Concerns regarding vaccine safety are increasing along with lack of compliance to vaccination schedules. This study aimed to assess vaccination-related risks and the impact of a Special Immunization Service (SIS) at the Pediatric Emergency Department (PED) of Padua on vaccination compliance among participants.st 2002 to December 31st 2015. The Service is divided into a clinic (SIS-C) where all referred children undergo a pre-vaccination visit and an area within the Pediatric Emergency Department (SIS-PED) where children are vaccinated if indicated. During each SIS-C visit, age, gender, admission criteria and scheduled vaccinations were recorded, with any vaccine-related adverse events captured during SIS-PED visits. Follow-up was conducted to evaluate vaccination plan completion.This retrospective cohort study included all children attending the SIS from January 1359 children received 560 vaccine administrations  at the SIS during the 14 year study. Admission criteria were adverse events after previous vaccination  in 27.2% of cases, non-anaphylactic allergies  in 42.7% and anaphylaxis in 10.3%. After vaccination, 15/560 (2.7%) mild adverse events were observed. 96.3% of children vaccinated at least once at the SIS-PED and available for follow-up completed their vaccination plan, in contrast to 55.5% of children referred to the SIS-C who were not vaccinated in SIS-PED.For children referred to SIS-C and available for follow-up, vaccination in SIS-PED was associated with more frequent completion of vaccination plans, indicating a benefit of the service to vaccine coverage. The low number and mild severity of adverse events reported after vaccination of high-risk children in SIS-PED attest to the safety of the service Vaccine administration is one of the greatest achievements of biomedical science and public health, and among the most effective medical procedures, preventing two to three million deaths every year . It alsoThe safety of vaccines in the pediatric population is of the utmost importance for parents and healthcare workers, especially in countries in which vaccine-preventable diseases are rare . As withSome children are at higher risk of vaccine-related adverse events (AEs) due to reactions to previous vaccinations, egg allergies, or anaphylaxis .With vaccination increasingly questioned by concerned parents and compliance to vaccination schedules falling –10, a SpSIS in other settings have played a central role in improving adherence to regional vaccination plans and increasing immunization coverage –13.The primary aim of this study is to describe SIS referrals and visits in Padua and AEs observed after vaccinations over a 14-year period.The secondary aim is to assess vaccination plan compliance after SIS attendance.st 2002 to December 31st 2015.This is a retrospective cohort study including all referrals and visits to the Padua SIS from January 1st 2002 in the PED of Padua University Hospital, is divided into a SIS-Clinic (SIS-C) in which a pre-vaccination evaluation takes place and an area within the PED (SIS-PED) where vaccinations are administered when indicated.The SIS, established on January 1Children are referred to our Service by primary care pediatricians or local Health Districts of Padua if considered at high risk for vaccine-related AEs or for non-adherence to vaccination schedules due to parental reluctance , whether to suspend the vaccination plan or to proceed. Patients advised to continue their vaccination schedule may be vaccinated in the SIS-PED or their local Health Districts according to their risk of AEs. Decisions are made following the most recent Guidelines of the Italian Higher Institute of Health  and NatiIn the SIS-PED, each child is evaluated by a pediatrician to exclude acute illness and preparations are made to respond to possible AEs prior to vaccination. Afterwards, children remain in the SIS-PED for a three-hour observation period, which can be extended as deemed necessary by the attending pediatrician.Following SIS-PED vaccination, each child is advised to complete their vaccination plan at the SIS-PED or local Health District, or to suspend their vaccination plan on the basis of any AE and accounting for parental preference ;AEs after previous vaccinations: immediate or delayed reactions and IgE mediated or not;other reasons: parents who chose not to vaccinate their children (until vaccination became mandatory in 2007), chronic conditions such as hemophilia, cutaneous mastocytosis, thrombocytopenia, epilepsy and rare genetic syndromes.Motivation for referral to SIS-C, including:-Type of vaccine(s) administered-AEs occurring during post-vaccination observation period or reported by parents via telephone after leaving SIS-PED.SIS-PED admissions and total vaccinations in the Padua urban area were compared using the ULSS 6 EUGANEA Prevention Department database, where all administered vaccinations in the metropolitan area are registered. This database was also used to follow up all children visiting SIS-C regardless of whether they were subsequently seen in SIS-PED.Compliance was categorized as vaccination plan completed at the SIS, vaccination plan completed at the Health District or vaccination plan not completed.Vaccination plan completion was defined as the accumulation of the required number of doses of all required immunizations by the specified age regardless of the timing of administration. Current Italian vaccination schedule at time of follow-up was used to determine required vaccines, doses and timelines .Influenza, rotavirus and Hepatitis A vaccination (HAV) vaccines were excluded in assessment of vaccination completion as they are not actively offered and are not free of charge.A descriptive analysis was performed to assess number of vaccines administered in SIS-PED and overall in the Padua region, motivations for SIS-PED admission, vaccine type and any associated acute adverse events. Time trends for the most requested vaccinations and related reasons for referral were also assessed. Results are summarized using frequencies and percentages for qualitative variables and median and standard deviation for continuous variables. Comparisons of categorical variables over time or based on SIS-PED admission was carried out using the chi-square test.Three-hundred and fifty-nine children attended the SIS-PED and 560 vaccine doses were administered between 2002 and 2015. Comparing the number of vaccines administered at the SIS-PED with those administered in the Padua Health District, SIS-PED vaccination was only a small percentage of overall vaccination in the region ).Allergies accounted for 53.0% (297/560) of SIS-PED admissions, 58/297 (19.5%) anaphylactic reaction and 239/297 (80.5%) non-anaphylactic. 186/560 (33.2%) SIS-PED admissions and 166/239 (69.5%) of non-anaphylactic allergy related admissions were for egg allergy, 31/560 (5.5%) were related to dairy allergy, 11/560 (2.0%) to drug allergies, and 59/560 (10.5%) to multiple allergies .AEs after previous vaccinations accounted for 27.2% (152/560) of SIS-PED admissions. 58/152 (38.2%) of these were immediate IgE mediated reactions, including important local reactions , urticarial rash or other spread cutaneous reactions , and bronchospasm ; 16/152 (10.5%) were immediate non-IgE mediated reactions such as vasovagal reactions  and hypotonic hypo-responsive episodes . 