT2 inhibitors  and a minimum study duration of 12 weeks are eligible. Controls contain placebo and other hypoglycemic drugs, such as metformin, sulfonylureas (SUs), ningestedglinide, thiazolidinediones (TZDs), α-glucosidase inhibitors (AGI), dipeptidyl peptidase-IV (DPP-IV) inhibitors, glucagon-like peptide 1 (GLP-1) receptor agonist and insulin. The dosages of the SGLT2 inhibitors and lipid levels  at baseline and after treatment were reported.2.3The related trails meeting the following criteria will be excluded:1.nonrandomized, cohort, case-control, descriptive, animal or laboratory studies;2.type 1 diabetes mellitus;3.aged<18 years old;4.pregnancy or lactation period;5.the study duration less than 12 weeks;6.the dosages of SGLT2 inhibitors and lipid levels at baseline and after treatment can not be obtained.2.42.4.1Lipid changes from baseline in different SGLT2 inhibitors dosage groups and control groups will be tested. We will investigate whether different categories or dosages of SGLT2 inhibitors have different effects on lipid levels.2.4.2The intensity of association between SGLT2 inhibitors dosage and lipid levels change.2.5Relevant studies will be identified by a literature search of PubMed, EMBASE, the Web of Science, the Cochrane Library, Chinese National Knowledge Infrastructure (CNKI), Chinese Biological Medical literature Database (CBM), Chinese VIP Information (VIP) and Wan Fang Database from database inception to March 31, 2020. This search will be divided into 3 conceptual groups. One group includes the term used to describe a “sodium-glucose transporter 2 inhibitor,” another encompasses the terminology with regard to “type 2 diabetes mellitus,” and the third contains a “randomized controlled trial”. We will retrieve Medical Subject Headings (MeSH) and equivalent control terms and keywords in all databases aforementioned. Table http://www.clinicaltrials.gov). There is no limit to study language.Furthermore, completed but yet unpublished studies with the drugs specified above will be searched at the clinical trials website , study characteristics , participants characteristics , intervening characteristics , curative effects and outcomes data. Two authors independently performed pre-extraction the files after prior training in relevant data extraction. On the basis of pre-extraction, the shortcomings of extraction table will be found and corrected. All included studies are going to be extracted according to the final data extraction table. If sufficient data information is not available in the literature, more detailed data will be obtained by contacting the corresponding author via email or telephone. The 2 authors shall resolve their differences through discussion. If no agreement can be reached, a final decision will be made in consultation with the third author.2.8 Two authors will assess 7 domains associated with the risk of bias in each study, including random sequence generation, allocation concealment, blinding , incomplete outcome data, selective reporting the outcomes, and other bias. And then, the evaluation results will be classified as low risk, high risk, and unclear risk. Unresolved discrepancies will be consulted with the third author.Each original study will be independently evaluated for risk of bias by 2 authors under the guidance of the Cochrane Handbook for Systematic Reviews of Interventions.2.9Statistical analysis will be performed using Review Manager Version5.3 software, The Nordic Cochrane Centre, The Cochrane Collaboration, 2014, Copenhagen. For the dichotomous variables, we will use the risk ratio (RR) and 95% confidence interval (CI) to evaluate the effect size. If it is a continuous variable, the effect size will be assessed with the weighted mean difference (WMD) and 95%CI. In the case of inconsistent data reported in the study, data conversion is necessary. The results will be presented in the form of forest plots. In addition, descriptive analyses will be carried out on data relating to demographic characteristics and baseline characteristics. The Chi-Squared test will be used to assess the inter-study heterogeneity among the trials, and the level of heterogeneity will be determined in combination with the I2 test. If P > .1 and I2 < 50%, the heterogeneity is considered to be small, and the differences between the included studies can be ignored. The heterogeneity cannot be neglected, and there is no homogeneity between studies, when P ≤ .1 and I2 ≥ 50%. Fixed-effects and random-effects models will be used with low and high levels of heterogeneity, respectively. If the heterogeneity between studies is small the fixed-effects model will be used, otherwise the random-effects model will be adopted. When the heterogeneity is too large, only descriptive analysis is going to be performed.It was found that heterogeneity analysis of the included studies was necessary before meta-analysis results were accepted. we hypothesized several subgroups based on possible influencing factors: race, age, sex, course of the disease, drug combination, etc. On the understanding that the number of included studies permits, a meta-regression analysis will be performed to further explore the sources of heterogeneity.On condition that heterogeneity is found to be substantial, we will conduct a subgroup analysis and meta-regression analysis to identify the sources of inter-study heterogeneity. Referring to the new criteria for evaluating the credibility of subgroup analyses,Sensitivity analysis will be carried out to determine the robustness of the meta-analysis results. We will eliminate some of the low-level ambiguities and then reaggregate and analyze the data to compare the differences between the reworked results and the original results. In addition, it is possible to reanalyze the data using different statistical methods, such as using the random-effects model instead of the fixed-effects model.In the event that more than 10 corresponding studies are included, visual inspection of funnel plot and Egger test of funnel plot will be accomplished to minimize the impact of reporting bias on the results of meta-analysis. When P value of intercept is less than 0.05 or the 95% confidence interval does not contain zero, the reporting bias is suggested.2.10 classify evidence quality into 4 levels: high, moderate, low and very low, based on 5 factors that may affect the quality of evidence: risk of bias, imprecision, inconsistency, indirectness and publication bias. According to GRADE, the outcome of the study will be evaluated for evidence quality.The Grades of Recommendations Assessment, Development and Evaluate system (GRADE) guidelines2.11Patient and public involvement will not be involved in this study because the data involved in this meta-analysis and systematic review has been previously published in the past.2.12As this is a systematic review and meta-analysis, ethical approval is not necessary. The study, which will evaluate the effect of different doses of SGLT2 on lipid levels in patients with type 2 diabetes mellitus, will be published in a peer-reviewed journal.3–25 Nevertheless, there is no doubt that SGLT2 inhibitors cause dyslipidemia as well as therapeutic effects. Meanwhile, the effect of SGLT2 inhibitors on lipid levels was inconsistent in different studies, of which some results confirmed that SGLT2 inhibitors could lead to an increase in LDL-C levels, but the opposite outcomes were also found.–14 Although statistical analyses by relevant meta-analyses and systematic reviews have shown that SGLT2 inhibitors are associated with an augment in HDL and LDL cholesterol,,16 the latest findings are not incorporated and evidence of a dose-related association is insufficient. This study will collect relevant literatures published and unpublished but with available data up to now, and analyze the correlation between different dosages of each category of SGLT2 inhibitors and changes on lipid levels in patients with type 2 diabetes mellitus. The results of this study will provide a basis for the clinical use of SGLT2 inhibitors and the management of blood lipids in patients with type 2 diabetes mellitus when taking these drugs, so as to better control blood glucose smoothly and prevent the occurrence of related complications.Type 2 diabetes mellitus is a frequent chronic disease that threatens human health and quality of life. Since SGLT2 inhibitors were used in clinic as hypoglycemic agents, considerable results have been achieved, such as lowering blood glucose, improving glycosylated hemoglobin, and reducing cardiovascular risk.Conceptualization: Tingyu Cai.Data curation: Yang Gao, Li Zhang.Formal analysis: Tingyu Cai, Yang Gao.Funding acquisition: Qiu Chen.Investigation: Li Zhang, Ting Yang.Methodology: Yang Gao, Ting Yang.Project administration: Qiu Chen.Resources: Tingyu Cai, Qiu Chen.Software: Tingyu Cai, Li Zhang.Supervision: Qiu Chen.Visualization: Tingyu Cai, Qiu Chen.Writing – original draft: Tingyu Cai.Writing – review & editing: Tingyu Cai, Qiu Chen.


