Pure iron has been confirmed as a promising biodegradable metal. However, the degradation rate of pure iron should be accelerated to meet the clinical requirements. In this work, two different designs of platinum disc arrays, including sizes of Φ20 μm × S5 μm and Φ4 μm × S4 μm, have been coated on the surface of pure iron. Corrosion tests showed the platinum discs formed plenty of galvanic cells with the iron matrix which significantly accelerated the degradation of pure iron. Simultaneously, due to the designability of the shape, size as well as distribution of Pt discs, the degradation rate as well as degradation uniformity of pure iron can be effectively controlled by coating with platinum discs. The cytotoxicity test results unveiled that Pt discs patterned pure iron exhibited almost no toxicity to human umbilical vein endothelial cells, but a significant inhibition on proliferation of vascular smooth muscle cells. In addition, the hemolysis rate of Pt discs patterned pure iron was lower than 1%. Moreover, Pt discs also effectively reduced the number of adhered platelets. All these results indicated that Pt discs patterning is an effective way to accelerate degradation and improve biocompatibility of pure iron. Biodegradable stents are considered to be the next generation of stents23456in vitro and in vivo investigations912131415Iron is an essential nutrient element in human body which plays a vital role in many biochemical reactions, such as induction and transportation of oxygen, electron transferring, catalyst, and so forth29Up to now, numerous methods have been tried to enhance mechanical properties and corrosion rate of pure iron, such as alloying3141718202122Surface patterning has been demonstrated as an effective way to adjust the adhesion, stretch and proliferation of cells, thereby improving the interaction between implants and the host282931In this work, platinum disc arrays were prepared on the surface of pure iron through photolithography and electron beam evaporation. Platinum has been confirmed to be a material with excellent hemocompatibilityAfter 3 days immersion, the surface of pure iron kept intact, only a few deposited salts were observed. As for Pt discs patterned pure iron samples, corrosion happened first on the iron matrix which closely surrounded Pt discs, and this phenomenon was more obvious on the Φ4 μm × S4 μm samples.After 7 days immersion, grain boundary could be observed clearly on the surface of uncoated pure iron. On the surface of Pt discs patterned pure iron, the corrosion range continued spreading outward.After 14 days immersion, the corrosion of uncoated pure iron relatively deepened. On the surface of Pt discs patterned pure iron, the corrosion extended to the whole exposed pure iron matrix. In a macro perspective, the corrosion was uniform.After 28 days immersion, on the surface of uncoated pure iron, it can be clearly observed that corrosion directions varied on grains with different orientations. Corrosion on the surface of Pt discs patterned pure iron started penetrating into the iron matrix covered by Pt discs.in situ. On the surface of Pt discs patterned pure iron with size of Φ20 μm × S5 μm, corrosion of pure iron matrix continued stretching to the center under Pt discs.After 42 days immersion, localized corrosion pits began emerging on the surface of uncoated pure iron. Pt discs with size of Φ4 μm × S4 μm fell off from the pure iron matrix and corresponding corrosion pits remained −1) > Φ20 μm × S5 μm (48.784 ± 1.215 μg∙mL−1) > uncoated pure iron (13.601 ± 0.5482 μg∙mL−1), which matched well with the corrosion results of static immersion tests in Hank’s solution. The corrosion rate of Pt discs patterned pure iron in DMEM was about 4 times faster than that of uncoated pure iron. Furthermore, because of the high chemical stability of Pt, Pt ion concentration was very low. According to 32335Accelerating degradation rate of pure iron has puzzled scholars for many years. In this study, Pt disc patterns were first adopted to regulate the corrosion behavior of pure iron for biomedical applications. It is widely believed that there are two main methods to accelerate corrosion of pure ironDue to the solution alkalization near Pt discs , iron hyin situ. Consumption of OH− on the tip of corrosion pits formed an acid atmosphere in this region, then pure iron in this region can act as anode. The corrosion current density on this tiny anode can be very large so that pure iron matrix in this region corroded rapidly. Therefore, the depth of corrosion pits increased gradually with time. In addition, as the existence of corrosion pits increased the contacting area of pure iron matrix with corrosive solution, then accelerate the corrosion rate. Therefore, the accelerating effect on degradation of iron matrix can be lasted even after the dropping of Pt discs  and Pt discs (A2) should be a constant value , which can be expressed as A1 + A2 = A. The corrosion current of Pt discs patterned pure iron can be calculated by Equation 5The phenomenon that the Pt discs patterned pure iron with size of Φ4 μm × S4 μm corroded faster than that with size of Φ20 μm × S5 μm can be explained by theoretical calculations. Pt discs covered corresponding surface area of pure iron, so the sum of area of exposed iron matrix , take the derivative of Based on A2/A1 = βc2/βa1, when βa1, βc2 can be obtained as 0.399 and 0.102, respectively. Hence, βc2/βa1 = 0.25564 and A2/A can be calculated to be 0.2036. That is, when the area of Pt discs occupies 20.36% of the total area, the corrosion current will achieve to its maximum value. By contrast, the bigger gap between 0.2036 and real value of A2/A, the smaller the corrosion current is. In terms of Pt discs patterned pure iron with size of Φ4 μm × S4 μm, the value of A2/A is 0.19635, which is very close to 0.2036. However, the A2/A value of Pt discs patterned pure iron with size of Φ20 μm × S5 μm is 0.50265, which is far away from 0.2036. Therefore, according to above mentioned theoretical calculation, Pt discs patterned pure iron with size of Φ4 μm × S4 μm can corrode faster than the one with size of Φ20 μm × S5 μm.2O3 composite (1.38 times)The corrosion rates of Φ20 μm × S5 μm and Φ4 μm × S4 μm Pt discs patterned pure iron in static immersion test were about 2.33 and 2.58 times as that of pure iron, respectively. Compare to the previous reports2336402+ and Fe3+ ions. According to the work of Zhu−1. The concentration of iron ions of uncoated pure iron, Φ4 μm × S4 μm and Φ20 μm × S5 μm Pt discs patterned pure iron were 13.601 μg∙mL−1, 52.494 μg∙mL−1 and 48.784 μg∙mL−1, respectively. Although the iron ion concentration was slightly higher than 50 μg∙mL−1 for Φ4 μm × S4 μm Pt discs patterned iron, yet no significant cytotoxicity was observed. All the experimental materials showed no obvious toxicity to EA. hy-926 cells. On the contrary, all of these materials strongly hindered the proliferation of vascular smooth muscle cells. Muller et al.et al.2+ and Fe3+ ions could repress the migration of smooth muscle cells at the concentration of 1 mM, while good endothelial coverage were found on iron wires.The biocompatibility of Pt discs patterned pure iron can be discussed from two aspects, including cytotoxicity and hemocompatibility. The cytotoxicity of biodegradable metallic materials are mainly attributed to the released metallic ionsIn terms of hemocompatibility, Pt was confirmed as a very good stent material and has been applied in clinic, such as platinum-iridium alloy stents454649Then the number of adhered platelets on the surface of Pt discs patterned pure iron was much less than that on the surface of uncoated pure iron. All these results proved that Pt patterning has the potential to decrease the risk of thrombosis of pure iron.in vitro was investigated by electrochemical tests and static immersion tests. The results indicated that coating platinum disc arrays on the surface of pure iron can form a plenty of galvanic cells to accelerate the degradation of pure iron. Simultaneously, due to the designability of the shape, size as well as distribution of Pt discs, the degradation rate as well as uniformity of pure iron can be effectively controlled by coating with platinum discs. The in vitro biocompatibility of Pt discs patterned pure iron has also been researched by indirect cytotoxicity tests, hemolysis tests and platelet adhesion tests. Pt discs patterned pure iron exhibited almost no toxicity to human umbilical vein endothelial cells (EA. hy-926), but performed a significant inhibition on proliferation of vascular smooth muscle cells (VSMC). In addition, the hemolysis of Pt discs patterned pure iron was lower than 1%. Moreover, the number of adhered platelets on pure iron coated with platinum discs was less than that on uncoated pure iron. Especially for pure iron coated by Pt discs with size of Φ4 μm × S4 μm, the number of adhered platelets was the lowest. These results indicated pure iron patterned by platinum discs have the potential to decrease the risk of thrombosis.In summary, two different designs of platinum disc arrays, including sizes of Φ20 μm × S5 μm and Φ4 μm × S4 μm, have been coated on the surface of pure iron in this work. The influence of Pt disc arrays on the degradation of pure iron matrix 2), then covered by a lithography mask. Afterwards, specimens were exposed to ultraviolet light and the part without the protection of lithography mask was rinsed in organic solvent (4 wt.% NaOH solution). After rinsing, a layer of photoresist with via-hole arrays was obtained. Electron beam evaporation was adopted to deposit Pt  Technology Co., China) on the surface of pure iron across the via-holes. Lastly, photoresist layer was removed using acetone.Two different designs of platinum disc arrays, including sizes of Φ20 μm × S5 μm and Φ4 μm × S4 μm, have been deposited on the surface of pure iron  Technology Co., China) through photolithography and electron beam evaporation. Pt disc arrays on the surface of pure iron was observed using environmental scanning electron microscope , with an energy dispersive spectrometer (EDS) attached. The energy dispersive spectrometer was employed for the analysis of chemical composition.2. The open circuit potential (OCP) measurement was set for 9000 s. The potentiodynamic polarization curves were carried out from  mV (vs. SCE) to  mV (vs. SCE) at a scanning rate of 0.33 mV·s−1. An average of at least three measurements was taken for each group. The corrosion rates were calculated according to ASTM-G102-89Electrochemical measurements were carried out using a traditional three-electrode cell at an electrochemical work station . The specimen, a saturated calomel electrode (SCE) and a platinum electrode were acted as working electrode, reference electrode and the auxiliary electrode, respectively. All the measurements were maintained at temperature of 37 ± 0.5 °C in Hank’s solutionVcorr (mm/yr) is the corrosion rate in terms of penetration rate, CR (mg·cm−2·day−1) is the corrosion rate in terms of mass loss rate, EW is the equivalent weight, K1 = 3.27 × 10−3 mm·g·μA−1·cm−1·year−1 and K2 = 8.954 × 10−3 g·cm2·μA−1·m−2·day−1.where Static immersion tests were performed in Hank’s solution for 3, 7, 14, 28 and 42 days, respectively. 