78/152 (51.3%) reactions occurred more than two hours after vaccination, most commonly cutaneous reactions , irritability and/or drowsiness and hypotonia , and febrile seizures .Other reasons for referral accounted for 19.8% (111/560) of SIS-PED admissions. Included in this group were parents who did not wish to vaccinate their children, and children with particular diseases such as neuromuscular disorders, cutaneous mastocytosis, thrombocytopenia, epilepsy, and rare genetic syndromes.From 2009, egg allergy was no longer considered an indication for referral to the SIS for MMR/MMRV vaccination ,15,16. AThe most requested vaccination in the SIS-PED was MMR/MMRV , followed by hexavalent vaccine . Other vaccinations were requested at a significantly lower rate .No serious vaccine-related AEs were observed over 14-years, with 15 mild vaccine-related AEs .The vaccinations related to these mild AEs were MMR/MMRV , hexavalent , influenza  and HPV .Eight children experiencing mild adverse events after SIS-PED vaccination  had been referred to the SIS for a previous vaccine-related AE, with six  referred for allergies. No vaccine-related AEs were recorded among children referred to SIS for parental concerns regarding vaccination.All 417 children who attended a pre-vaccination visit at SIS-C were followed up for vaccine schedule adherence, whether they had been admitted for immunization at the SIS-PED or not.Fifty-eight children  attending SIS-C were not referred to SIS-PED: 13/417 (3.1%) following recommendation of SIS-C pediatrician to suspend the vaccine plan (11/13 because of a neurologic disease under investigation and 2/13 for previous severe IgE mediated reactions) and 45/417 (10.8%) following recommendation of SIS-C pediatrician to continue vaccination plan with the Health District due to lack of identified risk for vaccine-related AEs. In this case, 25/45 (55.5%) completed their immunization schedules and 20/45 (44.5%) did not.Among patients vaccinated at SIS-PED, 65/359 (18.1%) continued the vaccination schedule at the Service and 294/359 (81.9%) received only one vaccination at the SIS-PED.Two-hundred eighty-three children  receiving a single vaccination at SIS-PED completed their vaccination plan at the Local Health District, while 11/294 (3.7%) interrupted the vaccinations for various reasons, including parental refusal. This percentage of completed vaccination plans is significantly higher than the 55.6% (25/45) observed among children receiving a SIS-C recommendation to continue their plan at the Local Health Districts without receiving a single vaccination in SIS-PED (p<0.001) .Concerns related to the safety of vaccines are increasing, and communication about relative risks and benefits is needed when advising parents on childhood vaccinations. Parents of children who experienced an AE after a previous vaccination, or who have a serious history of anaphylaxis may have particular difficulty in making the decision to complete their child’s vaccination plan. There is a paucity of information about the risk of subsequent vaccine-related AEs, and not all risk factors are completely understood. A SIS may help to address the need for more information, as indicated by increasing participation during the first three years of our service in Padua, Italy.One important consideration is the feasibility of extending SIS services to a broader segment of the population. From 2002 to 2014, 0.05% to 0.01% of vaccinations in the Padua Health Districts and SIS combined were administered at SIS-PED due to strict admission criteria.Although utilization of the SIS in Padua remained fairly constant during our study, reasons for referrals to the service varied. Admissions for non-anaphylactic allergies decreased (from 54.5% before 2008 to 31.3% after 2010), likely due to changes in recommendations for children with egg allergies, while admissions for anaphylaxis remained stable around 10%. Referrals for a past history of AE following a previous immunization increased over time, reaching 62.5% in 2015.We observed very few cases  of appropriate reason for referral to the SIS, such as an immediate reaction to a previous dose of the same vaccine  and a past medical history of anaphylaxis, but not related to any component of the vaccine .In our study, 38.8% of admissions with a past history of AE had an immediate IgE mediated reaction, while 10.5% were referred after immediate non-IgE mediated event, such as vasovagal reactions or hypotonic hyporesponsive episodes, and the remaining 51.3% after a delayed reaction, which may be of immunological and non-immunological type . In contOnly a few mild AEs after vaccination in SIS-PED were observed in our 14-year experience, confirming the high safety profile of vaccines and the low risk for complications.We observed a small number of children (2.7%) who experienced minor reactions to the vaccine administration. All were local or systemic reactions and quickly resolved, and all occurred among children referred to SIS for an appropriate reason. These types of vaccine-related AEs are well described in the immunization literature, with a reported incidence around 10% for local reactions and around 5–15% for systemic ones. In addition, the incidence of AEs after revaccination at our SIS-PED was similar to that reported in other studies ,19. The The difference in the proportion of children who completed their vaccination plan at the Health District after just a single vaccination at the SIS-PED (96.3%) and those who completed the plan at the Health District after a SIS-C visit not followed by a SIS-PED admission (55.5%) indicates a potential role of the SIS in promoting the immunization. The SIS addressed understanding the needs of vaccine hesitant parents and improving communication skills and interaction between parents and health care workers. Useful strategies from our SIS included solicitation of questions about vaccines, establishment of a trusting relationship, and provision of appropriate educational materials to parents .rd 2007 suspended mandatory vaccinations in the Veneto Region from 2008 onwards [The SIS became more important after Regional Decree n.7 of March 23 onwards .More and more frequently parents feel hesitant to vaccinate their children because of conflicting information from media, internet and other sources and were constantly influenced by their social group and anti-vaccination movements ,22. SubsIn response Italy's parliament has given final approval to a new program making vaccinations compulsory for school children up to age 16 . For thiThe high percentage of vaccination plans completed among children receiving at least one immunization at the Padua SIS over 14 years suggests a positive influence of the service on intention to vaccinate among concerned parents. A very small number of mild vaccine-related AEs were observed, with no moderate or severe vaccine-related AEs, and all children with an appropriate reason for SIS referral were successfully vaccinated. Specialized immunization services show potential for improving vaccination compliance, increasing immunization coverage and ensuring safe vaccination in high-risk children.S1 File(XLSX)Click here for additional data file.


Combining stay-green traits and environmental water-stress characterization, both standardized relative to anthesis, provides a powerful method to characterize and select for adaptation to well-watered and water-stressed environments. A stay-green phenotype enables crops to retain green leaves longer after anthesis compared with senescent types, potentially improving yield. Measuring the normalized difference vegetative index (NDVI) during the whole senescence period allows quantification of component stay-green traits contributing to a stay-green phenotype. These objective and standardized traits can be compared across genotypes and environments. Traits examined include maximum NDVI near anthesis (Nmax), senescence rate (SR), a trait integrating senescence (SGint), plus time from anthesis to onset (OnS), mid-point (MidS), and near completion (EndS) of senescence. The correlation between stay-green traits and yield was studied in eight contrasting environments ranging from well watered to severely water limited. Environments were each classified into one of the four major drought environment types (ETs) previously identified for the Australian wheat cropping system. SGint, OnS, and MidS tended to have higher values in higher yielding environments for a given genotype, as well as for higher yielding genotypes within a given environment. Correlation between specific stay-green traits and yield varied with ET. In the studied population, SGint, OnS, and MidS strongly correlated with yield in three of the four ETs which included well-watered environments (0.43–0.86), but less so in environments with only moderate water-stress after anthesis (−0.03 to 0.31). In contrast, Nmax was most highly correlated with yield under moderate post-anthesis water stress (0.31–0.43). Selection for particular stay-green traits, combinations of traits, and/or molecular markers associated with the traits could enhance genetic progress toward stay-green wheats with higher, more stable yield in both well-watered and water-limited conditions. ChangesThe