To highlight the contribution of belowground interactions to biomass and N and P yields, field bean and triticale were grown in a P-poor soil as sole crops and as replacement intercrops at two N levels. The shoots were always in contact, while the roots of adjacent rows were free to interact or were completely separated. This allowed simultaneous testing the intraspecific and interspecific competition between rows, which to our knowledge has not been studied before. Root biomass, distribution in soil, morphometry, and functional traits were determined, together with the nodule number and biomass. The Land Equivalent Ratio for shoot biomass and N and P yield were higher than 1 when roots were in contact, and markedly lower when they were separated. This demonstrates the positive contribution of root interactions, which in field bean, consisted of increased root elongation without changes in biomass and nutrient status; in triticale, of increased N and P uptake efficiency and reduced biomass partitioning to roots. The soil-plant processes underlying intercrop advantage led to complementarity in N sources with low N inputs and facilitated N and P uptake with high N inputs, which demonstrates that intercropping could be profitable in both low and high input agriculture. The ecological intensification of crop production improves the environmental and economic sustainability of agricultural systems by better exploiting the ecological processes occurring in plant-soil systems, thus obtaining comparable yields to industrial agriculture but with lower external inputs ,2. InterPoaceae (grasses) and Fabaceae (legumes) are frequently intercropped because they are complementary components in livestock diets and also because of their well-known complementarity in the use of nitrogen sources, which relies on the ability of legumes to establish a symbiosis with atmospheric-N fixing bacteria [2-fixation, indirect microbe-mediated facilitation and niche complementarity also appear to enhance the acquisition of P, Fe, and Zn in cereal/legume intercrops [bacteria ,9. Besidbacteria ,11. Thisbacteria ,12. Despbacteria . In cowpbacteria  found a tercrops . Liu et tercrops  also repThe biomass yield of intercrops compared to sole crops, together with their response to N levels, are priority goals in intercropping research ,18,19,20In legume/cereal intercrop research, it is therefore crucial to understand whether the benefits at the root level, such as the complementarity in the use of water and N sources and the facilitation in mineral acquisition, can overcome the negative effect of the aboveground competition for light, or vice versa . Above aInvestigations on belowground interactions are thus of huge interest in understanding the mechanisms underlying the agronomical and ecological advantages of intercrops over sole crops ,39. TherTo investigate the interactions occurring above and belowground between the partner species, we arranged a 1:1 field bean/triticale row-intercrop in growth boxes, in which the shoots of adjacent rows were always in contact, while the roots were separated  or free to intermingle (Full interaction). Field bean and triticale were chosen because of their wide use in livestock nutrition and their good yield and quality performance in intercrops ,49. A reThe research aimed at demonstrating the existence of positive interactions, such as complementarity and facilitation, between the roots of field bean and triticale, and at assessing the contribution of these interactions to shoot biomass and N and P yield. Sole crops of both species were arranged similarly to intercrops to compare inter- and intraspecific competition between shoots, and to reveal intraspecific competition between root rows, which to the best of our knowledge, has not been taken into account in previous intercropping studies ,34,38. TIn replacement intercrop designs, a Land Equivalent Ratio (LER) higher than 1 indicates that intercrops (IC) yield more than the cumulated sole crops (SC) on a land basis, while values lower than 1 indicate the opposite. In our research, the LERs calculated for the shoot biomass and N and P yields of shoots were always higher than 1 in the Full treatment and lower than 1 in the Aerial treatment. The only exception was for shoot N yield in Aerial-N120, thus demonstrating that root interactions played a primary role in advantaging intercrops over sole crops . The bioThe Competitive balance (Cb) index equals zero when intercropped species do not compete or have equal competitive abilities, while all the other values indicate that one species is less affected or benefits more from intercropping than the other. In our research, the Cb for shoot biomass showed positive values, indicating a greater advantage for field bean (Fb), especially with the lower N supply and in the Aerial treatments . Tritica−1 (N60) compared to 87.7 g row−1 (N120).Analysis of variance revealed a significant second-order Crop system × Row contact interaction for the shoot biomass of field bean. The highest shoot biomass was recorded in the Aerial SC and the lowest in the Full SC, suggesting that, in Fb, shoot growth was negatively affected by intraspecific root competition and by interspecific shoot competition, however the latter seemed to slightly benefit from root contact a. The ni−1. Pod biomass was markedly higher in the N60 IC and did not differ among the other treatments, which was essentially due to the higher pod number .The biomass of the entire root system changed in response to the third-order interaction. In SC, root biomass was lower in the Full than Aerial treatments, while in IC, it was higher in the Full treatment with N60 and did not differ between row contacts with N120 b. AccordA significant Crop system × N level × Row contact interaction was revealed for lateral root biomass and for root morphometric and functional traits. The distribution of lateral roots in the soil profile changed according to the Crop System × Row contact × Soil depth interaction, without differences in response to the N level.−1. Lateral root biomass was approximately 16 g row−1 without differences among crop systems and N levels when roots were separated, but was by 45% higher in the N60-Full IC than in the N120-Full SC when roots were free to intermingle  decreased in the order: 36.1 in pods, 24.0 in lateral roots, 14.9 in stems+leaves, and 10.6 in tap roots. The P concentration (mg g−1) was 2.7 in pods, 1.5 in lateral roots, 1.3 in stems+leaves, and 0.9 in tap roots. A slight response to N level was observed in the N and P concentrations of shoots in that the supply of 120 kg N ha−1 increased the average N concentration of the shoot by 4% and its P concentration by 7%. This did not affect the shoot N and P contents, because of the slight decrease in biomass recorded with the higher N supply (data not reported). Accordingly, averaged over all treatments, the N content was 2120 mg row−1 in shoots and 473 mg row−1 in roots, while the P content was 171 and 31 mg row−1, respectively.In field bean, the Crop system, Row contact, N level, and Soil depth treatments did not significantly affect the N and P concentration and content of aerial and hypogeal plant parts. On average, the N concentration  differed greatly among treatments. The NUEuS was higher in SC compared to IC, with the highest values recorded in N60 SC without differences between Full and Aerial treatments a. In theAnalysis of variance revealed significant third-order Crop system × N level × Row contact interactions for the shoot and root biomass of triticale. The shoot biomass responded positively to the increase in N supply, with a higher responsiveness to N in Full than Aerial treatments, especially in SC a.−1, with more pronounced differences in the Full SC  and that the N and P yields were higher with high N rates (120 kg ha−1). This suggests that intercropping could be profitable in both low and high input agriculture, giving primarily higher yields over than crops in low input agriculture, and better quality in terms of N and P concentration, in high input agriculture. The competitive balance indicated a slight advantage of field bean over triticale in terms of biomass production, while the opposite was reported by Sobkowicz [The LERs obtained with the above and belowground plant parts that were free to interact (Full), were higher than 1 for all the parameters analyzed. This demonstrated that field bean and triticale intercropped in a row replacement design led to a greater yield than sole crops, thus complying with the primary goals of intercropping ,18. In aobkowicz  in additHigher N and P concentrations in the forage and grain compared to sole crops are a common feature of cereal/legume intercrops, as they can be achieved by niche differentiation and facilitation processes , but alsThe comparison of the shoot biomass of sole cropped and intercropped field bean and triticale highlighted that, in both species and with both N levels, interspecific aboveground interactions were detrimental to shoot biomass, and that the negative effect of shoot competition was nullified when the roots were free to intermingle, thus highlighting that belowground interactions were favorable to both species. Previous studies have found that interspecific competition reduces the shoot growth of all species except vetch, when either barley or wheat were intercropped with lupin or vetch, but only the cereals benefitted from belowground interactions . SimilarThe analysis of the single shoot organs showed that, in both field bean and triticale, the lower shoot biomass in response to interspecific aerial competition depended essentially on reduced stem/culm growth, as the leaves did not change and the reproductive biomass, pods/spikes, were not or only slightly reduced. In addition, in both species, the reproductive organs received the greatest benefits from interspecific root contact, so that the partitioning of shoot biomass into pods or spikes tended to be higher in intercrops than in sole crops. These results could be explained by a different balance between competition and benefits during the crop cycle, however it is not clear at which growth stage belowground interactions become positive. In support of our findings, Benincasa et al.  reportedPositive belowground interactions largely rely