50 mL Hank’s solution was used for each sample following ASTM-G31-72CR (mg·cm−2·day−1) is the corrosion rate, m (mg) is the mass loss, S (cm2) is the surface area of the specimen exposed to the solution and t (day) is the immersion time.where −1 penicillin and 100 μg·mL−1 streptomycin at 37 °C in a humidified atmosphere with 5% CO2. According to ISO 10993-122·mL−1 in a humidified atmosphere with 5% CO2 at 37 °C for 72 h. After the extracts were centrifuged, the supernatant fluid was withdrawn and stored at 4 °C before cytotoxicity test. The control groups involved DMEM medium as the negative control and DMEM including 10% dimethyl sulfoxide (DMSO) as the positive control. The concentrations of metallic ions in the extraction medium were measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) . Cells were incubated in the 96-well plates at the density of approximately 5 × 103 cells per 100 μL medium in each well and incubated for 24 h to allow attachment. DMEM was then replaced by extraction mediums, and 10 μL serum was added to each well. After 1, 2 and 4 days incubation, 10 μL of cell counting kit (CCK-8) solution was added to each well, then continued incubating for 3 h. The absorbance of each well was tested using microplate reader (Bio-RAD680) at the wavelength of 450 nm. Viability of cells (X) was calculated using the following formula according to ISO 19003-5The cytotoxicity tests were performed by indirect contact methods. Human umbilical vein endothelial cells (EA. hy-926) and human vascular smooth muscle cells (VSMC) were used to evaluate the cytotoxicity of the Pt discs patterned pure iron. At first, cells were cultured in the Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U·mLOD1 is the mean absorbance of experimental sample groups and positive control group. OD2 is the mean absorbance of negative control group.Here Healthy human blood (anticoagulant was 3.8 wt.% citric acid sodium) extracted from volunteers was diluted by physiological saline based on volume ratio of 4:5. Uncoated pure iron and Pt discs patterned pure iron were separately put into centrifugal tubes with 10 mL physiological saline for 30 minutes, temperature was kept at 37 °C. Then 0.2 mL diluted blood was added to each tube and incubated at 37 °C for 60 minutes, 10 mL deionized water with 0.2 mL diluted blood as the positive control and 10 mL physiological saline with 0.2 mL diluted blood as the negative control. After completion of the above steps, samples were removed, and then these tubes were centrifuged at 800 g for 5 minutes. Supernatant was transferred to 96-well plates, the absorbance (OD) was determined by a microplate reader (Bio-RAD680) at the wavelength of 545 nm. Hemolysis of samples was calculated by the following formula:For platelet adhesion, whole blood from healthy human body was centrifuged at 1000 r/min for 10 minutes. Platelet rich plasma (PRP) was obtained from the upper fluid. Samples after ultraviolet disinfection were moved to 24-well plates and 0.2 ml PRP was added to each well, then incubated at 37 °C for 1 h. After gently rinsed by phosphate buffered saline (PBS), platelets on samples were fixed using 2.5% glutaraldehyde solution at room temperature for 1 h. Then dehydrated with gradient alcohol solution , each concentration for 10 min, and finally freeze-dried for 2 days. The morphologies of platelet adhered on the specimens were observed by ESEM (Quanta 200FEG).All quantitative data are expressed as mean ± standard deviations with n = 3. Statistical analysis was performed by one way analysis of variance (ANOVA) followed by Tukey’s post hoc tests using SPSS 19.0 and p-values less than 0.05 were considered statistically significant.How to cite this article: Huang, T. and Zheng, Y. Uniform and accelerated degradation of pure iron patterned by Pt disc arrays. Sci. Rep.6, 23627; doi: 10.1038/srep23627 (2016).


We assess the additional forest cover protected by 13 rural communities located in the southern state of Chiapas, Mexico, as a result of the economic incentives received through the country's national program of payments for biodiversity conservation. We use spatially explicit data at the intra-community level to define a credible counterfactual of conservation outcomes. We use covariate-matching specifications associated with spatially explicit variables and difference-in-difference estimators to determine the treatment effect. We estimate that the additional conservation represents between 12 and 14.7 percent of forest area enrolled in the program in comparison to control areas. Despite this high degree of additionality, we also observe lack of compliance in some plots participating in the PES program. This lack of compliance casts doubt on the ability of payments alone to guarantee long-term additionality in context of high deforestation rates, even with an augmented program budget or extension of participation to communities not yet enrolled. Payment for Environmental or Ecosystem Services (PES) programs have become increasingly important for biodiversity and ecosystems conservation worldwide, including in developing countries –3. ProofPES programs are voluntary transactions between at least one buyer and at least one seller, in which payments are transferred in exchange for the preservation of a well-defined environmental service ,7. PES aPago por Servicios Ambientales Hidrológicos (PSAH) scheme is managed by the National Forestry Commission (CONAFOR) and has been operational since 2003   30] pre-ejidos that received PES. We matched across biophysical characteristics: forest area in 2007, elevation, slope, deforestation risk index and the spatial lags for each of the four covariates. We also added an exact matching on the dominant soil in the cells, which served both as an index of relative soil productivity and as a way to control for the geographic location of the cells. Indeed, visual interpretation of the region's geomorphological map suggested that alluvial soils border rivers and protected areas while other soils are more present in remote areas. Our set of physical variables was assumed to capture opportunity costs at parcel levels. Because the control group included only parcels of ejidos treated in 2008 or 2009, we were neutralizing the influence of unobservable characteristics at ejido level such as decision-making processes and motivations. We present the covariate balance for all observable characteristics . In this case, non-treated ejidos correspond to those that had never received a PES contract between 2005 and 2012.However, we suspect specification A may be biased because each ejidos using some of the variables proposed by [ejido, we identified the most similar non-treated ejido in terms of socio-economic and demographic characteristics. In doing so, we used the following variables: distance to the nearest city, years since ejido creation, size, population with property rights, and other demographic information . The heterogeneity of ejido characteristics is very important, so only, eight non-treated ejidos were retained in our control group. The comparison of conservation outcomes between treated and matched ejidos would not be sufficient to measure program impacts. We then performed covariate matching with physical variables defined in the first specification. Therefore, for each treated parcel of a treated ejido, matched cells were more physically similar inside the more similar non-treated ejido , which corresponds to the average impact of the program on forest conservation for units that have received the program. In all our matching specifications, we employed a bias-adjustment procedure as proposed by  and we uejido level. These variables related to the evolution of total population, marginality index, proportion of active population (between 14 and 65 years-old) and proportion of people with secondary education.To test the robustness of each specification, we used a difference-in-difference (DID) estimator to control for time invariant factors that jointly affected control and treated units. We estimated the DID with i) an Ordinary Least Square (OLS) model and with ii) a Panel Fixed-Effects (FE) model. Our OLS specification contained our set of physical and socio-economic variables likely to have an influence on forest conservation independently of PES payments . Becauseejidos have a higher deforestation risk than treated parcels (respectively 0.074 against 0.066) . The similarity of these results can be an indication of the absence of leakage effect in our first specification.We estimated the ATT for our three above-described matching specifications  . In our ejidos, the additionality is relatively lower that if we only compare forest parcels that are similar in terms of biophysical characteristics. These differences are probably due to the fact that socio-economic and institutional factors have an impact on success of the program at community level.The last specification, which includes socio-economic variables, provides an estimation of the ATT that is substantially lower than the estimate from the second specification by nearly 0.2 ha by cell (but the avoided deforestation still represent about 12 percent of the enrolled area). Thus, if we compare similar parcels in similar We performed sensitivity analysis over three dimensions. First, we considered how the magnitude of the ATT estimate changed in response to modifying the matching parameters. Second, we check if the ATT estimate was sensitive to the threshold we outlined for the definition of minimum forest cover and minimum overlap with PES polygons. As observed in We check this result by analyzing how estimates of additionality resulting vary with changes in the minimum PES overlap. Our 8 ha threshold restricts the treated units to only those largely overlapping PES polygons and, when doing so, the additionality in all our specifications diminishes. We attribute this result to the fact that some cells imperfectly overlapping PES polygons were considered as treated with a less strict threshold but became considered as control now.Finally, we performed additional estimations to assess the robustness of the previous estimation of program impacts . In our Our results show that