aim of the current study was to determine the potential of the recently proposed component stay-green traits for crop improvement. Can stay-green traits be useful to select for high-yielding genotypes in a broad range of water-stress environments or, conversely, for adaptation to specific classes of water-stress environments? What is the magnitude of the effect of individual stay-green traits in different environments? A mapping population of doubled-haploid lines segregating for stay-green traits was studied over 3 years in a total of eight trials where rainfall, irrigation, and/or rain-out shelters were employed to generate a wide range of water-stress environments encountered in the TPE. Correlations between stay-green traits and yield, and the apparent effect of traits on yield, were examined to identify traits suitable for selection of high-yielding, stay-green genotypes.Triticum aestivum) cultivars SeriM82 and Hartog  in 2010, and Kingsthorpe  and Warwick  in 2011 and 2012. Crops were grown either in natural rain-fed conditions (‘rf’ treatment), in irrigated conditions (‘ir’) where irrigation was used to ensure little water-stress throughout the season, or under a rain-out shelter (‘ro’) which excluded rainfall from anthesis onwards. Some rain-fed treatments were also watered to near field capacity immediately after sowing to ensure uniform establishment. Experiment names are derived from a combination of location, year, and treatment such that the rain-fed trial at Warwick in 2011 is designated ‘WAR11rf’  at three sites in southern Queensland, Australia: Gatton , suggesting that transpiration patterns and water-deficit responses were well approximated. Data from all trials were centred at anthesis, and averaged every 100degree Celsius days (oCd) between emergence and 450 oCd after anthesis. The trials were classified according to their similarity to the four previously identified main ETs from the Australian TPE  stored in the soil prior to sowing . In-crop GAT11rf .The seasonal water-stress patterns for each of the eight environments were classified into one of the four water-stress ETs previously identified ; Fig. 1.As anticipated, water availability was a major yield constraint in the studied environments. There was a general trend to decreased yield as the water-stress pattern became more severe from the least stressed ET1 through to the most stressed ET4 ; Fig. 2.–2) and GAT10rf (1741 MJ m−2) were well below those for other environments, which ranged from 2157 MJ m−2 to 2649 MJ m−2 .Other factors probably influenced yield in certain environments. For example, environments at Gatton 2010 had the highest in-crop rainfall and potentially available water, but yield was lower than environments in 2011 . The hig9 MJ m−2 , particuoC probably experienced heat stress around anthesis and during the grain-filling period . Prolonged temperatures above 30 oC pre- and post-anthesis are known to affect wheat productivity . Environments in ET1 as well as KTP11rf in ET2 did not reach these temperatures during the early- or mid-grain filling period, or reached them only late in development when starch deposition would have been nearly complete . Thus, heat stress is less likely to have affected yield in these environments.In addition, crops in some environments with a weekly mean of the daily maximum temperatures reaching or exceeding 30 per se.Yield rankings for genotypes varied between environments, indicating crossover G×E. Genetic correlations between pairs of environments varied from −0.45 to +0.87, and the mean correlation for all eight environments was +0.3. In the overall analysis, the variance component due to G×E was 1.6 times greater than that for genotype. These results are not uncommon for multienvironment trials in the Australian TPE and highlight the need to seek adaptation indicators with higher heritability and lower G×E than yield Variation between genotypes for yield tended to decrease with increased water-stress and lower yield from ET1 to ET4 . The treWater-stress tended to shorten the thermal time period from sowing to anthesis, but only in some environments . There w−2 for each oCd increase in OnS (oCd) during the early grain-filling period in the TPE, this corresponds to an ~13g m−2 increase for a 1 d delay in senescence onset, representing a 3.2% yield increase for a trial with a mean yield of 400g m−2. Change in MidS had an impact of a similar order of magnitude on yield, and EndS slightly higher  but not in ET2 . In ET4, the correlation between SGint and yield was close (0.73), but not statistically significant (P>0.05). The lower number of genotypes tested in ET4 (76) and reduced yield variation due to severe stress .High correlations between stay-green traits and yield were observed across genotypes within most trials . The deg P>0.05; . The SGie stress  may haveP<0.05) and significantly negatively correlated with height at the other radiation-limited environment  and at one ET2 environment . Including these traits as covariates in the statistical model for the yield prediction (BLUPs) did not significantly affect the results for any environment, so the analysis without covariates was used.In contrast to OnS, MidS, and SGint, correlations between yield and SR or Nmax were more varied across water-stress environments. The maximum leaf canopy greenness (Nmax) was correlated with yield for both environments classified as ET2 and one in ET3 (WAR12rf). The SR was significantly correlated with yield in both ET3 environments, but in other ETs only at WAR11rf (ET1). Height and time to anthesis were not significantly correlated with yield in most environments, probably reflecting the previous selection of the population to reduce variation for these traits . Similarly, there was no clear difference in the pattern of genetic correlations for the potentially radiation-limited environments at GAT10ir and GAT10rf compared with the other likely non-radiation-limited environments .No clear pattern in the genetic correlations between stay-green traits and yield was observed in sites affected by heat stress  compared with others  and non-significant for all environments .It is unlikely that yield contrast between high- and low-yielding groups result from differences in anthesis date. Differences between groups in the mean period from sowing to anthesis were small , the studied population was also pre-selected to reduce variation for height and anthesis date , but sigOverall, Nmax, OnS, MidS, EndS, and SGint all decreased, while SR increased, as water-stress increased from environments in ET1 through to ET4 , 3. All To enable selection for yield, traits correlated with yield in the relevant environments are preferred.b; Stay-green traits OnS, MidS, and SGint could be used as proxy traits to select for adaptation in a broad range of environments. Stay-green phenotype and related traits have frequently been linked to improved yield in crops experiencing a terminal water stress  or more severely stressed environments (ET3 and 4), but not ET2 environments. In contrast, Nmax appeared more promising in the ET2 environments examined.Overall, SGint appears the most useful trait to characterize the stay-green phenotype, as it was highly or significantly correlated with yield in all but one of the ET2 environments KTP11rf; . This isSGint had the closest relationship with yield in most environments and will probably be useful for selection in a broad range of environments , 6. HoweThe eight environments tested in this study reflect the variability in water-stress environment types (ET1–ET4) encountered across the Australian cropping region in current and future climates, not only in subtropical northern Australia but also in temperate and Mediterranean climatic regions , which oTechnologies to determine NDVI for a large number of genotypes over the full senescence period are improving rapidly with development of drones and ‘phenomobiles’  and plotted against adjusted mean yield  estimated from measurements at eight trials in south east Queensland during 2010, 2011, and 2012.Figure S2. Weekly average of daily maximum temperatures and cumulative incident radiation throughout the growing season plotted at dates relative to anthesis of the reference cultivar Hartog for the eight studied environments.


Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer; few treatments exist for patients with advanced disease. Once tumors metastasize to distant sites, patients generally receive chemotherapy, but response duration and progression-free survival (PFS) are typically short. Few studies have assessed the efficacy of second-line chemotherapy for metastatic MCC. Here, we studied outcomes in patients who received ≥ 2 lines of chemotherapy for metastatic MCC.Patients in an MCC-specific registry diagnosed with stage IV MCC between November 1, 2004, and September 15, 2015, and treated with second-line or later chemotherapy were analyzed retrospectively. Patient records, including baseline characteristics, immunocompetent status, and responses to prior chemotherapy, were evaluated. Patients meeting eligibility criteria were followed through December 31, 2015.n = 34), the ORR was 8.8% . The median duration of response was 1.9 months . In the immunocompetent population, median PFS and overall survival were 3.0 months  and 5.3 months , respectively.Of 29 patients with metastatic MCC and immunocompetent status who had received ≥ 2 lines of chemotherapy, 3 achieved a partial response, for an objective response rate (ORR) of 10.3% . In the overall population including patients with immunocompetent and immunocompromised status (The low response rates and limited durability confirm previous reports of the ineffectiveness of second-line or later chemotherapy in patients with metastatic MCC and provide a benchmark for assessing clinical benefit of new treatments. Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer that is more prevalent in elderly and immunocompromised patients , 2. MCC MCC is associated with a poor prognosis. MCC-specific 5-year survival rates reported in patients with distant metastatic (stage IV) disease, defined by metastasis beyond regional lymph nodes, range from 0% to 18% . The morn = 30), the objective response rate (ORR) was 23%, median duration of response (DoR) was 3.3 months, and median PFS was 2 months.Recent FDA approval of avelumab represents the first and only approved treatment option for metastatic MCC . HistoriCurrently, there are no prospective studies of outcomes following second-line treatment of distant metastatic MCC in European patients. Because of the rare and aggressive nature of metastatic MCC, the lack of benefit with standard chemotherapy treatments, and the emergence of promising new treatment options –24, it iin situ, or cervical carcinoma in situ. Patients with immunocompromised status due to specific hematologic diseases  or immunosuppressive treatments were eligible, although the main analysis included only immunocompetent patients. Other criteria suggestive of immunocompromised status, such as organ transplant or HIV infection, were not recorded in the MCC registry and thus not available as a screening factor in this analysis.Patients included in this analysis were adults aged ≥ 18 years diagnosed with distant metastatic MCC and treated with ≥ 2 lines of systemic chemotherapy for metastatic disease. Patients were excluded if they had a history of any other solid tumor within 3 years before the start of treatment for MCC, except for basal or squamous cell carcinoma, bladder carcinoma Retrospective anonymized patient-level information was extracted from an observational, real-world MCC-specific registry that was established in 2005 in German-speaking countries. Patients were identified through a collaboration between IMS Health and the German Cancer Research Center (Deutsches Krebsforschungszentrum). Data in the registry were collected from 56 clinical sites , including data on demographics, medical history of skin cancer and immunosuppression, clinical characteristics, treatment, and outcomes. Informed consent was given by all patients who enrolled in the MCC registry. Records from November 1, 2004, through September 15, 2015, were searched, and qualifying patients were followed through December 31, 2015.Best overall response (BOR) was assigned to each patient based on clinical judgment by the reporting physician. Because reporting according to Response Evaluation Criteria In Solid Tumors (RECIST) , 27 was The primary objective was to determine the ORR achieved with second-line or later chemotherapy in immunocompetent patients. Secondary objectives included assessment of DoR, PFS, OS, and DRR. Time to progression (TTP) was also analyzed for patients who had disease recurrence or progression. Safety was not assessed in this study. All study objectives were analyzed in the main (immunocompetent) and overall (immunocompetent plus immunocompromised meeting eligibility criteria) populations. Responses to prior first-line chemotherapy were also recorded.n = 34). Two patients were excluded from the analysis of responses to first-line chemotherapy because distant metastatic MCC had not been diagnosed when their first-line therapy was initiated. These patients did qualify for analysis of outcomes with second-line or later treatment because the requirement for any chemotherapy for MCC in the first-line setting was met.Data from 971 patients with MCC registered between November 01, 2004, and September 15, 2015, were available for analysis , median age was 67 years , and 62.1% of patients were male (Table Baseline patient and disease characteristics were similar in the main (immunocompetent) and overall (immunocompetent and immunocompromised) populations. Of the 5 immunocompromised patients, 4 had B-cell chronic lymphocytic leukemia and 1 had received immunosuppressive treatment, and all 5 patients had visceral metastases at initiation of first-line therapy. There was no association between immunocompromised status and a history of other non-melanoma skin cancers.Among patients in the main analysis population who had received at least second-line chemotherapy, the median treatment duration was 4.5 months  with first-line chemotherapy, 2.6 months  with second-line chemotherapy, and 2.5 months  with third-line chemotherapy. All patients had discontinued first-line treatment because of disease progression. Second-line treatment was discontinued because of disease progression (93.1%) or death (6.9%).Chemotherapy regimens for MCC across all treatment lines are presented in Table No patient had a complete response to second-line chemotherapy, whereas 3 patients  had a partial response, resulting in an ORR of 10.3%  in the main analysis population  were classified as immunocompetent and qualified for inclusion in the main analysis group Table . No patin = 34) eligible for this study may limit the confidence in response evaluation, this study represents a carefully selected group of patients with distant metastatic MCC and allows for indirect comparisons with modern clinical studies in metastatic MCC. Additionally, this study represents the largest retrospective series reporting on outcomes of second-line or later chemotherapy.This observational, real-world-data study investigated the efficacy of chemotherapy in patients with distant metastatic MCC. Although prospective trials of chemotherapy have not been conducted, retrospective and real-world-data analyses of heterogeneous advanced disease populations have suggested that MCC is a chemosensitive malignancy –20. In tPatients in this study were also analyzed for response to first-line treatment administered prior to second-line chemotherapy. The ORR to first-line treatment was 46.4%, although responses were also of short duration , and the median PFS from the date of first-line treatment initiation was only 4.7 months . An obvious limitation of the first-line analysis is that all patients received second-line or later treatment per inclusion criteria; thus, those who were unable to receive second-line therapy after first-line therapy, eg, due to rapid deterioration, were not eligible for this study. These excluded patients may also have been less healthy in general than the patients able to receive second-line or later treatment. Thus, these findings might not be generalizable to the first-line setting for distant metastatic MCC.A recent retrospective analysis of 30 patients with distant metastatic MCC enrolled in a US-based repository also found that responses to second-line chemotherapy were of very short duration  . In addiThe literature characterizing outcomes of patients treated with chemotherapy for both regional and distant metastatic disease is scant and limited to summaries of retrospective case studies or anecdotal case reports. Due to the potential for reporting bias and reliance on summary data for evaluable patients only, actual ORR and DoR data may be lower than what is reported in the literature. Provided these limitations, it can be summarized that MCC is described as a chemosensitive tumor with a short DoR, although rare cases of prolonged response duration have been reported. Current treatment guidelines similarly acknowledge evidence for chemosensitivity while also noting the lack of response durability and high toxicity in elderly patients , 16. OveThese reports, combined with this study and other recent retrospective analyses in distant metastatic MCC described above, highlight the high unmet need for effective treatment options providing durable benefit in patients with distant metastatic MCC. Because of the rarity and aggressiveness of MCC, together with a rapidly changing clinical landscape in which immune therapy is emerging , a largeMCC is characterized as an immunogenic cancer based on the presence of various antigens created by viral infection or UV-induced mutations and neoantigens, which can be recognized by the immune system; furthermore, unknown primary-tumor status, assumed to be related to a prior robust antitumor immune response, is the only reliable predictor of positive outcomes in patients with MCC –6, 30. M