on the ability of mixed roots to avoid competing for the same soil space and mineral resources, and a greater proliferation of mixed root systems associated with a complementary redistribution in the soil profile is reported as a major effect of intercropping ,9,33. Wa−1 in Fb and 22–33 g row−1 in T, huge differences were recorded in root length and surface, which were, on average, 7 and 2.6 times greater in triticale. This did not seem to impair the uptake ability of field bean, as the uptake efficiency per unit root surface was approximately 6 and 3 times greater than in triticale in terms of N and P uptake, respectively.In field bean, shoot and root biomass changed with similar patterns in response to treatments, with only a slightly higher partitioning of biomass to roots in IC compared to SC. In triticale, conversely, we found marked variations in the shoot to root ratio, revealing a great plasticity of biomass allocation in this crop, which is probably due to the different plant architecture of cereals and legumes. In fact, cereals are composed of a sum of nearly independent culm and root units, whereas legumes by branched but unique shoot and root systems. Low root:shoot ratios were found in triticale not only in the N120 SC, which is considered a typical response to high nutrient levels , but als2 fixation to competition for mineral N uptake when its availability in soil increased [Root morphometric traits responded differently to root neighboring. In triticale, both intra and interspecific row contacts were detrimental to root elongation, whereas in field bean, root length was only reduced by intraspecific root contact. Root traits also changed in contrasting ways in response to mineral N. In fact, high N supply slightly decreased root length in triticale but increased it in field bean, with marked increases prompted by interspecific root competition. We found that enhanced root elongation was associated in field bean with lower nodule initiation and growth, suggesting a shift from biological Nncreased ,55. Reduncreased  and Cardncreased , which qncreased ,56,57. Hncreased . On the ncreased ,36,43. Pncreased ,58. In cncreased ,35,37. Ancreased  reportedAlthough a great promotion of soil enzyme activity in intercrops was not found, field bean sole crops showed higher soil enzyme activity compared to triticale sole crops, as also reported by Siczek et al.  in faba The root functional traits, Specific Root Length and Root Tissue Density, have rarely been examined in root interaction studies, but are widely used to describe the economy of root growth in response to soil conditions, and both parameters are negatively related to soil fertility and soil moisture, on a large-scale gradient ,63. HoweOur study considered both intra and interspecific interactions, which revealed that in field bean, the primary negative determinant of shoot growth and pod yield was intraspecific belowground competition, followed in second order by the interspecific aerial competition with triticale, which occurred irrespectively of N level. The contact between Fb roots from adjacent rows was detrimental, especially to nodule initiation and growth and to root elongation, whereas root biomass changed in proportion to shoot biomass, as confirmed by the small changes in the root:shoot ratio and by the low Specific Root Length. Our findings contrast with those of Murphy and Dudley , who repSurprisingly, we found that field bean roots were markedly longer in the Aerial IC than in the Aerial SC, and thus changed in response to intra and interspecific aboveground interactions without root contact. In addition, while the root elongation prompted by root competition was associated with reduced nodulation, shoot interactions did not affect nodule initiation and growth, thus suggesting that these were independently regulated processes. Previous studies on the response of legumes to aboveground competition found that root biomass and biomass partitioning within the plant did not depend on the type of competition (inter or intra), and that shading inhibited root elongation in soybean intercropped with maize ,66,69. WIn triticale, unlike field bean, shoot growth responded primarily to N levels, and intra and interspecific interactions only modulated the positive response to the increasing N supply. In line with the characteristic of Poaceae to grow in dense populations, intraspecific competition was not observed between the aerial parts of plants in adjacent rows, while it was revealed between roots with a low N supply. Thus, in triticale, the lower shoot biomass coupled to a higher root:shoot ratio recorded in the Full compared to the Aerial sole crop with N60 is in agreement with the “tragedy of the commons”, which was driven by the competition of roots for mineral N .The research was carried out in 2018 at the experimental station of the Department of Agricultural, Food and Environmental Sciences of the University of Pisa, Italy, which is located at a distance of approximately 5 km from the sea  and 1 m a.s.l. According to Köppen, the climate is classified as Csa, humid temperate with dry and hot summers. Meteorological data were obtained from a station located within 100 m of the trial site. Over the research period, the average daily temperature was 13.3 °C, the average maximum and minimum daily temperatures 18.2 and 8.3 °C, respectively, and the cumulated rainfall 427 mm .Triticosecale Wittmack cv. Trismart  and Vicia faba var. minor Beck cv. Vesuvio  grown as sole crops and intercrops in alternate rows within growth boxes that enable the roots of adjacent rows to be separated if necessary. The three crop plots, hereafter named crop systems, were field bean sole crop (Fb SC), triticale sole crop (T SC), and field bean/triticale intercrop (IC). All crop systems were split into two subplots with different levels of nitrogen supply, which were further split into two sub-subplots differing in the contact between plants in adjacent rows. The resulting arrangement was a split-split-plot design, with Crop system as the main plot, N level the subplot, and Row contact the sub-subplot, with three replicates.The experiment was arranged with −1, which comprise low and medium rates, respectively, for cereal sole crops in the cultivation area. Both rates positively affected the yield parameters of field bean, although the higher rate decreased root length and nodule development [The N levels corresponded to 60 (N60) and 120 (N120) kg N haelopment ,72. The elopment , which helopment ,74,75.3 of soil. The physical and chemical properties of the soil were: 69.1% sand, 26.6% silt, and 4.3% clay (USDA method); pH 8.2 (soil:H2O 1:2.5), organic matter 17.3 g kg−1 , total CaCO3 5.4 g kg−1, total N 1.3 g kg−1 , available P 7.4 mg kg−1 (Olsen method), available K 127 mg kg−1 (ammonium acetate method), conductivity 339 μS cm−1, and cation exchange capacity 8,0 meq 100 g−1 (BaCl2-TEA method).For the experiment, 40 stainless-steel boxes with 1.0 m length, 0.2 m width, and 0.45 m depth, with large removable sides, were filled with 80 dmPlants were grown in 1 m long rows and each box contained two rows spaced 15 cm apart both for the sole crops and intercrops. This spacing was optimal for triticale, but narrow for field bean. Boxes were aligned on the long side, spaced 10 cm apart to maintain the row distance throughout crop plots and embedded in expanded clay to prevent daily fluctuations in soil temperature. To eliminate the border effect, four additional boxes were distributed at the beginning and end of the box arrangement and between crop system plots. The intercrop was a replacement design obtained by alternating rows of field bean and triticale.−2, in the respective sole crops and of approximately 300 seeds m−2 in the intercrop. Field bean seeds were inoculated immediately before sowing with a specific commercial rhizobia inoculant using Rhizobium leguminosarum bv. viciae.Sowing took place on 9 February 2018, at densities of 30 seeds per linear meter of field bean and 60 seeds for triticale, corresponding to a seed density of 200 and 400 germinable seeds m2SO4 at a rate corresponding to 100 kg K ha−1 in all the treatments. Nitrogen fertilizer was applied in two rates, corresponding to 15 and 45 kg N ha−1 in N60 and to 30 and 90 kg N ha−1 in N120. The first rate was applied at sowing, as ammonium sulphate, and the second on 8 May, as urea. On this date, triticale was at the pseudo-stem erection stage  and fiePlant harvest started on 4 June (123 DAS), in correspondence with the pod ripening stage of field bean  and the flowering stage of triticale . This harvest time was chosen in order to prevent root decay and leaf loss in field bean. Within each species, no appreciable differences in the phenological stage were recorded among the treatments.Shoots were manually cut 2 cm above ground level. Plant height was measured, and the biomass of each species was divided into stems, leaves, and spikes or pods. The number of stems and pods, or spikes, was recorded. In field bean, the number of seeds was also determined, while this was not possible in triticale, due to the earlier growth stage. For dry weight determination, the aerial plant parts were oven-dried at 65 °C to constant weight.The performance of the intercrop, in terms of shoot biomass and N and P yield per unit area, was expressed as the Land Equivalent Ratio (LER), as reported in Bedoussac and Justes (2011):The competitive ability of one species in intercropping, relative to the other, was measured by the Competitive Balance Index (Cb), as reported in Mariotti et al. :Cb=ln[. The washed roots were transferred to jars filled with water and stored at 4 °C until further cleaning, which was completed within one week under a magnifying glass. In roots of field bean, nodules were removed, counted, and weighed. A sub-sample of about 10 g from the root samples of all treatments and soil depths was cleaned to remove residual materials, such as weed roots, small woody fragments, and debris, and in the Full contact intercrop, to separate field bean and triticale roots based on the different morphological aspect and color of roots. The clean root sub-samples, residual materials, remainder of the root sample, and the nodules were gently dried between two filter papers to remove surface water, until no more water tracks remained on the paper, and weighed to obtain the fresh weight of each fraction [The next day, roots were extracted by gently washing them under a water flow and, to prevent the loss of root fragments, the washing water was filtered through a fine glass-fiber net .Before scanning, the roots of triticale were stained by immersion in Toluidine blue 0.05% for 5 min, and then rinsed with water to eliminate the color from the root surface. Staining was not necessary for field bean roots, which were of a dark brown color at this growth stage. The fresh root sub-sub-samples  were spread with minimal overlapping into 20 × 25 cm trays filled with a 0.5 cm layer of deionized water, and then scanned at 400 dpi. Fishing wires of 0.1 and 0.5 mm were added to the triticale and field bean roots, respectively, for the manual calibration of images. Roots with a diameter below 0.05 mm were not detected. After scanning, the fresh weight of the measured root sub-sub-samples was determined.