Mexico's PES program for biodiversity conservation has been effective in enrolling areas that generally show high deforestation risk. We also have shown that on average, treated parcels in recent cohorts and un-enrolled parcels have similar deforestation risk, which suggests that participation in PES cannot be explained only by land opportunity costs but, as other studies have suggested, also by factors related to collective decision-making and local governance . The proejidos could enroll in programs was 20 ha, but in recent years the complementary Special Program for the Lacandon rainforest allows this minimum area to be split between several parcels.The program's additionality diminishes to around 20% when taking into account socio-economic variables. Given the small number of observations, however, it has not been possible to determine the relative contribution of leakage effects to such diminished additionality. Robustness analysis confirms additionality but variations are found when testing for different classification thresholds for determining land use units comparison groups with respect to the size of the forest parcel and the level of overlap with the PES polygons. Lower additionality in PES lands with smaller forests suggests additionality could be lower in already fragmented forests surrounding agricultural or pasture lands. Further analysis is needed to confirm this relation but it raises doubts about the possibility to maintain additionality for current cohorts under contract. Indeed, in 2008, the minimum area that de jure commonly held and managed land; de jure commonly held land but de facto divided by the assembly and household managed; and de jure household-owned land. Additionally, we observed that un-enrolled parcels of treated ejidos have been less deforested than parcels of non-treated ejidos despite a higher deforestation risk index for the former can imply the presence of a positive spillover of the program. For example, some treated ejidos in the area have been able to successfully develop ecotourism projects in parallel with the enrollment of their forest program [The analysis also reveals that additionality can be achieved despite low compliance levels in some PES areas (i.e. plots under PES lands have been losing around 10% of forest cover during the contract). We argue that the lack of compliance can be explained by constrained forest governance at collective or household levels coupled with deforestation inertia, as well as by the government's insufficient enforcement of program rules. Regarding the former, it is important to highlight that non-compliant PES areas can encompass three types of tenure arrangements, namely:  program . Howeverejido level, and lack of enforcement of the PES program by CONAFOR. The long-term permanence of current high additionality levels in the studied municipalities is likely to be dependent upon sustaining payments over time and keeping PES targeted areas sufficiently large, in combination with alternative conservation strategies ranging from improved command and control approaches to investment into more diversified forest agricultural economies. A comprehensive conservation and development strategy appears as the necessary next step to take into account all together conservation priority zones, biodiversity corridors and agricultural development hot spots [Overall then, given that deforestation has proceeded apace in non-PES and a minority of PES targeted areas during the study period, we think that the PES program has been insufficient to halt deforestation at municipal level. This outcome is explained by the existence of incentives to convert forest lands to more profitable land uses such as oil palm cultivation ,37, resoot spots .S1 FileTo perform this analysis, we have used databases provided by different organizations. We provide information on the availability for each of these sources.(DOC)Click here for additional data file.S2 File(ZIP)Click here for additional data file.


The community research fellows training (CRFT) program is a community-based participatory research (CBPR) initiative for the St. Louis area. This 15-week program, based on a Master in Public Health curriculum, was implemented by the Division of Public Health Sciences at Washington University School of Medicine and the Siteman Cancer Center.We measure the knowledge gained by participants and evaluate participant and faculty satisfaction of the CRFT program both in terms of meeting learning objectives and actively engaging the community in the research process.We conducted analyses on 44 community members who participated in the CRFT program and completed the baseline and follow-up knowledge assessments.Knowledge gain is measured by a baseline and follow-up assessment given at the first and final session. Additionally, pre- and post-tests are given after the first 12 sessions. To measure satisfaction, program evaluations are completed by both the participants and faculty after each topic. Mid-way through the program, a mid-term evaluation was administered to assess the program’s community engagement. We analyzed the results from the assessments, pre- and post-tests, and evaluations.p < 0.0001). Post-test scores were higher than pre-test scores for 11 of the 12 sessions. Both participants and faculty enjoyed the training and rated all session well.The CRFT participants’ knowledge increased at follow-up as compared with baseline on average by a 16.5 point difference (The CRFT program was successful in increasing community knowledge, participant satisfaction, and faculty satisfaction. This success has enhanced the infrastructure for CBPR as well as led to CBPR pilot projects that address health disparities in the St. Louis Greater Metropolitan Area. The benefits of community-based participatory research (CBPR) have been well established; however, there is limited information on developing the infrastructure and increasing community capacity to partner in CBPR projects. Participating in public health research training can prepare community members for collaborative work with academic researchers and empower them to act as equal partners in the research process , 2. In aIn 2010, a similar training program, Community Alliances for Research Empowering Social Change (CARES), was initiated on Long Island, New York , 4. ThisThe CARES academic-community collaboration developed a training program for community members based on the standard Master of Public Health curriculum, designed to implement culturally appropriate ways to increase research literacy among community members . The CRFCommunity-based participatory research emerged from research traditions of 1980s and 1990s that focused on engaging stakeholders affected by the public health concerns at hand . CommuniSeveral studies suggest that medical mistrust and negative encounters with health care personnel are closely linked to racial disparities in health , 18. AttEnhance community knowledge and understanding of researchCreate a pool of trained community members who can serve on Institutional Review Boards and community research advisory boardsDevelop CBPR pilot projects that address health disparities in the St. Louis Greater Metropolitan AreaEnhance the infrastructure for CBPRProvide community members with skills to engage as equal partners in every phase of the research process.The CRFT program was a pilot project of the Program to Eliminate Cancer Disparities at the Siteman Cancer Center (SCC), Barnes Jewish Hospital, and WUSM. The goal of CRFT was to train community members to serve as the bidirectional conduit between WUSM/SCC and communities in St. Louis. A community advisory board (CAB) was formed to help guide all aspects of the CRFT program including recruitment and acceptance decisions, program implementation, selection of pilot projects, and evaluation of the program. The objectives of this training were toThe CRFT program sought to involve community members in research methods training designed to help them to become good consumers of research; understand the utility of research in improving health outcomes in their communities, increase their understanding of how to work with academic researchers, and develop skills that increase capacity for organizations and communities to engage in research. To this end, a 15-week training course, adapted from the CARES program, was designed and implemented . It is iTwelve CAB members, 17 faculty members, and 10 research assistants were involved in the conception, planning, and implementation of the CRFT program . While pThe training uses multiple teaching approaches  to explain topics in ways that reach a variety of learning styles . ConsistThe Human Research Protection Office at WUSM classified this study as program evaluation and non-human subjects’ research. Analysis was completed using SAS/STAT 9.4 . Of the 50 fellows enrolled in the CRFT program, 45 (90%) completed the 15-week training program and 44 (88%) completed both the baseline and follow-up assessments. The majority of the fellows who completed both the baseline and follow-up assessments were female (84%) and African-American/Black (86%). The fellows ranged in age from 28 to 72, with a mean age of 51. The cohort of fellows was comprised of community members (30%), those affiliated with community-based organizations (23%), health care workers (21%), government workers (11%), those affiliated with faith-based organizations (9%), and those in academia (7%). Education attainment ranged from junior high school to those with a graduate degree. Over half (57%) of fellows self-reported previously taking a research course  were well organized.The facilitator(s) seemed knowledgeable about the subject.Overall, how would you rate this session?At the end of each of the 12 didactic training sessions, fellows completed evaluations. Evaluations consisted of six statements with Likert-scale response options and four free response questions; eight of the sessions had two parts, and therefore, two evaluations, making for a total 20 evaluation questions. We focus on the quantitative Likert-scale response questions for this analysis. Fellows were asked to rate the following statements from 1 to 5, with 1 being “Strongly Disagree” and 5 being “Strongly Agree” for evaluation questions 1–5, and 1 being “Poor” and 5 being “Excellent” for the sixth evaluation question:The mean for each statement is computed, as well as a range of within-session means.Community Research Fellows seemed well prepared for today’s training sessionCommunity Research Fellows frequently took notes in the training sessionCommunity Research Fellows contributed to discussions in the training sessionCommunity Research Fellows provided comments that were insightful