To document clinical outcomes and women’s experiences following the introduction of mifepristone into South African public sector second-trimester medical abortion services, and compare with historic cohorts receiving misoprostol-only.Repeated cross-sectional observational studies documented service delivery and experiences of women undergoing second-trimester medical abortion in public sector hospitals in the Western Cape, South Africa. Women recruited to the study in 2008 (n = 84) and 2010 (n = 58) received misoprostol only. Those recruited in 2014 (n = 208) received mifepristone and misoprostol. Consenting women were interviewed during hospitalization by study fieldworkers with respect to socio-demographic information, reproductive history, and their experiences with the abortion. Clinical details were extracted from medical charts following discharge. Telephone follow-up interviews to record delayed complications were conducted 2–4 weeks after discharge for the 2014 cohort.The 2014 cohort received 200 mg mifepristone, which was self-administered 24–48 hours prior to admission. For all cohorts, following hospital admission, initial misoprostol doses were generally administered vaginally: 800 mcg in the 2014 cohort and 600 mcg in the earlier cohorts. Women received subsequent doses of misoprostol 400 mcg orally every 3–4 hours until fetal expulsion. Thereafter, uterine evacuation of placental tissue was performed as needed. With one exception, all women in all cohorts expelled the fetus. Median time-to-fetal expulsion was reduced to 8.0 hours from 14.5 hours (p<0.001) in the mifepristone compared to the 2010 misoprostol-only cohort . Uterine evacuation of placental tissue using curettage or vacuum aspiration was more often performed  for those receiving mifepristone; major complication rates were unchanged. Hospitalization duration and extreme pain levels were reduced (p<0.001), but side effects of medication were similar or more common for the mifepristone cohort. Overall satisfaction remained unchanged (95% vs. 91%), while other acceptability measures were higher (p<0.001) for the mifepristone compared to the misoprostol-only cohorts.The introduction of a combined mifepristone-misoprostol regimen into public sector second-trimester medical abortion services in South Africa has been successful with shorter time-to-abortion events, less extreme pain and greater acceptability for women. High rates of uterine evacuation for placental tissue need to be addressed. Accessible and effective provision of second-trimester abortion is particularly important in settings where a high proportion of women seek abortion in the second trimester, as is common in low- and middle-income countries (LMICs) , 2. SoutBoth surgical and medical methods for second-trimester abortion are considered safe and effective when performed by skilled providers, and major complications are rare events –13. HoweMedical methods for abortion have been shown to be acceptable to women in many settings, both for first- and second-trimester procedures, as well as for managing incomplete abortion , 25–28. With a shortage of physicians in the South African public sector who are skilled in D&E and willing to provide the service , medicalPrevious research among women undergoing second-trimester abortion in South Africa using the misoprostol-only regimen reported safe abortion provision; however, the study reported delays in accessing care, and prolonged hospitalization beyond 2 days in many cases . The mifImplementation of new second-trimester abortion services into public sector teaching hospitals in LMICS s can be challenging as limited capacity, high patient-to-staff ratios, extensive referral areas, complicated referral pathways, high rates of staff rotation are commonplace , 32–34. The historic cohorts receiving misoprostol-only were recruited from February through July 2008 and April through August 2010 at Obstetric and Gynecology departments of two public sector teaching hospitals . Other clinical outcomes included drug administration, time-to-fetal expulsion, rates of uterine evacuation for placental tissue and time-to-abortion completion . In addition, hospitalization duration, analgesia, major complications  and post-abortion contraception were recorded. Women’s experiences included presence of side effects (yes/no), and level of pain, overall satisfaction  and acceptability (would you recommend the method to a friend?) measured on 5-point scales.a priori,  and stratified by quartiles of total misoprostol dosage. HRs were also calculated for the majority subgroup of participants receiving their first misoprostol dose via the vaginal route of administration (PV).Data were analyzed using Stata v.13. Data for the 2008 and 2010 misoprostol-only cohorts were combined to improve statistical power of the historical data. Missing data were not imputed, and only valid percentages were reported. Descriptive statistics were reported for participant characteristics and study outcomes. Five point scales were collapsed to three categories for analysis, and groups were compared using Chi-squared tests for proportions or Kruskal-Wallis tests for medians. Kaplan-Meier analyses and log-rank tests were used to compare unadjusted time-to-fetal expulsion between mifepristone and misoprostol-only groups. To adjust for confounding and differences in drug administration, hazard ratios (HRs) for time-to-expulsion were calculated for exposure to mifepristone, adjusted for significant covariate differences identified The desired sample size was based on fetal expulsion rate, as the more direct measure of abortion effectiveness compared to abortion completion, which is dependent on physician practice with regard to curettage. Reported fetal expulsion rates within 24 hours range between 93–97% for the mifepristone-misoprostol regimen , 15 and For the 2008 and 2010 misoprostol-only and mifepristone cohorts respectively, 86% (84/97), 62% (58/94) and 85% (208/244) of women screened for participation were enrolled into the study. Overall, enrolled women constituted 50% (350/700) of all women undergoing medical abortion at study facilities during the study periods. Participant enrolment for all cohorts, including reasons for non-participation are shown in Significant differences in participant characteristics between the 2008 and 2010 misoprostol-only cohorts were gestational age at initiation of abortion (median (interquartile range): 18.1 weeks (17.6–19.9) and 16.5 weeks (14.9–17.6) respectively; and the proportion reporting prior abortions (7% [5/74] in 2008 compared to none in 2010). Participant characteristics for the combined 2008/2010 misoprostol group and the mifepristone group are shown in All women in the mifepristone cohort were instructed to self-administer 200 mg mifepristone orally at home, 24–48 hours prior to their booked admission date. Women with gestations within 1–2 days of the legal limits for medical abortion were given priority bookings, and admission was arranged in order to manage bed availability. Only 1 participant was admitted directly following assessment for mifepristone administration in the hospital, as her travel time home and back would have been excessive. The median and inter-quartile range (IQR) for the mifepristone-misoprostol interval was 49 hours (IQR 4–53). Intervals were <48 hours for 44% (91/208) participants and >72 hours for 2% (4/208). All participants reported they took the mifepristone at home, and none aborted prior to hospital admission.Following admission, administration of misoprostol was incorporated into the general ward routine. Misoprostol doses and administration routes are shown in Fetal expulsion occurred in all participants except one in the 2008 misoprostol-only cohort, who was transferred to another facility for D&E. Median time-to-fetal expulsion was shorter , and the proportion with fetal expulsion within 24 hours higher  for the mifepristone cohort compared to the misoprostol-only 2010 cohort. Kaplan-Meier survival curves and log rank tests for unadjusted time-to-fetal expulsion demonstrated significantly shortened intervals for the mifepristone cohort p<0.001, . Hazard Uterine evacuation of placental tissue was performed according to physicians’ judgement, and there was higher rate of uterine evacuation of placental tissue among the mifepristone group over the misoprostol-only group . The rate of major complications was similar for the two groups . Complications included hemorrhage requiring blood transfusion, infection treated with IV antibiotics and possible seizure . All theMore participants in the mifepristone group experienced nausea , vomiting  and tiredness . Other symptoms experienced similarly by both mifepristone and misoprostol-only groups, respectively, were diarrhea , dizziness  and headache . There was no significant linear association between pain levels and groups ; howeverCompared to the previous misoprostol-only regimen, the new service regimen reduced time-to-abortion events and hospitalization. The 93% fetal expulsion rate within 24 hours in our mifepristone group is slightly lower compared to the 94–98% reported in RCTs and case series , 22–24. Other research has reported much lower rates of placental retention for second-trimester medical abortion with mifepristone , and ourThe introduction of self-administration of mifepristone at home prior to admission for second-trimester medical abortion allowed for planning for admission while eliminating additional hospital visits for mifepristone where admission was delayed by more than 48 hours. Although women’s convenience was not specifically catered to in this study, flexible timing of admission without requiring mifepristone to be taken on-site gives women time to arrange their personal affairs, if needed, and we consider it a beneficial service delivery option. Most women stayed at least one night in hospital as incorporation of abortion care into the general ward routine delayed women receiving their misoprostol while this study