−1 dw) and Root Tissue Density , were calculated [The root parameters length, surface, volume, and average diameter, were recorded with WinRHIZO separately for each soil layer. As root samples do not have a constant diameter, the surface and volume were obtained by adding the values obtained for the different diameter classes . Parametlculated  in orderThe dried leaves, stems, pods, and spikes were separately ground to powder . The roots were milled at 18,000 rpm using a 0.5 mm Conidur distance-sieve . The nitrogen and phosphorus concentrations of the powders were determined by the modified Kjeldahl method and the ammonium–molybdophosphoric blue color method. The total N and P yields of separate plant parts were calculated by multiplying the N, or P, concentration per dry matter. The N and P uptake efficiency of roots (NUEuS and PUEuS) was estimated for both field bean and triticale as the amount of N and P absorbed per unit root surface, using the formula:−1 2 h−1) at 490 nm. The dehydrogenase activity (Deh) was determined by Tabatabai’s method [−1 soil per hour. The alkaline phosphatase activity  was determined by the colorimetric assay based on the hydrolysis of p-nitrophenyl phosphate added to soil samples. This phosphate releases p-nitrophenol, which can be detected colorimetrically at 410 nm, and Pal was expressed as µmol of p-nitrophenol g−1 soil per hour [At plant harvest, soil samples were collected close to roots, sieved, and stored at 4 °C. Enzyme activities were assayed within one week of sampling. The hydrolysis rate of fluorescein diacetate (FDA-ase) was estimated as reported by Dick et al. , by detes method  based onper hour . All datData on field bean and triticale were analyzed separately by analysis of variance (ANOVA), using JMP . For shoot parameters, the response to the Crop system, N level, Row contact, and their interactions was analyzed by arranging data in a split-split-plot design with three replicates, in which the crop systems (SC and IC) were designed as whole plots, the N levels (N60 and N120) as subplots, and the row contacts  as sub-subplots. For the root parameters, the treatment Soil depth was added as a nested sub-sub-subplot to the previous design, to test the effects of three soil depths . For soil enzyme activities, only Full data were collected and analyzed, arranged in a split-plot design, with Crop System as the main plot, and N level as the sub-plot. Finally, the significance of the LER and Cb indices was tested by arranging data in a split-plot design with three replicates, in which N levels were the main plot and row contacts were the subplots. Significantly different means were separated at the 0.05 probability level by Tukey’s test .−1, and in N and P yield with the supply of 120 kg N ha−1. In triticale, the benefits from interspecific root interactions relied primarily on the greater partitioning of resources to the shoot with the lower N supply and on the higher efficiency in N and P uptake per unit root surface with the higher N supply. In field bean, benefits of intercropping were essentially related to higher root biomass and length and also to higher nodulation with the lower N input. Conversely, our results demonstrated that field bean competed with triticale for mineral N, when its availability increased in the soil.Our study demonstrated that belowground interactions play a primary role in determining the aboveground biomass and the N and P yield of field bean/triticale intercrops. The advantage of intercrops over sole crops was in biomass production with the supply of 60 kg N ha−1. With the supply of 120 kg N ha−1, facilitation in N and P uptake could be hypothesized, which was probably related to the great proliferation of field bean roots, while specific soil enzyme activities were not increased or only minimally.Thus, the observed intercrop benefits could be explained by the niche complementarity for N source only with the supply of 60 kg N haWe found that both in field bean and triticale, intra and interspecific competition and N level affected root biomass and length with different patterns, which highlights that both root parameters should be of concern in intercropping studies.


Escherichia coli genome has an estimated 34 RR genes in total, including 14 genes of OmpR family genes. To reveal the contribution of TCSs for fast growth as an adaptive growth strategy of E. coli, we isolated a set of gene knockout strains by using newly developed genome editing technology, the HoSeI (Homologous Sequence Integration) method, based on CRISPR-Cas9. The statistics of single cell observation show a knockout of an arbitrary pair of phoP, phoB, and ompR genes, stably expressed by positive feedback regulation, dramatically inhibit the optimum adaptive growth of E. coli. These insights suggest that the adaptive growth of bacteria is fulfilled by the optimum high intracellular level of regulators acquired during growth under environmental conditions.Bacteria survive in the environment with three steps: a sensing environmental conditions, a responding to sensed signals, and an adaptation for proper survival in the environment. An adapting bacterial cell occurs cell division to increase the number of sister cells, termed adaptive growth. Two-component systems (TCSs), representing the main bacterial signal transduction systems, consist of a pair of one sensor kinase (SK) and one response regulator (RR), and RR genes are abundant in most bacterial genomes as part of the core genome. The OmpR gene family, a group of RR genes, is conserved in 95% of known bacterial genomes. The  Transcription factors (TFs) are classified as those that facilitate direct sensing and indirect sensing of environmental signals. The Lac repressor and Crp activator are good examples of direct sensing TFs and sense lactose and cAMP, respectively. A response regulator (RR) is a typical example of an indirect sensing TF and is activated by phosphorylation by the cognate sensor kinase (SK), detecting environmental signals at the membrane2. Direct sensing TFs temporally support bacterial growth because extracellular nutrients stimulate intracellular metabolism and produce signal metabolites that activate those TFs.Indirect sensing TFs, such as RR, are known to be important for temporal survival but are not known for aiding continuous growth.Bacteria survive and then increase their population by binary cell division in a timely manner. Individual bacterial cells sense changing environmental signals, transduce those environmental signals into biological responses, and adapt to the environmental change by biological responses. Successful integration of these biological stress responses must induce adaptive growth to obtain a continuous chance for the production of offspring. Bacteria are able to survive in various environments by changing the expression pattern of their genome, which takes place by controlling the promoter recognition properties of RNA polymerase by transcription factors3. Within the core genome, there is a set of genes involved in central and secondary metabolism, cell cycle, and gene expression, many of which are essential for growth3. In addition to genes coding for essential biological functions, regulatory genes are conserved in the core genome but are not essential for growth3. One of the core-genome regulatory genes is the RR gene, which is a unique signal transduction component that is only conserved among prokaryotes. Two-component systems, known as the common signal transduction systems of prokaryotes, basically consist of a pair of one SK and one RR. SK autophosphorylates upon stimulus and then transfers a phosphate group to a cognate RR. The typical phosphorylated form of a given RR is active and controls the cellular system to survive in changing environments by gene expression regulation because most response regulators harbour structural domains for DNA binding to regulate genome expression4. Several TFs are autoregulated during the response to environmental signals. In Escherichia coli, more than 50% of identified TFs in the genome are positively or negatively autoregulated5. Positive autoregulation leads to a fast response and impacts the response dynamics7. Among ~30 positively autoregulated TFs in the E. coli genome5, 10 of those are RRs of TCSs, and 34 RRs have been characterized in the E. coli genome. On the other hand, negatively autoregulated RRs are less common than positively autoregulated RRs in TCSs8. A recent report showed that coupled positive and negative feedback allowed both a fast response and optimal RR protein levels in the PhoB/PhoR system in E. coli6. However, the contribution of coupled autoregulation for continuous bacterial growth is poorly understood.Recent massive amounts of genomic information show the core genome, a set of species-specific genes, and pan genome, representing non-conserved genesphoP, phoB, ompR) affect E. coli growth. To gain insight into the effect of RR genes on growth, we isolated the set of all combinations of single-, double-, and triple-gene knockout strains by using the newly developed HoSeI method for the E. coli genome based on CRISPR-Cas . The differences of elapsed time until the first cell division and initial growth rate of the isolated strains were measured and analysed with statistics, indicating that a particular pair