and constructiveCommunity Research Fellows asked insightful and constructive questionsCommunity Research Fellows listened attentively and seemed interested when I presented materials and informationOverall, how would you rate your experience teaching this session?The CRFT CAB felt it was important to also assess satisfaction with teaching in the program. A main goal of the CRFT program is to create a bridge between the St. Louis community and researchers; to foster this connection, faculty satisfaction is imperative. Fifteen faculty members taught the 12 didactic training sessions and completed evaluations. The CRFT faculty evaluation consisted of seven Likert response items:Would you be willing to teach again for the CRFT program in the future?Did you learning anything from the Fellows during the training session?Are you willing to collaborate with Fellows on a CBPR pilot project?Items 1–6 have Likert-scale responses 1 to 5, with 1 being “Strongly Disagree” and 5 being “Strongly Agree” and item 7 has Likert-scale responses with 1 being “Poor” and 5 being “Excellent.” In addition, there were three yes/no items:This analysis only examines the 10 close-ended questions on the faculty evaluation which also included 3–4 opened ended response questions depending on answers to the close-ended questions. The mean, SD, minimum and maximum for each of the 7 Likert response items were computed. The three dichotomous response questions were examined by the percent reporting yes to each question.t-test for this knowledge change was highly significant (p < 0.001). Of the 29 questions, on average fellows answered about 7 (23.5%) questions essentially correctly at baseline  and 16 (54.8%) questions essentially correctly at follow-up . We then looked at fellows’ responses to see if each individual question’s score increased, decreased, or remained the same from baseline to follow-up . This corresponds to an absolute percent increase of 28.4% . Only three fellows  decreased their scores from baseline to follow-up. The paired For each of the 29 questions, we examined which fellows scored “essentially correct” on the baseline assessment as compared to scoring “essentially correct” on the follow-up assessment. Using McNemar’s test, we found that 24 of the 29 questions had statistically significant results, suggesting that there was evidence of improvement from baseline to follow-up Table . This fup-value of 0.778 and 0.783, respectively. We performed a two-sample t-test and found that the score differences are statistically significant, with a p-value of 0.038, indicating that although both groups had significant overall increase of knowledge from the baseline to the follow-up assessment, fellows who had not previously taken a research course had a significantly higher increase in knowledge. This suggests that both fellows who had taken a research course previously and those who had not, gained knowledge from the CRFT program, and that the knowledge gained was more substantial for those who had never taken a research course. This further emphasizes that this course is well designed, both for those who have some previous knowledge about research, and for those who lack it completely.We then examined the sample stratified by whether or not the fellow had taken a previous research course. Twenty-five of the 44 fellows (56.8%) self-reported previously taking a research course. This group had a mean difference between the baseline and follow-up assessment scores of 13.4, or 23.1% absolute increase. The remaining 19 fellows (43.2%) had a mean difference between baseline and final assessment scores of 20.6, or a 35.5% absolute increase in score. The scores in both groups are normally distributed, with a Shapiro–Wilk p  = 0.001, <0.001, <0.001, <0.001, and 0.028, respectively); the post-test score for session 3, Public Health Research, was significantly lower than the pre-test score (p  = 0.003) , 8 (CBPR), 9 (Research Ethics), 10 , and 11  post-test scores were significantly higher than pre-test scores (3) Table .Overall, session evaluations were high, with all session mean evaluation scores between 4 and 5 on a five point Likert-scale, with 4 indicating “Good” or “Agree” and 5 indicating “Excellent” or “Strongly Agree” depending on the question Figure . The aveFaculty evaluations are an integral part of the CRFT program’s evaluation. To note, 15 (100%) of the faculty members responded that they would be willing to teach again for the CRFT program. Thirteen (87%) responded that they learned something from the fellows during the training session and 15 (100%) responded that they are willing to collaborate with fellows on a CBPR pilot project. The mean overall faculty rating of their teaching experience is 4.8 (Table n = 44), we chose robust tests to evaluate all aspects of the CRFT program.We assessed participant knowledge gain and evaluated participant and faculty satisfaction. The CRFT program evaluation results build upon and enhance the positive results demonstrated in the CARES program. The completion rate in the CRFT program was much higher than that in the CARES program; 90% of the fellows completed the 15-week training, as opposed to 68% in the CARES program, and 88% completed the baseline and follow-up assessments, as compared to 58% in the CARES program . AdditioThis quantitative evaluation analysis is just one component of the comprehensive (formative and summative) mixed-methods  evaluation of the CRFT program. The use of multiple items to assess participant knowledge and satisfaction allows for triangulation of results. For example, Session 3 Public Health Research was the only session to see a significant decrease in score on the post-test compared to the pre-test. However, in the session evaluation, participants said that the information learned in the session was helpful; more so than any other session (highest mean). This suggests a need to refine items on assessment instruments, specifically pre- and post-tests. Session pre- and post-tests consisted solely of multiple-choice items to help reduce participant burden and time of administration. However, we needed to increase the number of questions (from 5 to 10) to increase the variability of question types  thus increasing variability in scores across participants; a major limitation is some participants felt like they were taking a test. Inconsistency between pre-test and post-test scores could be due to differences in items used on the assessments. In future work, we examine the use of the same items (in a different order) on the pre-/post-test.The participant and faculty evaluations were similarly positive, with all averages above four on a five point Likert-scale, averaging between “Good” and “Excellent” or “Agree” and “Strongly Agree” depending on the question. The overall satisfaction, both from the participant and the faculty member’s perspective, is crucial in evaluating a CBPR program. Our assessment of the knowledge gained by fellows through the CRFT program confirms that the first, second, and fifth CRFT program goals have been met. Our evaluation of participant and faculty satisfaction suggests the fourth program goal has been met. In terms of the third program goal, the CRFT program has funded two pilot projects, led by participants in the program, and two more projects have been funded by other sources; we believe that CRFT has been instrumental in enhancing the CBPR infrastructure in St. Louis, opening the doors to collaboration in the future. CRFT pilot projects extend Fellows learning beyond the classroom setting to implementing CBPR projects in real community-based settings, demonstrating the ability of the course to create community–academic partnerships that address community driven research questions .The CRFT program was successful in increasing participant knowledge in public health topics, such as epidemiology, CBPR, research ethics, and clinical trials, as evidenced by the increased scores from the baseline to final assessment and positive difference in pre- and post-tests administered after each session. At the initiation of this program, we set out to achieve five goals.The first goal was to enhance community knowledge and understanding of research. From a quantitative perspective, there is evidence of knowledge gained with an improvement from 20.6 on the baseline assessment to 37.1 on the final assessment (mean change of 16.5) and the mean increase in post-test scores compared to pre-test scores. This demonstrates that the course content (Table The second goal was to create a pool of trained community members who can serve on Institutional Review Boards and community advisory boards. While the quantitative data here suggest that the fellows, on average, would have the knowledge base to serve on these boards, the actual outcomes more clearly support this objective. CRFT alumni have formed their own Patient Research Advisory Board and are currently serving on at least five advisory boards and community partnerships across the WUSM medical center; five CRFT alumni current serve on the CRFT community advisory board.The third goal was to develop CBPR pilot projects that address health disparities in the St. Louis Greater Metropolitan Area. The knowledge gained, as demonstrated here, was a springboard for 9 groups consisting of a total of 30 fellows, to submit brief proposals for pilot projects. Four of these were funded, and are currently underway in the St. Louis Greater Area .The fourth goal was to enhance the infrastructure for CBPR. The gathering of community members and faculty for bidirectional learning was successful in developing CBPR infrastructure, and the positive participant and faculty evaluations suggest both groups enjoyed this approach to partnership development. Additionally, the four pilot projects demonstrate that the foundation laid by this course created a conducive environment for community members to become involved in the research process.Finally, our fifth goal was to provide community members with skills to engage as equal partners in every phase of the research process. Our course covers the spectrum of the research process, from research methods, to quantitative and qualitative analysis, to research ethics, synthesis, and evaluation. We demonstrate here, through the increase in scores, that knowledge was gained in these areas, giving the participants the information needed and pilot projects provide an opportunity for this goal to be realized. Overall, the CRFT program was successful, as evidenced by the knowledge gained, the positive participant and faculty evaluations, and the movement toward initiating fellow-inspired community-centric pilot projects.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.