was in progress. To facilitate same-day discharge, a day ward administering misoprostol immediately at early morning admission is recommended as the most cost-effective model of service provision for medical abortion , 36. ThiThe number of women experiencing hemorrhage requiring transfusion was similar in both the mifepristone and misoprostol-only groups and . The proIn terms of women’s experiences, both the mifepristone and the misoprostol-only groups reported a high prevalence of common side effects. The greater proportion in the mifepristone group experiencing nausea, vomiting and tiredness may be due to the higher initial dose of misoprostol, or possibly to mifepristone. In contrast, this group also reported less extreme pain levels and more received analgesia than the misoprostol-only cohorts; however, pain levels in all groups remained high, and more research on pain management is needed to better guide practice. The improved acceptability in the mifepristone group, despite the higher proportion who underwent curettage for retained placental tissue, is encouraging. Reduced hospitalization and less extreme pain may have contributed to a better experience. However, unmeasured differences between facilities and over time may also have played a part, and we cannot make conclusive statements in this regard from our data.Limitations of this study are that the majority of participants in the mifepristone cohort groups were from a different facility from the historic cohorts due to changes in service delivery. Study groups were non-randomized. For example, gestational age was different in the two groups; however, we adjusted for known confounders, including gestational age, in the proportional hazards analysis . The comThis study reports clinical and acceptability improvements during introduction of a mifepristone-misoprostol regimen for medical abortion at 12–20 weeks gestational age at busy public sector hospitals in South Africa. Specific aspects that were successful included self-administration at home of mifepristone which allowed for admission planning, shortened procedure times, reduced hospitalization which increased the capacity of facilities to serve more women compared to previously, and increased acceptability.S1 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Traumatic brain injury (TBI) is a major brain injury type commonly caused by traffic accidents, falls, violence, or sports injuries. To obtain mechanistic insights about TBI, experimental animal models such as weight-drop-induced TBI in rats have been developed to mimic closed-head injury in humans. However, the relationship between the mechanical impact level and neurological severity following weight-drop-induced TBI remains uncertain. In this study, we comprehensively investigated the relationship between physical impact and graded severity at various weight-drop heights.The acceleration, impact force, and displacement during the impact were accurately measured using an accelerometer, a pressure sensor, and a high-speed camera, respectively. In addition, the longitudinal changes in neurological deficits and balance function were investigated at 1, 4, and 7 days post TBI lesion. The inflammatory expression markers tested by Western blot analysis, including glial fibrillary acidic protein, beta-amyloid precursor protein, and bone marrow tyrosine kinase gene in chromosome X, in the frontal cortex, hippocampus, and corpus callosum were investigated at 1 and 7 days post-lesion.Gradations in impact pressure produced progressive degrees of injury severity in the neurological score and balance function. Western blot analysis demonstrated that all inflammatory expression markers were increased at 1 and 7 days post-impact injury when compared to the sham control rats. The severity of neurologic dysfunction and induction in inflammatory markers strongly correlated with the graded mechanical impact levels.We conclude that the weight-drop-induced TBI model can produce graded brain injury and induction of neurobehavioral deficits and may have translational relevance to developing therapeutic strategies for TBI. Traumatic brain injury (TBI) is one of the most common brain injuries caused by an external mechanical force, such as crushing, rapid acceleration or deceleration impact, and projectile penetration . TBI is Previous studies have demonstrated that TBI induces sensorimotor and cognitive impairments in weight-drop-induced TBI models , 8. AlthFor translational purposes, identifying and comparing the mechanical or kinematic properties during impact and the induced brain injury level between animals and humans is crucial. Previous studies have demonstrated that the level of mechanical responses such as linear acceleration or velocity during impact is associated with traumatic axonal injury or severity of behavior at 24 h post-injury , 18. HowFifty-two male Sprague—Dawley rats  weighing between 364 and 425 g  were used for the present experiment. All experimental procedures were preapproved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University (TMU) and followed by the TMU IACUC guidelines to treat animals humanely and reduce animal suffering by use of appropriate anesthesia and analgesics . Rats were housed on a 12-h light/dark cycle in a temperature- and humidity-controlled animal center until experimental use at TMU. Animals were monitored twice daily prior and post-lesion and animal were humanely euthanized according to accepted pre-established endpoint criteria: loss of >20% body weight, labored breathing, hunched body, and lethargic animal. Furthermore, at the end of the study, all animals were humanely euthanized via carbon dioxide inhalation followed by cervical dislocation.To minimize animal suffering and distress during surgery, animals were anesthetized using intraperitoneal injection of tiletamine—zolazepam  and xylazine  30 min prior to impact. To record the mechanical impact of the head during weight-drop, pressure was recorded using a load cell sensor  fixed to the central portion of the rat skull vault between the bregma and lambdoid sutures. To induce TBI, Marmarou’s impact acceleration model was modified and applied; rats were placed prone on flexible foam and were secured in place by using two elastic belts. A Plexiglas tube was then positioned vertically, and the lower end of the tube was centered directly above the pressure sensor. TBI was induced using a 450-g brass weight falling from 1, 1.5, and 2 m through a vertical transparent Plexiglas tube. The impact response was recorded by the load cell fixed on the rat skull. All the signals were measured using the data acquisition system . Sham control TBI rats underwent the same surgical procedures, but did not receive weight-drop induced TBI. Body temperature was monitored with a rectal probe throughout surgery, and the temperature was maintained at 37.0 ± 0.5°C using an adjustable heating pad during recovery from anesthesia.To record the kinematic changes in the head during impact, the impact event was captured using a high-speed video camera  . The hig(x) of the rat head during impact were determined by tracking the attached impactor and helmeted head by using an image analysis program written in MATLAB. The relationship among displacement (x), velocity (v), and acceleration (a) of the rat head with time (t) during impact can be formulated as follows:To analyze the changes in displacement, velocity, and acceleration during impact, a high-speed digital camera was used to record the displacement of the rat head during impact. Black and white line markers were painted on the impactor and helmet for subsequent image tracking analysis through video recordings (see v) of the rat head was calculated by differentiating displacement-time histories of the head from the recorded video data. In addition, the acceleration-time history was calculated using velocity-time histories. Moreover, the acceleration calculated using digital image analysis was compared with that measured by the accelerometer. Thus, peak instantaneous acceleration and deceleration of the rat head were determined using the acceleration-time curve recorded using the digital camera and accelerometer. After surgery, animals were daily monitored for pain and distress. Animals were administered Ketorolac analgesics for 48 h after surgery if pain and distress behaviors were observed.Change in velocity  is a multifunctional evaluation scale that comprises motor, sensory, reflex, and balance tests. The rats receive one point when they are unable to perform the test or lose the tested reflex. Therefore, the score is proportional to the injury severity. The test scale ranges from 0 to 18 .The beam walking test was used to evaluate fine motor coordination and balance function . Rats weWe extracted brain tissue samples from the rats 1 day and 7 days post TBI to quantify the injury severity. The rats were rapidly anesthetized with sodium pentobarbitone  and then decapitated. Samples were extracted from the frontal cortex (FC), corpus callosum (CC), and hippocampus (H). The samples were then lysed with radioimmunoprecipitation assay buffer. For Western blot analysis, protein cell lysates (30 μg) were resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes. Finally, the membranes with blots were incubated overnight with primary antibodies  at 4°C. The densities of the protein bands were digitized, and the ratio of the ERT density to the beta-actin or alpha-tubulin density was compared among the groups.n = 3 at each timepoint in each group) were sacrificed for western blot analysis  between the mechanical impact level , the severity of brain injury and neurobehavioral function. Biochemistry comparisons between rats in the sham-lesion group and various fall height groups were performed by one-way ANOVA with LSD multiple comparison post hoc test. Data were analyzed using SPSS version 17.0  with the significance level set at p < 