of RR genes of E. coli K-12 are required for adaptive growth via genetic epistasis.We found a correlation of the RR family with bacterial genome size, speculating that the main RR genes  database has been designed to classify proteins on the basis of orthologyrve Fig. . Bacterirve Fig. . Bacterirs Fig. S, suggestE. coli K-12 genome was evaluated in detail. The genome sequence of E. coli K-12, which is 4.6 Mbp in size, has 34 predicted response regulator genes, of which 6 are classified as RR COGs13. Among all 34 RR genes, OmpR family genes are the most prevalent, with 14 genes, arcA, phoB, cusR, kdpE, torR, phoP, rstA, hprR, basR, qseB, ompR, cpxR, creB, and baeR, and the CriR family and LytT family have the smallest number, at 2 genes, with citB and dcuR and with btsR and pyrR, respectively  method, to overcome the limitations of antibiotic resistance marker genes. It is readily feasible for us to design and isolate multi-gene knockout strains of E. coli. The designed sgRNA (single-guide RNA) containing the recognition sequence for the E. coli genome specifically digests the genome, resulting in a dead phenotype . The addition of the designed DNA fragment, including the nonsense codon for the target gene, recovered the transformation of E. coli harbouring pCas by psgRNA. The proper introduction of the nonsense codon, TAA, was confirmed by Sanger sequencing in all cases  and showed a growth rate of 0.6–1.4 divisions/hour (79.3%), as did the parent strain  and showed a growth rate of 0.4–2.0 divisions/hour (74.1%)  Figs. o and S7.E. coli, while the disappearance of any two EnvZ, PhoR, and PhoQ, the cognate SKs for OmpR, PhoB, and PhoQ, proteins slightly affect the adaptive growth of fast-growing E. coli.Taking all these observations together, the disappearance of any two OmpR, PhoP, and PhoB proteins seriously disrupts the adaptive growth of fast-growing phoP Δ phoB, Δ phoP Δ ompR, and Δ phoB Δ ompR, almost disrupted the adaptive growth. We examined whether double RR gene knockout of other combinations. Cluster analysis with all RRs of E. coli K-12 showed that PhoP, PhoB, and OmpR were highly similar RRs   and showing a growth rate of 0.6–2.0 divisions/hour (69.6%), as did the Δ phoQ Δ phoR Δ envZ strain  Fig. S. Most ofrs Fig. S. Three dur Figs.  and S7. ur Figs.  and S7. phoP Δ phoQ, Δ phoB Δ phoR, and Δ ompR Δ envZ, for adaptive growth. Most of the cells of the three cognate pair double gene knockout strains grew significantly at the end of observation after 8 hours  and 1.3–2.7 divisions/hour (70%), respectively, whereas 70% of cells of the Δ phoB Δ phoR strain showed growth rates of 0.4–1.4 divisions/hour, similar to the parent strain  or larger (P. aeruginosa) genomes than that of E. coli. A genome comparison of E. coli strains reveals that the predicted pan-genome comprises 15,741 gene families and that the core genome comprises only 993 (6%) of the families, as represented in every genome25. OmpR family genes are included in the E. coli core genome and increase their number in the genome. We found that the OmpR family includes conserved TCS genes withing the bacterial genome. The number of the OmpR family gene is also correlated with the size of the genome . We isolated all of 14 OmpR family single RR gene knockout strains by HoSeI method, indicating that none of them was essential for growth under the used conditions in good agreement with previous works27. Time-lapse live cell imaging with microscope indicated that 14 single RR gene knockout strains were divided into three groups, the faster growth rate mutants, the normal growth rate mutants, and the slow growth rate mutant . Despite different genetic backgrounds, the genetic defect of an arbitrary pair of phoP, phoB, and ompR largely delays initiation of growth and dramatically reduces the initial growth rate, the results of which were entirely different from not only phoP, phoB, and ompR RR single knockouts but also their triple knockouts  and a C-terminal DNA-binding domain (DBD), which are connected by a linker. To date, the molecular structure of the truncated RD and DBD and the full-length active and inactive form of OmpR family proteins have been solved by X-ray crystallography and NMR spectroscopy36. Among these proteins, dimerization or higher-order multimerization is commonly observed, and at least three surfaces have been observed for intermolecules. Two surfaces, the α1α5 and the α4β5α5 faces, conserve the RD and one surface the DBD. PhoP, PhoB, and OmpR are known to bind to at least 15 promoters in vivo, as denoted in RegulonDB (http://regulondb.ccg.unam.mx/index.jsp), indicating that the intracellular level of approximately 1,000 copies of PhoP, PhoB, and OmpR each per genome is clearly too high, and thus, these proteins could play a role except in the stress response. These are in good agreement with the observation of the adaptive growth of E. coli of a knockout of an arbitrary pair of envZ, phoR, and phoQ genes, the cognate SK genes for OmpR, PhoB, and PhoP , suggesting that PhoP, PhoB, and OmpR each basically function as a DNA-binding protein. Genome-wide binding profiles have indicated the recognized DNA sequences TTTAnnnnTTTA as the PhoP-binding consensus37, TTTGTTACAT as the OmpR-binding consensus38, and TGTnAnAAAnnTGTnA as the PhoB-binding consensus39. The tandem triplet of A or T, found as a common sequence among the genes, could be weakly recognized by every one of PhoB, PhoP, and OmpR. Thus, E. coli cells could show adaptive growth when a total of 3,000 intracellular proteins of PhoP, PhoB, and OmpR have become associated with the genome. E. coli cells, however, could not properly grow, if insignificant occupation of the genome by 1,000 intracellular proteins of either PhoP, PhoB, or OmpR.As unexpected, we found that knockout of the arbitrary pair of wth Fig. . When thn Table S, only dooli Fig. . When thn Table S, only dooli Fig. . Besidests Figs.  and S8. oP Figs. . PhoB anE. coli. One possible reason that normal growth of the triple RR gene knockout strain is the existence of excess intracellular RRs in place of PhoP, PhoB, and OmpR. We did not, however, find a significant difference in the profile of cellular proteins, with more than 200 proteins detected with LC-MS, between the parent strain and the triple RR gene knockout strain (data not shown). Additionally, the promoter activities of all TCS genes in the E. coli genome were measured using the luciferase reporter system, indicating that three double RR gene knockout strains and a triple RR gene knockout strain were mostly similar to the parent strain with respect to these promoter activities (data not shown).The triple RR gene knockout strain, however, recovered the elapsed time and the growth rate to values similar to those for the parent strain Fig. . This obphoP, phoB, and ompR is important for the bacterial cell cycle, genome replication and/or cell division but not for general metabolism and stress responses. The epistatic inhibition for E. coli adaptive growth by an arbitrary pair of phoP, phoB, and ompR was obscure on LB agar (data not shown). All of the isolated knockout strains also showed a similar adaptive growth to the parent strain on LB agar (data not shown). One possible is that only either PhoP, PhoB, or OmpR at a high intracellular level bias genome expression profile and inhibit the optimum adaptive growth in E. coli grown in M9 glucose that contains less amino acids than LB broth. Another is that a high concentration of only one species of OmpR family severely affect bacterial growth by an unknown molecular mechanism, which could be suppressed by a high concentration of another species of OmpR family. These findings suggest that RRs contribute to the optical high level of the intracellular RRs, resulting in a fitness advantage for growth. The results are in good agreement with the idea of fitness advantages being conferred for both positive and negative feedback in both dynamic and stable environments. Taken together, in E. coli, PhoP, PhoB, and OmpR are autoregulated at the optimal intracellular level and balance of their intracellular level play an important role in survival for repeatable growth fitness under the evaluated experimental conditions.These findings suggest that the mode of action for inhibition of adaptive growth by knockout of the arbitrary pair of E. coli strains, plasmids, and oligonucleotides are listed in Tables SE. coli K-12 W3110 typeA strain was used as a parent type40.The used E. coli cells were grown at 30 °C in M9-glucose medium.21. The designed 227 bp DNA sequence introducing the Not I recognition site immediately upstream of tracrRNA (trans-activating CRISPR RNA) was synthesized and cloned into the pEX-A2 vector by Eurofin genomics , resulting in the construction of psgRNA . The inserted DNA sequence on the resulting plasmids was confirmed by DNA sequencing  using a 100x/NA 1.4 objective lens  and a Retiga EXi Fast1394 CCD camera (Q Imaging). The temperature was maintained at 37 °C using a closed circulation system (EYELA). Image acquisition and microscope control were performed with Image Pro Plus (Nippon roper). The elapsed time until cell division and growth rate of each 41. In brief, the protein-coding regions of phoP, phoB, and ompR gene with an artificial SD sequence were amplified by PCR using specific primers . The inserted DNA sequence on the resulting plasmid was confirmed by DNA sequencing . The ratio of luminescence to OD600 (LUX/OD) was evaluated as a specific activity of the promoter.We performed luciferase reporter assay in g Table S. The conCluster analysis was performed with R software. The obtained data of the elapsed time and the growth rate were prepared for Ward’s method. A hierarchical clustering dendrogram was calculated using Euclidean distances.We used all of the genome sequences of 628 species of bacteria registered in the COG databank. TCS genes and RNA polymerase genes were isolated, and the number of these genes was counted for each bacterial genome. The number of COGs involved in TCSs and the RNA polymerase subunit was analysed in comparison with the genome size, and the average number of COGs was calculated for each genome size of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, and 15 Mb. The calculated data were fitted by the Gompertz curve Supplementary Information.