As a result of whole genome duplication in the ancestor of the salmonid fish family, Atlantic salmon  possess two highly similar Ig heavy chain gene complexes (A and B), comprising two μ genes, two δ genes, three intact τ genes and five τ pseudogenes. The μA and μB genes correspond to two distinct sub-populations of serum IgM. The IgM-B sub-variant has a characteristic extra cysteine near the C-terminal part of the heavy chain and exhibits a higher degree of polymer disulfide cross-linking compared to IgM-A. The IgM-B:IgM-A ratio in serum is typically 60:40, but skewed ratios are also observed. The IgT isotype appears to be specialized to mucosal immune responses in salmonid fish. The concentration of IgT in serum is 100 to 1000 times lower than IgM. Secreted forms of IgD have been detected in rainbow trout, but not yet in Atlantic salmon.There are three major immunoglobulin (Ig) isotypes in  There are three major types of immunoglobulins in teleost fish: IgM, IgD and IgT/IgZ, the latter of which is unique for teleost fish . The ortThe IgM monomer has been relatively stable and maintained the primordial structure in all jawed vertebrates, but the primary amino acid sequence and the overall polymer structure has diverged considerably . The IgDsalmonid fish) is specialized to mucosal immune responses [The major systemic antibody in teleost fish is IgM, whereas IgT  has a fundamental role in the transport of IgA (and IgM) across the epithelial cell layer into the mucus. A part of the pIgR (secretory component) is bound to the antibody and protects it from degradation in the hostile mucosal milieu. A molecule homologous to pIgR has been identified in teleosts and shown to be involved in transport of IgM . HoweverThe first identifications of immunoglobulin genes in teleost fish about 25 years ago were based on immunoscreening, DNA-cross-hybridization and PCR-homology cloning, and later, by large-scale sequencing and searches of sequence databases. Molecular characterization of IgM provided an explanation to a previous observation: that membrane bound IgM in teleost fish appeared to be shorter than the mammalian counterparts . In conti.e., an IgM to IgG1 switch).The Ig heavy chain genes in teleost fish have a translocon type of organization, and the δ gene is located immediately downstream of the μ gene like in higher vertebrates. However, the τ genes are located between variable gene segments upstream of μ and δ . AccordiThe antibody repertoire in teleost fish is generated by V(D)J recombination of variable gene segments, enzymatic deletions or additions of nucleotides in the junctions, combinations of heavy and light chains, and subsequently, somatic mutations. Theoretically, there is a potential of creating an almost unlimited number of different antibodies, but it has been shown in humans that the usage of variable gene segments is not generally random. Some gene segments are used as little as 0.1%, whereas other are used more than 10%. As an outcome of evolution the expressed Ig repertoire is to some degree genetically determined , and it Teleost fish have two primary lymphoid organs; the anterior part of the kidney  and the thymus. The kidney serves as a secondary lymphoid organ as well, together with the spleen. The abundance of μ, τ and δ mRNA in teleosts is typically highest in head kidney and spleen, and correlates to a large degree with blood content in other tissues . In a stFish possess a secondary capillary system (homologous to lymph vessels) but lack lymph nodes . In mamm+ cells were found in the region that was examined [salmonid fish, and that this is an arena for B and T cell communication. In mammals, mucosal surfaces harbor aggregations of organized lymphoid cells, i.e., the tonsils in the pharynx and the Peyers patches of the intestines. The localization, architecture and arrangement of these immune tissues are of fundamental importance. In a study of rainbow trout it was found that the ratio of IgD to IgM in the gills was higher than in other tissues, indicating a significant role for IgD in the gills [The gill is an extremely important entry point for pathogens. The gill constitutes more than 90% of the surface area of the fish and represents a semi-permeable barrier to the surrounding environment. In sea bass, the gill was identified as a major organ for antibody secreting cell production . An intrexamined ,23,24. Hhe gills , and reche gills . The invhe gills ,26.Teleost fish is a very heterogenous group of animals; the number of species is estimated to be higher than 30,000. A whole genome duplication, which occurred early in the evolution of teleosts, more than 300 million years ago, has contributed to this diversity .salmonid fish family [salmonid fish. Due to highly similar stretches of DNA in the haploid genome it has been a challenge to assemble the complete genome sequences of species like rainbow trout and Atlantic salmon. However, the genome sequence of rainbow trout was published recently [A second whole genome duplication event, estimated to approximately 96 million years ago, occurred in the ancestor of the h family ,29. In ssalmonid fish may benefit from their partially tetraploid genome, for example in their adaptation to an anadromous life cycle and various environments [There has been much speculation on how ronments . It is pronments .In agreement with ancestral tetraploidy there are two highly similar Ig heavy chain gene complexes, named A and B in the Atlantic salmon genome . The A aversus IgM-A on the surface [The μA and μB genes correspond to two distinct IgM subpopulations in Atlantic salmon (IgM-A and IgM-B), which can be isolated from serum by gel filtration followed by anion exchange chromatography ,33,34,35 surface .Salmo genus, i.e., Atlantic salmon and brown trout [An extra cysteine residue near the C-terminal part of μB characterizes this sub-variant ,34. Compwn trout . As illuwn trout ,38.The fact that two Ig heavy chain gene complexes have been preserved in Atlantic salmon and brown trout during a long time in evolutionary terms, addresses questions regarding the biological significance of this phenomenon. Sequence analyses of the Atlantic salmon and brown trout μ genes indicate that recombination between the paralogous genes has occurred ,35. RecoBased on scanning electron microscopy photographs of IgM from some teleosts and estimation of the molecular weight of the polymers, IgM is regarded to be a tetramer in teleosts ,39,40. IThe 3D structure of teleost IgM is open to speculation. In contrast to mammals, where IgM cannot be secreted if it is not fully disulfide cross-linked, IgM tetramers of teleost fish are more loosely linked by disulfide bonding. In Atlantic salmon the degree of inter-heavy chain disulfide bonding appears to be higher in IgM-B than in IgM-A , presumaOur working hypothesis is that salmon IgM-A and IgM-B can be separated by anion exchange chromatography due to (1) distinct exposure of charged residues related to the polymer structure/post-translational modifications , and/or (2) a hitherto unidentified associated component. SDS-PAGE analyses have not revealed any additional bands that could indicate the presence of an associated molecule. However, an additional band might have been shaded by the Ig heavy or light chains. The Ig light chain bands vary extensively in size, and usually a spectrum of Ig light chains are seen by SDS-PAGE. Additional faint bands in the same region as the major Ig bands might represent posttranslational modifications.i.e., that high affinity B-cells produce more highly polymerized IgM, and that high-affinity highly polymerized antibodies possess longer half-lives than lower-affinity antibodies [In rainbow trout it has been shown that there is a correlation between greater antibody affinity and increased disulfide polymerization, tibodies ,45,46. Ietc. (Although knowledge about Ig concentrations in fish is somewhat anecdotal there is no doubt that there are huge differences between species. The reported serum concentrations of IgM in fish vary to a great extent, depending on infection and vaccination status, and with several other parameters like, age/size/weight, gender, season, environment, etc. .In some recent reports it has been shown that Atlantic salmon that have received commercial oil-adjuvanted vaccines have IgM concentrations that are two- to fourfold higher than in un-vaccinated fish. The depot effect of the mineral oil seems to trigger a continuous higher production of IgM which persists to the fish are slaughtered ,52. As sMagnadottir  did a cosalmonid fish have attracted much focus these last years [Exciting discoveries of IgD and IgT responses in mucosal tissues of st years ,8,25,26.st years . However