0.05 for each assessment. All data are presented as the average standard error of the mean (SEM).For statistical analysis of all behavioral measurements, a two-way repeated measure analysis of variance (ANOVARM) with group  and time factors  was performed. Multiple within-subject comparisons were performed using post hoc least significant difference (LSD) analysis when the main effect of time was significant. Furthermore, we used linear Regression test to determine the coefficient of determination  mortality rate was observed following weight-drop induced TBI from 2 m. In the 1.5-m fall height group, a mortality rate of 12.5% (2/16) was observed. No animal deaths occurred following injury in the 1-m fall height group. All of the deaths of experimental animals occur immediately (< 30 min) followed by the induction of brain injury.The response of impact force and acceleration waveforms of 1, 1.5, and 2 m are shown in The impact displacement trace was captured by the high-speed video camera and is presented in 6,72 = 4.60, p = 0.001) as well as significant time  and group  effects. Post hoc LSD analysis revealed that the mNSS in the 1-m, 1.5m and 2-m group was significantly increased on day 1 and remained consistent up to day 7 after the weight-drop experiment compared with the pre-lesion level.For behavioral tests, neurological and balance functions were assessed 1 day prior to the weight-drop treatment and 1, 4, and 7 days after the weight-drop treatment. Group results for the three fall height groups are presented in 3,66 = 9.218, p < 0.0001) but no significant time × group interaction . Compared with the pre-lesion level, the beam walking test in 2-m group showed a statistically significant increase on day 1 and remained consistent up to day 7 after the weight-drop lesion, but the 1.5 m group did not reach statistical significance up to day 4 post-lesion. In the 1-m group, no significant differences were found in beam balance test when compared to pre-surgery baseline data   .2 = 0.76, p <0.001), acceleration  and impact height  , acceleration  and impact height   . We also <0.001) .p < 0.05, one-way ANOVA, post hoc LSD analysis). To further identify the relationship between physical impact and induced brain injury level at various weight-drop heights. We also performed the correlation tests between the inflammatory expression markers  on day 7 post-injury and impact force, peak to peak acceleration and impact height. The linear correlation analysis showed a significant positive correlation between the protein expression in APP and impact force , acceleration  and impact height  , acceleration  and impact height   at 1 , S1 Fig  <0.001) . A signi <0.004) . These rIn the present study, we modified Marmarou’s impact acceleration model and characterized the kinematic parameters during the weight-drop impact. The changes in impact force on the head, linear acceleration, and displacement of the rat head during impact injury of various severities were recorded and analyzed. Furthermore, we conducted a detailed experiment to identify the relationship between the impact kinematics and the changes in neurological function and motor behaviors by using the modified weight-drop TBI model. Our results demonstrate that controlling impact height and pressure can reliably induce injury of graded severity. The results also showed a highly positive correlation between the behavioral tests and recorded impact parameters. This model may serve as a translational platform for bridging human and animal studies and establishing new therapeutic strategies for TBI.Although Marmarou’s impact acceleration model has been widely used in various TBI studies, it has been criticized for not providing highly reproducible results because of the lack of precise control over impact force and animal biomechanics and the lack of precise recording during impact , 18, 22.When a suitable diseased animal model is used, understanding the neurological outcomes after injury is crucial. We employed a time-course analysis of behavioral and biochemical recordings, which highly correlated with the impact force and the kinematic results during impact. We selectively used three traditional fall heights, which were obtained from conditions identical to those in the original impact acceleration model by Marmarou , 5. The Functional behavior analysis is crucial in TBI research and we demonstrated that the modified weight-drop model may induce the severity-dependent neurological and motor behavioral disturbances. The multiple behavioral and biochemical testing during the course of trauma progression are warranted to reveal the levels of brain injury and motor performance, as well as to determine whether the weight-drop model induced functional deficits are stable or spontaneously recover over time. Whereas previous research has examined the behavioral alterations arising from various falling height of weight-drop method, the time-course changes of neurological and balance function of varying levels of brain injury remained not fully characterized. Thus, we applied general neurological and beam balance test following weight-drop at different time points to observe the changes of functional outcome. One day after TBI lesion, except for the 1-m fall height group, 1.5 and 2-m groups caused temporary motor balance deficits. Over 7 days, although there were spontaneous recoveries for the three fall height groups following TBI lesion, the neurological and balance function still did not reach the pre-surgery level in the 2-m weight-drop group. This implies that following severe weight-drop induced TBI, the neurological and balance function were profoundly altered. The neurological and motor deficits produced by our modified weight-drop methods are consistent with the previous reports demonstrating significant motor impairments following lateral fluid percussion and controlled cortical impact brain injury model –28. In aAnimal models are essential for studying the biomechanical, cellular, and molecular aspects of human TBI that cannot be addressed in a clinical setting and for developing and characterizing novel therapeutic interventions. We used three markers  to represent inflammation and axon injury following TBI. Our results support the association of BMX, APP, and GFAP upregulation with trauma severity in rats. Following weight-drop-induced brain injury, an increase in the expression of these proteins in the injured brain area was founded. The association between the BMX, APP, and GFAP expression levels and severity of injury was demonstrated using various fall heights. We found that the amount of neural inflammation and axonal injury varied with impact force and acceleration; a low impact height (1 m) produced little or no axonal injuries and a higher impact height (1.5–2 m) significantly increased these markers of inflammation and axonal injury. Notably, the changes in the levels of these markers were apparent early at day 1 and remained elevated for at least 1 week post TBI-lesion, indicating that the inflammation and axonal injuries appeared in acute or subacute stages of TBI. Furthermore, with the spontaneous recovery, the level of balance function was not significant in the 1 and 2 m weight-drop groups. However, the biochemistry biomarkers appear to be still sensitive detection methods when compared with the behavioral tests, indicating that the subsequent increases in the levels of TBI biomarkers on day 7 post-lesion also validate the brain injury level. These results suggest that neuroinflammation or axonal damage following weight-drop-induced TBI may become detectable at acute, subacute and chronic time points following injury and may develop in a progressive manner.Consistent with earlier studies, the increased APP, GFAP and BMX metabolites are observed following brain injury using similar Marmarou acceleration weight drop model , 29, 30 The present study provides such evidence and shows a significant correlation between the neurologic dysfunction severity and the mechanical impact magnitude. It is noted that the severity of neurological function, beam balance and brain damage at 1 day or 7 days post-lesion have been shown to strongly correlate with the increased impact force, acceleration and fall height during impact, as determined by several specific sensors. Furthermore, the impact force and peak to peak acceleration showed better correlation with neurobehavioral function than impact height. Although initial impact height were consistent during impact, this result indicates that traditional weight-drop protocol for inducing graded injury severity by adjusting fall height could still have high variability. It is suggested that the graded severity of brain injury can be predicted by the real time measurement of impact force and acceleration. Otherwise, our results demonstrate that stringent control over impact force and acceleration can reproducibly and reliably induce graded severity of brain injury.In the present study, we developed a characteristic analysis method for detecting kinematic data for various severities of TBI in the weight-drop-induced TBI model. By using this model, we showed that various graded impact forces and accelerations can produce graded neurological and motor impairments as well as axonal inflammation and injury. This method entails using newly developed quantitative measures to reduce the variation and increase the reproducibility of the weight-drop-induced TBI model. This method would be useful for studying the pathophysiology of TBI and developing therapeutic strategies for TBI.S1 Data(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 VideoThe demonstrated video was recorded at 300 frames/s during weight-drop experiment. The acceleration, impact force, and displacement during the impact were measured using an accelerometer, a pressure sensor, and a high-speed camera, respectively.(WMV)Click here for additional data file.