PLOD family members are associated with tumor progression and metastasis in most human cancers. However, the prognostic and biological roles of PLODs in HCC remain largely unknown.Hepatocellular carcinoma (HCC) is one of the most common malignancies with rising incidence and persistently high mortality. Previous researches have demonstrated that some PLOD family members in HCC.ONCOMINE, HPA, UALCAN, GEPIA, cBioPortal, GeneMANIA, NetworkAnalyst, Metascape, DAVID 6.8, and TIMER were used to determine the prognostic values and biological function of PLOD family members were noticeably upregulated in HCC compared to normal tissue. The high expression levels of PLOD1 and PLOD2 genes were significantly correlated with higher tumor grades in HCC patients. In addition, the high expression levels of PLOD1–3 were remarkably associated with poor overall survival in HCC patients, while high PLOD1 and PLOD3 expression were markedly associated with worse disease-free survival. In the co-expression gene analysis, 20 genes were primarily associated with the differentially expressed PLOD family members in HCC cases. Through functional enrichment analysis, the biological functions of PLODs in HCC were mainly involved in collagen fibril organization, lysine degradation, collagen biosynthesis, and modifying enzymes. Furthermore, the expression levels of PLOD1–3 were positively correlated with the activities of tumor-infiltrating immune cells, including macrophages, neutrophils, CD4+ T cells, and dendritic cells. Besides, the expression levels of PLOD2 and PLOD3 were positively correlated with the infiltrating levels of B cells.The mRNA and protein expression patterns of The findings of this study could provide novel insights into the identification of prognostic biomarkers for HCC patients. PLOD1–3 genes can promote tumor progression and metastasis, and it is reasonable to speculate that PLODs are the potential targets for HCC treatment  in various types of cancers were identified from ONCOMINE database using the analysis of “Gene summary view” and “Dataset view”; P values were obtained using the Student’s t test. The parameters were set as follows: P value < 0.05; FC, >2; and gene ranking, top 10%. Analysis type: cancer vs. normal analysis; data type: mRNA. Cancers, genes, datasets, sample sizes, fold change, t test, and P value were obtained from studies that showed statistical differences.ONCOMINEpression . The exp2 is a user-friendly website that comprises immunohistochemistry-based expression profiles for the top 20 most common forms of cancer (n = 12 for each cancer type) er type) . The pro3 is a comprehensive web tool that provides analysis according to both MET500 cohort and the Cancer Genome Atlas (TCGA) datasets  projects  and disease-free survival (DFS) of patients at risk. Hazard ratio (HR), 95% confident interval (CI), and P value were calculated accordingly.GEPIAprojects . In this5 is a comprehensive web resource that can visualize and analyze multidimensional cancer genomic data  network, gene co-expression network, and gene regulatory network  of <0.05.DAVIDet genes . In this10 is an intuitive software that can be used to systematically evaluate the infiltration of various immune cells and their clinical impacts  was obtained from the ONCOMINE database. Next, the expression levels of the three PLOD family genes were compared between 20 different types of human cancers and adjacent normal tissues. According to the ONCOMINE data, the hepatic expression levels of PLOD2 and PLOD3 were remarkably elevated in liver cancer tissue compared to normal liver tissue (PLOD1 and PLOD3 (P < 0.05) were significantly elevated in HCC tissue compared with normal tissue  and PLOD2 (P < 0.0001) expressions and tumor grade was observed , PLOD2 (P = 0.00043), and PLOD3 (P = 0.012) were markedly associated with shorter OS in HCC patients. The prognostic roles of differentially expressed PLOD members in the DFS of HCC patients were also explored. It was found that high transcriptional levels of PLOD1 (P = 0.013) and PLOD3 (P = 0.0015) were remarkably associated with shorter DFS in HCC patients  compared to those with unaltered PLOD gene expression , macrophages , neutrophils , and dendritic cells . PLOD2 expression was negatively correlated to the infiltrating B cells , while positively correlated to the infiltrating CD4+ T cells , macrophages , neutrophils , and dendritic cells , while positively correlated to the infiltrating CD4+ T cells , macrophages , neutrophils , and dendritic cells  is of great importance to predict clinical outcome and develop immunotherapy . NataliyPLOD gene expression, tumor prognosis, and cancer immune microenvironment, indicating that PLOD family members may mediate tumor progression and exert immunotherapeutic effects on HCC. Thus, a better understanding of how this gene is regulated during tumor progression may pinpoint the potential prognostic and therapeutic roles of PLODs in HCC.In summary, our findings demonstrated a significant correlation among The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.BY and YoZ contributed to the study concept and design of this study. LWa, YuZ, and LWe contributed to the acquisition, analysis, interpretation of data, and the drafting of the manuscript. DC and ZC contributed to the review and the revision of the manuscript. All authors gave final approval to this manuscript for publication.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.


Pseudomonas aeruginosa (PA) is a common bacterial pathogen in chronic wounds known for its propensity to form biofilms and evade conventional treatment methods. Early detection of PA in wounds is critical to the mitigation of more severe wound outcomes. Point-of-care bacterial fluorescence imaging illuminates wounds with safe, violet light, triggering the production of cyan fluorescence from PA. A prospective single blind clinical study was conducted to determine the positive predictive value (PPV) of cyan fluorescence for the detection of PA in wounds. Bacterial fluorescence using the MolecuLight i:X imaging device revealed cyan fluorescence signal in 28 chronic wounds, including venous leg ulcers, surgical wounds, diabetic foot ulcers and other wound types. To correlate the cyan signal to the presence of PA, wound regions positive for cyan fluorescence were sampled via curettage. A semi-quantitative culture analysis of curettage samples confirmed the presence of PA in 26/28 wounds, resulting in a PPV of 92.9%. The bacterial load of PA from cyan-positive regions ranged from light to heavy. Less than 20% of wounds that were positive for PA exhibited the classic symptoms of PA infection. These findings suggest that cyan detected on fluorescence images can be used to reliably predict bacteria, specifically PA at the point-of-care.  Pseudomonas aeruginosa (PA) [It is well established that bacterial proliferation in wounds contributes to infection and delayed wound healing ,2,3,4. Tosa (PA) ,7. This osa (PA) ,9,10. Imosa (PA) . In a muosa (PA) , while aosa (PA) ,14,15. Tosa (PA)  and highosa (PA) . Similarosa (PA)  and a hiosa (PA) . PA is nosa (PA) ,20,21,22osa (PA) . Currently, point-of-care identification of wounds infected with PA is typically through evaluation of clinical signs and symptoms. Wounds grossly colonized or infected with PA may present with a malodorous, greenish crust, a greenish tinge on removed dressings or may emit a characteristic sweet odor ,25. ThesPseudomonas spp. produce a unique cyan fluorescent signature under violet light illumination [One way to detect PA more rapidly in wounds is by taking advantage of its intrinsic fluorescence properties. Studies have shown that mination ,29,30, amination ,32. Pyovmination . It is omination . Pyoverdmination , and itsmination ,37,38,39mination . As PA imination ,27, the i:X, Toronto, ON Canada). Compelling in vitro data have reported the detection of cyan fluorescence from PA (ATCC 9027) as early as 24 h after being incubated in blood agar plate media [Preclinical and clinical studies have consistently reported detection of cyan fluorescence correlating with the presence of PA when imaged with violet light  determine the range of bacterial loads detected from cyan-positive regions of the wound. In line with preclinical studies, numerous observational studies and clinical trials have detected cyan fluorescence in wounds positive for PA c,d 27,4,49,50,51Participants were recruited from the Scarborough Health Network  as part of a non-randomized, single-center evaluation. Ethical approval was granted by the Scarborough and Rouge Hospital Research Ethics Board . All patients 18 years or older who present with a chronic wound (>4 weeks duration), were able to provide consent and were receiving standard treatment, were eligible to participate. Patients were excluded if they had any known contraindications to routine wound care, had received treatment with an investigational drug within the previous month or were unable to provide consent. All patients provided written consent for participation and medical photography release. 