BCR-ABL1 at the Major Molecular Response (MMR) level is a well-established method used by clinical laboratories world-wide. However, the quality of cDNA provides sensitivity challenges and consequently affects the detection of Minimal Residual Disease (MRD).The synthesis of complementary DNA (cDNA) for use in the detection of ABL1 copy number. Based on our results, the Invitrogen SuperScript® III Reverse Transcriptase kit performed better, among the ones used in this study, for the cDNA synthesis, followed by the First Strand cDNA Synthesis Kit for RT-PCR (AMV), available from Roche Applied Sciences.Herein, we evaluated six commercially available kits for the synthesis of cDNA according to amplification success rate, linearity and BCR-ABL1 detection. We propose that the Invitrogen SuperScript® III Reverse Transcriptase kit is the most suitable, among the ones used in this study, for the cDNA synthesis to be used for the detection of BCR-ABL1 at the MMR level in a CML MRD assay.Accurate and sensitive testing for the detection of abnormal transcripts, allows the correct stratification and treatment of patients. Hence, the use of a suitable kit for the cDNA synthesis is of great importance. This study provides a comprehensive point of reference for clinical laboratories in an attempt to optimize The online version of this article (doi:10.1186/s12575-015-0014-x) contains supplementary material, which is available to authorized users. BCL-ABL1 fusion genes in hematopoietic progenitor cells, resulting from a reciprocal translocation between chromosomes 9 and 22, leading to a shortened chromosome 22, the Philadelphia (Ph) chromosome [BCR-ABL1 and its active protein tyrosine kinase product p210BCR-ABL [BCR-ABL1 after bone marrow transplantation is indicative of disease relapse [BCR-ABL1 transcripts present in the blood of patients, its detection is achieved by Real Time Quantitative Polymerase Chain Reaction (RQ-PCR), which detects abnormal transcripts with a sensitivity of 1 in 104 or 1 in 105 cells [BCR-ABL1 leukemia-specific targets, indicating patients with highest risk of relapse [Chronic myelogenous leukemia (CML) is a myeloproliferative disorder that accounts for about 20 percent of newly diagnosed leukemia cases in adults ,2, with romosome . In CML,0BCR-ABL -7. Recur relapse ,9. Follo05 cells . Minimal relapse . Such qu relapse ,14. It i relapse . Hence,  relapse .BCR-ABL1 transcript levels must be carried out every 3 months until a MMR has been achieved and then repeated at least every 6 months [BCR-ABL1 to ABL1 (or other housekeeping genes) x 100%; converted to the international scale, the ratio ≤ 0.1% defined MMR [BCR-ABL1 transcript levels ≤ 10% at 3 months, < 1% at 6 months, and ≤ 0.1% from 12 months onward are set to define optimal response, whereas > 10% at 6 months and > 1% from 12 months onward indicate failure, and call for a change of treatment (second-line therapy) [BCR-ABL1 transcript levels ≤ 10% at 3 and 6 months, < 1% at 12 months and ≤ 0.1% from 12 months onward are set to define an optimal response, whereas > 10% at 6 and 12 months indicate failure [According to the European LeukemiaNet (ELN), RQ-PCR assessment of 6 months ,18. Resuined MMR . BCR-ABLBCR-ABL1/ABL1 on a log scale [BCL-ABL1 quantitation by RQ-PCR defining an MMR [BCR-ABL1 ratio lower than 0.0032% on the international scale (IS) provided copy numbers for the control gene are at least 32 000 (i.e. a 4.5-log reduction)” [Existing guidelines, define an optimum response to therapy by the attainment of MMR by 18 months of the start of therapy ,19, withog scale ,22. Integ an MMR ,15. “MR4uction)” .BCR-ABL1 at the MMR level. In this study, we compare six commercially available cDNA synthesis kits  protocol for the detection of BCR-ABL1 and ABL1 gene using the TaqMan® method. The results are analysed and scored according to failure rate, linearity and ABL1 copy number.Currently, clinical laboratories world-wide are using different commercially available kits, based on different primers and enzymes, for the synthesis of cDNA. This can potentially influence the results and consequently the detection of MRD. Yet, no study is available comparing these kits in order to define whether these are suitable for the detection of BCR-ABL1 transcripts has been proven to be the most sensitive available method with a great prognostic impact and serving for an early assessment of MRD [ABL1 copy number and c) linearity.Serial measurement of leukemia-specific transcripts is a valuable approach for monitoring individual patients and in some cases for indicating the need to reassess therapy . Followit of MRD . Hence, BCR from the cDNA. The success rate was calculated based on the number of wells that were successfully amplified divided by the total well number for BCR amplification, with the enzyme with the highest success rate achieving the highest score (with the highest score being 6 and the lowest 1). All kits detected 0.32% BCR-ABL1 copy numbers. Using the SuperScript® III Reverse Transcriptase kit the BCR was successfully amplified from the cDNA at all levels, achieving a score of 6 (18/18). QuantiTect® Reverse Transcription, First Strand cDNA Synthesis kit for RT-PCR and the Transcriptor First Strand cDNA Synthesis kit achieved 15 successful reads at the 0.0032% level, attaining a score of 5 (15/18). AffinityScript Multiple Temperature cDNA Synthesis kit achieved 13 successful reads at the 0.0032% level, attaining a score of 2 (13/18) and the iScript™ Select cDNA Synthesis kit recorded 5 successful reads at the 0.032% level and one successful read at the 0.0032% level (12/18), attaining the lowest score of 1  and ratio of 0.0032% − (ratio of 0.032% divided by 10). Finally, a score of 1 to 6 was given to each kit based on the absolute values observed. Smaller absolute values were given higher scores, with the highest being a score of 6, assigned to the kit with the highest final score after adding the two individual scores. The highest ratios were observed by the SuperScript® III Reverse Transcriptase and AffinityScript Multiple Temperature cDNA Synthesis kit, both achieving a score of 6. Whereas the iScript™ Select cDNA Synthesis kit and the Transcriptor First Strand cDNA Synthesis kit achieved the lowest scores  with 2O. cDNA was stored at - 30°C overnight and then used for RQ- PCR.Total RNAs were extracted from bone marrow or peripheral blood from healthy individuals and Ph-positive CML patients using the QIAamp® RNA Blood Mini Kit (QIAGEN) on the QiaCube automated platform (QIAGEN). cDNA was synthesized following the manufacturers’ protocols. The amount of input RNA used was 10 μg and the final volume for all reactions was adjusted to 50 μl with ddHBCR-ABL1 [A standardized protocol established by the EAC program was used for the quantitation of BCR-ABL1 . 5 μl cD


Tobacco mosaic virus (TMV) is located at the 3′-untranslated region (UTR). It plays an important role in virus replication and translation. To determine the importance of UPD and 3′-UTR, and the effects of introduced RNA elements in TMV 3′-UTR, a series of TMV mutants with internal poly(A) tract upstream of UPD was constructed for structural analysis by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). TMV(24A+UPD) and TMV(42A+UPD) formed a similar structure as that of TMV 3′-UTR, but TMV(62A+UPD) structures altered by the introduced poly(A) tract. In addition, TMV(24A+UPD) had a higher viral RNAs accumulation than TMV in N. benthamiana protoplasts, and induced lethal symptoms in the infected plants. TMV(62A+UPD) showed a drastically reduced accumulation, its coat protein was undetectable in protoplasts, and the inoculated plants remained symptomless. This study analyzed the structures of 3′-UTR of TMV and found that the longer poly(A) tract introduced upstream of UPD reduced viral RNAs accumulation and induced milder symptoms in N. benthamiana. In conclusion, different lengths of the internal poly(A) tract introduced into the TMV 3′UTR lead to structural variations that affect virus accumulation and symptom expression.The upstream pseudoknots domain (UPD) of  Tobacco mosaic virus (TMV) is a positive-sense single-stranded RNA virus that belongs to the genus Tobamovirus. TMV has a wide host range and the symptoms vary with plant species and environmental conditions. In the field, tobacco