4 CFU/g appear red or cyan [The fluorescence imaging procedure was performed as part of routine wound assessment. The procedure was explained to all patients before imaging. All wound dressings were removed, and initial cleaning and debridement was performed to remove surface contaminants. Both standard and fluorescence images were acquired for each wound. The imaging was performed as follows: First, a standard image was taken with the imaging device under ambient lighting. The room lights were then turned off or a DarkDrape  was used to create the darkened environment required to capture a fluorescence image. The 405 nm excitation light emitting diodes (LEDs) were then turned on, the device was placed 8–10 cm away from the wound (guided by the device rangefinder) and a fluorescence image was captured. Specialized optical filters on the device ensure that only relevant fluorescence from tissue and bacteria are captured and the violet excitation light is filtered out. Under violet light illumination, background tissues appear various shades of green, while bacteria at loads >10 or cyan ,52,53. TTo determine if bacteria were present in the regions with cyan fluorescence, a wound sample was collected via curettage sampling . This sun = 25 to achieve a 10% margin of error at a 90% confidence level. The sample size calculation is based on asymptotic normal approximation, which is given by: p is the planned estimate of positive predictive value, and zα⁄2 is the 100(1 − α/2) percentile of the standard normal distribution.A priori sample size calculation was performed. With a hypothesized positive predictive value of 0.90, the sample size needed is A curettage scraping obtained from a cyan fluorescence positive area, as indicated by fluorescence imaging, resulting in a microbiology finding confirming the presence of PA (light growth or higher) was considered a true positive (TP). A biopsy or curettage scraping obtained from a cyan fluorescence positive area, as indicated by the imaging, resulting in a microbiology report negative for the presence of PA or with scant growth only was considered a false positive (FP). The positive predictive value—the probability that a region of cyan fluorescence within or around a wound will contain PA—was calculated as follows:A total of 28 patients (Fitzpatrick skin type 1 to 6) with at least one wound emitting cyan on the fluorescence image were recruited into the study . Most stAll wounds included in the study fulfilled the criteria for cyan fluorescence outlined in Bacterial load of curettage scrapings taken from cyan-positive regions of the wound and analyzed by semiquantitative analysis ranged from scant to heavy growth but were predominantly moderate-to-heavy. Culture analysis confirmed presence of PA in 26/28 wounds. PA was the only Pseudomonad detected in all culture reports. Eighteen (64.3%) wounds had heavy growth of PA, corresponding to cyan-positive regions, while in six (21.4%) wounds, moderate growth of PA corresponding to cyan fluorescence was observed . In two Enterococcus faecalis (34.6% of wounds), Staphylococcus aureus (19.2%) and Escherichia coli (15.4%). Culture analysis of wound samples revealed antibiotic resistant PA present in three patients, one of which had multi-drug resistant PA. Consistent with previous studies ,7,55, PAspp. symptoms  in these wounds was surprisingly rare. The study clinician found these symptoms could not be used as a preliminary screening tool to determine which wounds should be imaged for potential study inclusion. Notably, the greenish tinge on the bandage was observed in less than 20% of study wounds, including the example in The presence of the “classic” Pseudomonas Though not a pre-defined study outcome, the cyan signal from PA after targeted treatments was monitored using fluorescence imaging as part of the patients’ standard of care. In some wounds, once PA presence and location(s) were detected, cleansing and debridement were able to remove the cyan signal, entirely or in part, during the patient’s visit. Other wounds had antimicrobial dressings prescribed, and at the next visit a reduction in cyan signal was typically, but not always, noted. The case in The need for definitive methods to improve bacterial detection at point-of-care is indisputable, given that >80% of wounds with high bacterial loads are missed by routine assessment ,56. The In this study, a high variation in the intensity of cyan fluorescence was observed across wounds, in some cases appearing blue–green and in others, appearing a glowing white. The case in It was not surprising that PA was the only Pseudomonad detected in this study; larger clinical trials have shown that PA is the overwhelmingly predominant Pseudomonad in chronic wounds ,27. Pseu3 to 106 CFU/g in wound samples [There are several limitations of this study which should be noted. This trial was designed to test the positive predictive value of cyan on fluorescence images, thus it does not provide information on negative predictive value, sensitivity or specificity of the images. These have been assessed by other studies, which demonstrate high sensitivity and specificity of the images for detecting high bacterial loads ,44,46,49 samples . Underes samples ,70, thisPyoverdine, the virulence factor and source of cyan signal, plays an essential role in establishing infection and biofilm formation . HoweverPseudomonas spp. symptoms to aid early detection of PA [PA infection is a common occurrence in chronic wounds  and is mon of PA . In pracTreatment of PA continues to be a challenge given the lack of information available at the point-of-care on the efficacy of treatments used to eradicate PA, propensity for antibiotic overuse to manage skin and soft tissue infections  and the


To describe and evaluate epidemiological investigation results and containment measures implemented in Busan, where 108 cases were confirmed with coronavirus disease 2019 (COVID-19) between February 21, 2020 and March 24, 2020.Any individual who tested positive for COVID-19 was classified as a confirmed case. Measures were taken to identify the source of infection and trace and quarantine contacts. Serial intervals were estimated and the effective reproduction number was computed.t) remained below 1 in Busan.Of the total 18,303 COVID-19 tests performed between January 16, 2020 and March 24, 2020 in Busan, 108 yielded positive results . All confirmed cases were placed in isolation at hospitals. Of the 108 confirmed cases, 59 (54.6%) were female. The most common age group was 20-29 years with 37 cases (34.3%). Regarding symptoms at the time of diagnosis, cough  and fever  were most common; 12 cases (11.1%) were asymptomatic. The source of infection was identified in 99 cases (91.7%). A total of 3,223 contacts were identified and quarantined. Household contacts accounted for 196, and the household secondary attack rate was 8.2% . The mean serial interval was estimated to be 5.54 days . After February 26, (RThe early containment strategy implemented in Busan shows that control is possible if outbreaks are of limited scope. In preparation for future outbreaks, public health and healthcare systems should be re-examined and put in a ready state. Shincheonji) was reported in Daegu city, reports of confirmed cases started increasing rapidly, and the number of cases without an identified source of infection rose [As of March 31, 2020, the global number of cases confirmed to have coronavirus disease 2019 (COVID-19) had surpassed 750,000, with the death toll at 36,000 [ion rose . On Febrt).Busan is a Korean metropolitan city, located at the southeastern coast of the Korean Peninsula, with a population of approximately 3,400,000. Busan is approximately 88 km away from Daegu, the city where 68% of the total confirmed cases in Korea were reported. The first case in Busan was reported on February 21, 2020 and as of March 31, 2020, a total of 119 cases had been reported. Here, we describe the epidemiological investigation results on the 108 confirmed cases reported in Busan as of March 24, 2020 and the containment measures that were implemented. Additionally, we evaluated the containment measures used in Busan by using important epidemiological indices  and the effective reproduction number  were classified as confirmed cases, irrespective of the presence or absence of symptoms, and required to report to a public health center. Public health centers and local autonomous governments were mandated to perform epidemiological investigations on all reported cases to identify the source of infection and find all contacts to place them in self-quarantine. A contact was defined as anyone who was in contact with a confirmed case from a day before the symptoms occurred, in a manner that offered the potential for transmission through respiratory droplets. The decision was made in consideration of mask wearing, contact duration, and environmental factors. For example, if both individuals were wearing masks and talked only briefly, the person was not classified as a contact. In contrast, anyone who had meal with a case at the same table was classified as a contact.The investigation to identify the source of infection as well as contacts was initially based on patient statements. Immediately after a case was reported, the investigation began. It typically took about 1 hour to listen to a patient’s report. Because memories can be incomplete and patients sometimes hide information, objective data  were additionally used to complete and confirm information.Those classified as a contact and placed in self-quarantine were forbidden to leave home. Public workers at the corresponding local government called these individuals at least twice a day to check on symptom onset and body temperature. If symptomatic, these contacts were instructed to get tested. Contacts with high exposure levels, such as family members, were instructed to get tested regardless of the presence or absence of symptoms. The duration of self-quarantine was 14 days, counting from the day of the last contact with a confirmed case.If a case belonged to a group/facility at high risk for transmission, like church members, hospital staff, or school teachers, a complete enumeration regarding symptoms was performed in the group/facility. Either all group/facility members or only those members who were symptomatic were tested.f(t)=G(t+0.5)-G(t-0.5), with G(t) representing a cumulative gamma distribution. The likelihood function was as follows:Serial intervals were calculated using the data of infector-infectee pairs for whom the dates of symptom onset were reported. The distribution of serial intervals was assumed to follow a discretized gamma distribution. The discretized gamma distribution was defined as μ and σ were the mean and the standard deviation, respectively, of the serial interval distribution. For a total of m infector-infectee pairs, i-th infector and i-th infectee , and tj indicated the number of symptomatic cases that imported from another city or a foreign country on the same day (imported cases). According to the parameter α, which represents the relative contribution of secondary transmission of imported cases, tR can be computed in consideration of two scenarios. First, new cases can be infected only by locally confirmed cases in the Busan area . Second, if α≠0, imported cases from another city or foreign country can generate new cases. Finally, estimates of tR were computed over a time window τ to analyze the dynamics of COVID-19 [Here, COVID-19 -9. The 9This study was conducted by provincial government in accordance with the law in a situation where urgent measures are