plants infected by TMV show a typical mosaic pattern of light and dark green colour on the leaves, and distortion of newly emerged leaves. In the greenhouse, TMV infected N. benthamiana plants show severe leaf necrosis and stem collapse, leading to plant death within a few days. Similar to other tobamoviruses, the genome of the TMV-U1 strain13467911Bamboo mosaic virus interacts with secondary structural elements upstream to form a pseudoknot structure, and disruption of this interaction reduces virus accumulation1720. Dulcamara mottle virus possesses an internal poly(A) tract in the 3′-UTR which lacks a 3′-terminal tRNA-like sequence (TLS). This is different from other tymovirusesPolyadenylation is known to function in regulating RNA stability and translation. The importance of the poly(A) tail to viral infectivity has been reported in some viruses and the poly(A) length could also affect virus replication131415Hibiscus latent Singapore virus (HLSV)Hibiscus latent Fort Pierce virus23N. benthamiana. Moreover, HLSV is unable to replicate if the internal poly(A) tract is substituted by TMV UPD sequences, indicating that the internal poly(A) tract is not replaceable. This is different from previous studies that the UPD can functionally substitute for a poly(A) tail in plant and animal cellsIn recent years, two tobamoviruses isolated from Hibiscus spp. were reported to contain an internal poly(A) tract upstream of 3′-TLS. They are In vitro competition experiments reveal that the TMV-TLS has the highest binding affinity for RNA polymeraseN. benthamiana. TMV(42A+UPD) induced necrosis in the inoculated leaves, and systemic movement to the upper leaves was slower than that of TMV(24A+UPD). The accumulation of viral RNAs of TMV(62A+UPD) declined rapidly in N. benthamiana protoplasts and infected plants were symptomless after inoculation. The RNA structures of the mutated TMV fragments were subjected to selective 2′-hydroxyl acylation analyzed by the primer extension (SHAPE) method. Structural change in the 3′-UTR was dependent on the length of the poly(A) tract that was introduced upstream of the UPD in TMV genomes, and the longer the tract, the lower the viral RNAs accumulation and its infectivity.In general, the 3′-UTR is considered as a modular element that largely provides self-contained functions. The TLS mimics the tRNA structure, and it enhances virus translation and promotes minus-strand synthesis27N. benthamiana protoplasts and the accumulation of CP was higher than that of TMV in transfected protoplasts (N. benthamiana plants inoculated with in vitro transcripts of TMV(24A+UPD) showed water-soaked lesions on the inoculated leaves and developed severe necrosis 5 days post inoculation (dpi) (TMV24A+UPD) contained an internal poly(A) tract of 24 nt which was introduced upstream of TMV UPD at the 3′-UTR . It repltoplasts . N. benton (dpi)  and 4. TA+UPD conN. benthamiana protoplasts. Although the coat protein of TMV(42A+UPD) could be detected after 48 hours post transfection (hpt) in transfected protoplasts, its expression level was lower than that of TMV(24A+UPD). Unlike TMV(24A+UPD) which produced lethal symptoms in N. benthamiana, TMV(42A+UPD) produced necrotic lesions on the inoculated leaves at 5 dpi, but the upper leaves showed no symptoms  tracts increase viral infectivitysymptoms  and 4. Asymptoms .TMV(24A+UPD) infection caused lethal symptoms with a higher accumulation level of viral RNAs in the upper leaves. However, after introducing a longer poly(A) tract, the accumulation of viral RNAs was significantly reduced in the upper leaves of TMV(42A+UPD)-infected plants. In order to test whether TMV(42A+UPD) systemic movement was inhibited by the introduced longer poly(A) tract of 42 nt, we compared the viral RNAs accumulation in both inoculated and upper leaves from TMV(24A+UPD)- and TMV(42A+UPD)-inoculated plants. As TMV(24A+UPD) replicates faster than TMV(42A+UPD) in protoplasts, we tested two different amounts of viral RNA transcripts  to compare viral RNAs accumulation of TMV(42A+UPD) and TMV(24A+UPD).N. benthamiana plants at 7 dpi, the side view of TMV(24A+UPD) infected plants  in the upper leaves was lower than that of TMV(24A+UPD). The viral RNAs accumulation of TMV(42A+UPD) in the upper leaves increased by using 2.5 μg inoculum compared with that of using 250 ng. Despite using ten times the amount of TMV(42A+UPD) as inoculum, the viral RNAs accumulation in the upper leaves was still significantly lower than that of TMV(24A+UPD) . In addid plants  clearly d plants .N. benthamiana protoplasts at 24, 48 and 72 hpt was also monitored. The qRT-PCR results showed that the accumulation of TMV(62A+UPD) genomic RNA did not increase, in comparison to TMV and two other mutants (TMV(62A+UPD)-inoculated plants were symptomless at 7 dpi  and 4a.  mutants . Althoug mutants . No CP w mutants . These rSince there is no internal poly(A) tract present in the TMV genome, we propose that the introduced internal poly(A) tract in the TMV mutants would remodel the RNA structure of the 3′-UTR. The structures of the TMV 3′-UTR fragment and its mutants were analyzed by the SHAPE probing method . For TMV97UUCUGU101 turned to be reactivate in TMV(24A+UPD). In TMV(42A+UPD) structure  , the inttructure , the apiThe structure of TMV(62A+UPD)  was diffThe SHAPE analysis of those TMV poly(A) tract mutants showed that the stability of PK1, PK2 and PK3 was not greatly affected by the introduced internal poly(A) tracts of different lengths. Besides the large internal loop contributed by the internal poly(A) tracts, the obvious alteration in TMV(62A+UPD) was the disruption of the stem-loop that originally located at the 5′-end of TMV sequence. Also, the structure of TLS in TMV(62A+UPD) was altered. The TLS was proven to be important for TMV replicationThe SHAPE values of each nucleotide in TMV sequence and its mutants were listed in bar chart to compare the reactivity changes after introducing internal poly(A) tract . Compare11-O-ribose adduct, while the 2′-hydroxyl group of structurally constrained residues shows reduced nucleophilic reactivity. In contrast to conventional chemical and enzymatic probing techniques, SHAPE provides structural information at every nucleotide positionAttempts have been made to predict the secondary structure of TMV 3′-UTR using a variety of chemical and enzymatic tools93334N. benthamiana protoplasts over time  tract upstream of UPD in the TMV genome, we found that the altered virus killed the plants more rapidly than the wild-type TMV, indicating that an introduced internal poly(A) tract could enhance viral virulence. However, after increasing the introduced internal poly(A) length to 62 nt, there was no symptom observed on TMV(62A+UPD)-inoculated plants, and this mutant virus showed a rapidly diminishing accumulation of viral RNAs in transfected ver time . These rCoronavirus37Coronavirus with a longer poly(A) tail was enhanced. This result is contrary to our result in that the mutant viruses with a longer internal poly(A) tract showed reduced replication in N. benthamiana protoplasts and less virulence in the whole plants (The effects of poly(A) tail length on viral replication have been reported in e plants  and 4. Te plants .We have chosen 24 nt to be inserted into TMV genome (TMV-24A+UPD) because 24 nt internal poly(A) tract is the minimal length for HLSV replicationN. benthamiana protoplasts. However, the replication and accumulation of TMV(43A) were significantly reduced, as compared with that of TMV. Also, different from the lethal symptom caused by TMV infection, TMV(43A) induced a mosaic symptom and its infected plants remained alive after infection. These results indicated that the UPD of TMV alone could be replaced with an internal poly(A) tract but with a reduced replication efficiency.Before introducing the internal poly(A) tract to TMV genome, we tested whether the UPD of TMV could be replaced with an internal poly(A) tract. TMV(43A) has the UPD replaced by an internal poly(A) tract of 43 ntThe poly(A) tract of HLSV is located upstream of TLS and longer poly(A) tract upstream of 3′-TLS would result in higher accumulation of the viral RNA2526N. benthamiana. The internal poly(A) tracts of 24 nt, 42 nt and 62 nt caused symptoms of rapid plant death, localized tissue necrosis, and symptomless, respectively. TMV(42A+UPD) induced necrosis only in the inoculated leaves and petioles, and no symptoms were observed in the upper leaves at 14 dpi. Additionally, in the crude sap of TMV(42A+UPD)-infected