needed for public health.A total of 18,303 COVID-19 RT-PCR tests were performed in Busan between January 16, 2020 and March 24, 2020, with 108 yielding positive results . Daily test numbers and positive test rates are shown in All of the 108 confirmed cases were admitted to hospitals in isolation; 59 (54.6%) were female and 49 (45.4%) were male. The most common age group was 20-29 years , followed by 50-59 years  and 70 years or older  . RegardiOf the 108 cases, the source of infection was identified in 99 (91.7%) and not identified in 9 (8.3%) cases . The larBased on contact tracing of the 108 cases, a total of 3,223 individuals were identified as contacts and quarantined. The number of contacts linked to a group/facility was the highest in the church, at 1,089, followed by the long-term care hospital (n= 296), a hospital (n= 159), the Catholic Church (n= 131), the kindergarten (n= 71), and the educational institution (n= 61). Tests were performed on all contacts of cases in the long-term care hospital, hospital, kindergarten, and educational institution, irrespective of the presence or absence of symptoms. Additionally, 128 contacts of the church cluster cases with high exposure levels were tested, regardless of the presence or absence of symptoms. In all other contacts, tests were only performed if symptoms developed during quarantine.A total of 196 contacts were family members. Of those, 165 (84.2%) underwent COVID-19 real-time RT-PCR tests at least once before self-quarantine was over, and 16 tested positive. Thus, the household secondary attack rate (SAR) was 8.2% (95% CI 4.7 to 12.9).i= 1, 2, …, 28, m= 28). The mean serial interval was estimated to be 5.54 days  and the standard deviation 3.90 days .Serial intervals were estimated based on the data from 28 pairs of 56 cases in whom the infector-infectee relationship was clearly identified  ,11. PartIn Busan, Korea, where relatively small COVID-19 outbreaks occurred without the large-scale superspreading events (SSEs) that were observed in Daegu ,6, aggre0), indicative of the transmission speed of an infectious disease, measures the average number of cases infected by a single case during the infectious period in a population of susceptible persons. In other words, a value of R0 over 1 means that the infection is spreading, while a value under 1 means that the outbreak will eventually die out. We computed the Rt to assess the containment strategy implemented in Busan. In t values were very high initially, when spreading from the church cluster was not yet detected. After February 21, 2020, when the first confirmed case had been reported, an aggressive containment strategy was implemented, and Rt values decreased to levels below 1 from February 26, 2020, suggesting that the outbreak was under control.The basic reproduction number , a level similar to the household SAR of 7.6% among the first 30 cases in Korea . AlthougSo far, at least one cluster of asymptomatic cases has been reported , and folThe situation in Busan and in Korea overall shows that control is possible when cases are isolated and contacts are quarantined through aggressive case tracing and wide-ranging testing. However, it is difficult to predict up to what incidence level such a containment strategy will work as the number of persons traveling from overseas increases and small-scale outbreaks persist, and what will happen if infection management interventions at the level of healthcare facilities do not work well.The early containment measures described here show that control is implemented if an outbreak is of limited scope. This should be seen as having secured the time for preparation to develop sustainable containment strategies. Findings from epidemiological investigations of the early cases in Daegu suggest that if the number of cases increases and outbreaks continue, the likelihood for the measures currently implemented in Korea  to work well is low, and infection management interventions at community hospitals may only provide limited prevention of nosocomial infections. Until now, in Busan, approximately 30 contacts per case were quarantined, in spite of the burden on the local health authorities. This level comes very close to the 36.1 suggested for an effective containment strategy in a modeling study conducted in the United Kingdom . In ordeIf infected cases are not effectively isolated and infections spreading through hospitals are not blocked, containment strategies may not be as effective as they have been observed here. If hospitalbased SSEs are likely and if post-isolation infection prevention rates are not high, the effect of containment strategies will be limited. If such a situation occurs even though NPIs have been implemented and maintained for a long time, the drastic effect of outbreak control that we observed this time may not occur in the future ,27.Additionally, in preparation for an increase in the number of cases, planning should ensure that aside from the hospitals specialized for the treatment of COVID-19, mostly public hospitals, any institution with an internal capacity for infection management should be actively utilized to isolate and treat cases. As shown in Italy , the Uni


P < 0.05) were negatively correlated with survival time. Verification of these six DEmRNAs in the Pathology Atlas indicated that PDE2A was a possible biomarker for CESC patients. PDE2A might be a biomarker for early diagnosis and prognosis evaluation of CESC patients, but due to the lack of available data, further studies may be needed for confirmation.To reconstruct the ceRNA biological network of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and to select an appropriate mRNA as a biomarker that could be used for CESC early diagnosis and prognosis evaluation. We downloaded CESC data from the TCGA public database, and statistical analysis was conducted with the R software to find out differential expressed genes encoding for lncRNAs, miRNAs, and mRNAs. The differentially expressed mRNAs (DEmRNAs) screened in the ceRNA network were analyzed for survival to find the mRNAs with significantly linked to the survival prognosis. These mRNAs were searched in the Pathological Atlas to identify the final appropriate mRNAs. Differential expression analysis revealed 773 lncRNAs, 94 miRNAs, and 2466 mRNAs. Survival analysis of DEmRNAs in the ceRNA network indicated that ADGRF4, ANXA8L1, HCAR3, IRF6, and PDE2A ( Cervical cancers are a leading cause of mortality among women , especiaHowever, this is not an easy task to do, because the occurrence and development of CESC are a very complex biological process , involviGenerally speaking, lncRNAs mainly have a function in chromatin regulation, transcriptional regulation, and regulation of alternative splicing in the nucleus . HoweverThe Cancer Genome Atlas (TCGA) platform is a well-known open-source sequence database, which covers more than 30 human cancer types and contains a large amount of clinical and bioinformatics data . It has https://tcga-data.nci.nih.gov/tcga/). The screening condition was “Project: TCGA-CESC,” “Experimental Strategy: RNA-Seq,” and “Workflow Type: HTSeq-Counts,” containing 306 CESC Primary Tumor tissues and 3 Solid Normal tissues. lncRNA, miRNA, and mRNA transcription profiles and clinical information of CESC are publicly available and open access. Therefore, approval by a local ethics committee was not needed. Annotation information for lncRNA, miRNA, and mRNA was obtained from the human GENCODE project (https://www.gencodegenes.org/).Transcriptome RNA-sequencing data of count file of lncRNA, miRNA, and mRNA and clinical information of CESC patients were downloaded from the TCGA data platform (P less than 0.05) as determined using the Benjamini-Hochberg method [We used the edgeR package (R version 3.6.1) to normalize and analyze significantly differentially expressed lncRNAs, miRNAs, and mRNAs, the selection criteria being (|log2 fold change| ≥ 2.0 and FDR adjusted g method . DiffereWe used the weighted gene coexpression network (WGCNA) algorithm  implemenhttps://david.ncifcrf.gov/) was used to validate mRNAs in the ceRNA network, to identify enriched pathways in KEGG  PATHWAYS and biological processes and cell components in Gene Ontology . P values <0.05 were used in all enrichment analysis.DAVID  was used to analyze the DEmRNAs in the ceRNA network between CESC and normal samples. A https://www.proteinatlas.org) maps all the human proteins in cells, tissues, and organs. It can help to better understand the expression of different proteins in normal and pathological tissues and provide valuable information to further identify meaningful biological biomarkers.The Human Protein Atlas database  , 773 DElncRNAs , and 94 DEmiRNAs . Differentially expressed RNA-encoding genes are shown on volcano plots and heatmaps .The lncRNA-miRNA-mRNA ceRNA network was reconstructed based on the relationship between differentially expressed lncRNAs, miRNAs, and mRNAs determined with WGCNA and visualized by Cytoscape, the selected soft threshold power parameter were 5,6,5 in the subnetwork of lncRNA-mRNA, lncRNA-miRNA, and miRNA-mRNA  project  providesThe protein-protein interaction network among proteins encoded by DEmRNAs in ceRNA was constructed using the online tool STRING database, and the network was visualized using Cytoscape. The complete network contains 144 nodes and 285 edges, as shown in .P < 0.05. Survival analysis of all the clinical data shows that the expression of 13 DEmRNAs  was correlated to the prognosis of patients, among which ADGRF4, ANXA8L1, HCAR3, IRF6, and PDE2A were negatively correlated to survival time  algorithm  implemenPDE2A is highly and specifically expressed in the brain and in certain tumors and involved in the pathophysiology of related diseases . PDE2A iMost of the current researches on PDE2A are pursued in neuroscience. Conversely, there is very little literature on the direct relationship between PDE2A and tumors, and its involvement in the mechanism of appearance and development of tumors is not clear. Even in neuroscience, the research on PDE2A is still in its infancy. Some researchers  have proBased on the analysis of data from public databases, PDE2A might be a biomarker for early diagnosis and prognosis evaluation of CESC patients, although the lack of detailed clinical and experimental data requires further studies.