leaves, virus particles were observed as flexuous, rod-shaped structures after negative staining, indicating that the virus could assemble with a 42 nt internal poly(A) tract (TMV(24A+UPD) replicated more rapidly than TMV in A) tract . TMV viral RNAs in the cytoplasm. The structural analysis of TMV(62A+UPD) showed that the conformation of the original TMV TLS was altered. As TLS is important for TMV replication by enhancing viral RNAs stability during translation, the altered TLS in TMV(62A+UPD) could be the reason contributing to the lack of CP expression. The larger hairpin loop of the 62 nt poly(A) tract may lead to the absence of CP expression and symptom expression. Most of the cis-acting RNA elements in RNA viruses are local sequences or confined secondary or tertiary structures404142444546According to the SHAPE-probed structural analysis, TMV(62A+UPD) 3′-terminal structures were distinct from that of TMV. TMV(62A+UPD) showed a reduced viral RNAs expression level and its CP was undetectable in the transfected Since the large hairpin loop was located upstream of the pseudoknot domain, interaction between the UPD and host eEF1A may also be affected by the structural alteration. Although the negative- strand of TMV(62A+UPD) viral RNAs were detected from the transfected protoplasts by qRT-PCR, it showed a decreasing trend and the amount was significantly lower than that of TMV, TMV(24A+UPD) and TMV(42A+UPD). The altered RNA structure of TMV(62A+UPD) might affect viral translation efficiency, so the amount of RNA-dependent RNA polymerase would be reduced, leading to a reduced production of negative-sense viral RNA templates.N. benthamiana. Additionally, in order to monitor the variation of introduced poly(A) tract in TMV genome, we also verified the genomic sequence of TMV(24A+UPD) after its replication in N. benthamiana. The result showed that the internal poly(A) tract was also elongated as shown in HLSV. As our results showed that longer poly(A) tract upstream of UPD leads to reduced TMV replication, the elongation of internal poly(A) tract is likely to result from polymerase slippage during virus replication and it is an inherent property that exist in both HLSV and TMV mutants.In this study, instead of the entire genome sequences, the 3′-terminal 14 nucleotides of the CP ORF plus the 3′-UTR region of TMV and its mutants were used for SHAPE analysis. As the long range interaction of TMV 5′- and 3′-UTRs is mediated through host proteins, instead of direct RNA-RNA interaction424748505152In conclusion, this study analysed the 3′-UTR structure of TMV with introduced internal poly(A) tracts of different lengths through SHAPE analysis. It revealed that the longer the introduced poly(A) tract, the lower the virulence of the mutants. This appears to be associated with structural variations and dynamic changes. This research work represents the first application of SHAPE analysis to TMV 3′-UTR and comparison of the length effect of introduced internal poly(A) tracts on viral accumulation. Our results provide a better understanding of the functions of UPD+TLS in relation to RNA structures in TMV. In addition, the new knowledge on the effects of different internal poly(A) tract lengths on virus accumulation has potential application in engineering TMV and its mutants into high-efficiency expression vectors.DpnI was added into the PCR products and incubated at 37 °C for 30 min to remove the original DNA template. The treated PCR products (5 μl) were transformed into competent E. coli cells by heat-shock treatment. Colonies were screened by sequencing the transformed plasmids on an Ampicillin-LB medium to confirm the introduced internal poly(A) length. The same approach was used to construct TMV(42A+UPD) and TMV(62A+UPD). The sequences of primers were listed in Using a TMV full-length cDNA clone as template, primers 24AUPD-F and 24AUPD-R were designed to introduce a 24 nt internal poly(A) tract upstream of its UPD. High fidelity enzyme (KAPA Biosystems) was used for over-lapping PCR. KpnI. After phenol/chloroform extraction and ethanol precipitation, the purified DNAs were used as templates for in vitro transcription by the mMessage mMachine in vitro transcription kit . RNA transcripts were precipitated by LiCl, following the manual instructions and dissolved in RNase-free water.Full-length cDNA clone of TMV and the mutants were linearized with N. benthamiana were mechanically inoculated with 2 μg in vitro transcribed viral RNA in GKP buffer . Total RNAs were extracted from the inoculated and upper leaves by TRIzol reagent. qRT-PCR was performed using the KAPA SYBR Fast qPCR kit on a CFX384 Real-Time PCR system (Bio-Rad). The N. benthamiana actin gene was used as an internal control. Error bars (SD) were obtained from nine replicates that included three biological replicates and each biological replicate contains three technical replicates. Primer-R and Primer-R-negative-strand were used for positive- and negative-strands reverse transcription, respectively. Primers qPCR-F and qPCR-R were used for target fragment amplification and primer Actin-F and Actin-R were used to amplify the actin gene. The sequences of primers were listed in Fully expanded leaves of 4-week-old Nicotiana benthamiana protoplasts were isolated and transfected with in vitro transcripts of TMV and its mutants by polyethylene glycol transfection. Total RNAs were extracted from transfected protoplasts with TRIzol reagent at 24 h, 48 h and 72 h, respectively, followed by cDNA synthesis with primer R using the SuperScript III Reverse Transcriptase kit (Invitrogen). TMV-CP gene was amplified using primers TMV-CP-F and TMV-CP-R. Actin was used as internal control. Total proteins isolated from the transfected protoplasts at different time points were used for Western blot to detect CP expression using TMV-CP antibody.14+UPD)-F and primer R (14+AAA)-F and primer R (® SV Gel and PCR Clean-up System (Promega) and used as templates for in vitro transcription using AmpliScribe T7-Flash transcription kit. The RNA transcripts were purified by denaturing PAGE gel purification. After ethanol precipitation, RNA was dissolved in RNase-free water.Primer TMV-T7 reagent reaction and a sequencing reaction were mixed for one capillary. Similarly, the (−) reagent reaction was mixed with an identical sequencing reaction.pmol of RNA were mixed with RNA folding buffer (50 mM HEPES (pH 7.0); 100 mM KCl; 2.5 mM MgCl2 and denatured at 65 °C for 5 min. The RNA was then slowly cooled at room temperature for 30 min. Reactions were divided into two aliquots of 4.5 ul each and 0.5 ul NMIA (0.001 g in 125 ul DMSO) was added to one as the (+) reaction. Equal volume of DMSO was added to the other aliquot as the (−) reaction. Both (+) and (−) reactions were incubated at 37 °C for 40 min and precipitated at −20 °C with 60 ng/μl glycogen, 0.3 M sodium acetate, pH 5.2 and three volumes of cold ethanol. Precipitated RNA was collected by centrifugation, washed once in 70% ethanol and resuspended in RNase-free water. Two pmol of VIC or NED-labeled primer P6 were annealed to the RNA at 90 °C 3 min, 55 °C 10 min, on ice 3 min. RNA was reverse transcribed at 55 °C for 50 min with the Invitrogen superscript III transcriptase kit. Sequencing ladders were prepared using the cycle sequencing kit according to the manufacturer’s instructions and primers labeled with NED or VIC dyes which was different from that used for the (+) and (−) reactions. Sequencing reactions were divided into two and mixed with (+) and (−) reactions. RNA was precipitated as above, dried and resuspended in 15 μl Hi-Di formamide. Primer extension products were analysed on an Applied Biosystems 3100 xl DNA analyser. Electropherograms were processed using the QuSHAPE programmeTwo Molecular modeling.  The RNAstructurehttp://rna.urmc.rochester.edu/software.html) at University of Rochester Medical Center, was used to generate the secondary structures based on the 3′-UTR SHAPE-probed secondary structure information. The “Force-Read SHAPE Reactivity-Pseudo energy Constraints” option was chosen for the RNA modeling under default setting of Slope  1.8 and Intercept  −0.6 with the analyzed sequence SHAPE file. The secondary structures and the corresponding SHAPES values were visualized using the VARNAHow to cite this article: Guo, S. et al. TMV mutants with poly(A) tracts of different lengths demonstrate structural variations in 3'UTR affecting viral RNAs accumulation and symptom expression. Sci. Rep.5, 18412; doi: 10.1038/srep18412 (2015).