Across the participants, reduced FPN connectivity was associated with greater cardiovascular capacity (p = 0.05). In the AT group, reduced FPN connectivity was significantly associated with improved mobility performance, as measured by the Timed-Up-and-Go test . These results suggest progressive AT may improve mobility in older adults with SIVCI via maintaining intra-network connectivity of the FPN.Impaired mobility is a major concern for older adults and has significant consequences. While the widely accepted belief is that improved physical function underlies the effectiveness of targeted exercise training in improving mobility and reducing falls, recent evidence suggests cognitive and neural benefits gained through exercise may also play an important role in promoting mobility. However, the underlying neural mechanisms of this relationship are currently unclear. Thus, we hypothesize that 6 months of progressive aerobic exercise training would alter frontoparietal network (FPN) connectivity during a motor task among older adults with mild subcortical ischemic vascular cognitive impairment (SIVCI)—and exercise-induced changes in FPN connectivity would correlate with changes in mobility. We focused on the FPN as it is involved in top-down attentional control as well as motor planning and motor execution. Participants were randomized either to usual-care (CON), which included monthly educational materials about VCI and healthy diet; or thrice-weekly aerobic training (AT), which was walking outdoors with progressive intensity. Functional magnetic resonance imaging was acquired at baseline and trial completion, where the participants were instructed to perform bilateral finger tapping task. At trial completion, compared with AT, CON showed significantly increased FPN connectivity strength during right finger tapping ( Impaired mobility is a major concern for older adults and is associated with increased risk for disability, institutionalization, and death . The preCurrent evidence supports the recommendation of targeted exercise training to improve mobility, prevent major mobility disability, and reduce the risk of future falls . The widThese data suggest that exercise may reduce falls in older adults via several mechanisms, not just via improved physical function.We previously proposed that cognitive and neural plasticity may be an important, yet under-appreciated mechanism by which exercise promotes mobility and reduce falls . This hyDespite the growing recognition that targeted exercise training may promote mobility outcomes in older adults via central mechanisms , few intOlder adults with subcortical ischemic vascular cognitive impairment (SIVCI), the most common form of vascular cognitive impairment (VCI) , are at Among the relevant neural networks, most notably, is the frontoparietal network (FPN). The FPN is involved in top-down attentional control and allocation of available neural resources that contribute to executive processes, such as response anticipation and conflict processing , as wellThus, we propose FPN connectivity as one of the neural mechanisms by which exercise promotes mobility in older adults with mild SIVCI. Using functional magnetic resonance imaging (fMRI) data from a 6-month single-blind randomized controlled trial , we conducted a planned secondary analysis to assess the impact of moderate-intensity aerobic exercise training on functional connectivity of FPN among older adults with mild SIVCI. We hypothesized that aerobic exercise-induced increases in FPN connectivity would correlate with improved mobility. The primary results from the parent study have been published , which pThis is a secondary analysis of neuroimaging data acquired from a 6-month proof-of-concept RCT (NCT01027858) of aerobic exercise in older adults with mild SIVCI , 2016. TAs the current study was a secondary analysis, we sought to recruit as many eligible and consenting individuals from the parent study as possible. To briefly describe the recruitment process, we recruited from the University of British Columbia Hospital Clinic for Alzheimer’s Disease and Related Disorders, the Vancouver General Hospital Stroke Prevention Clinic, and specialized geriatric clinics in Metro Vancouver, BC. Recruitment occurred between December 2009 and April 2014 with randomization occurring on an ongoing basis. Study participants were clinically diagnosed with mild SIVCI as determined by the presence of cognitive syndrome and small vessel ischaemic disease . Small vFigure 1). Of the 38 participants (54% of parent sample) that completed baseline MRI scanning, 7 (18% of the sample) dropped out from the study and 10 (26% of the sample) failed to correctly perform the motor finger tapping task . Consequently, 21 participants who completed MRI at baseline and trial completion were included in this secondary analysis (30% of parent sample). Ethical approval was provided by the University of British Columbia’s Clinical Research Ethics Board (H07-01160). All participants provided written informed consent.The Consolidated Standards of Reporting Trial flowchart shows the number and distribution of participants included in this secondary analysis . A research team member not involved with the study held this sequence at a remote location. After the completion of consent and baseline testing, the research coordinator contacted the team member holding the list to determine the next allocation.The randomization sequence was generated using the web application For the AT group, AT consisted of supervised thrice-weekly 60-min classes of walking for the 6-month intervention period. All AT group classes were led by instructors certified to instruct seniors and were delivered in a group setting. Each 60-min class included a 10-min warm-up, 40-min of walking, and a 10-min cool down. Both the warm-up and cool-down included passive and active stretches, as well as range of motion exercise.Walking occurred outdoors and followed predetermined routes around local areas. The intensity of the AT program was monitored and progressed using three approaches: (1) heart rate monitoring with an initial intensity of 40% of age specific target heart rate . HRR was calculated by subtracting resting heart rate from maximum heart rate [using the formula: 206.9 – 0.67 × Age ] and recThe AT group was also given a pedometer to serve as both an incentive and monitoring tool. Participants recorded the number of steps each day taken outside the AT classes on standard logs provided by the research team.Participants in the CON group received usual care, in which they were provided with monthly educational materials about VCI and healthy diet. However, no specific information regarding physical activity was provided. In addition, research staff phoned the CON participants on a monthly basis to maintain contact and to acquire research data.All participants were instructed to report any adverse effects due to the AT exercises to our research coordinator, such as falls or musculoskeletal pain persisting longer than 48 h. Participants were also questioned about the presence of any adverse effects, such as musculoskeletal pain or discomfort, at each exercise session. All instructors also monitored participants for symptoms of angina and shortness of breath during the exercise classes. External experts from our safety monitoring committee reviewed all adverse events reported on a monthly basis.At baseline, participants underwent a clinical assessment with study physicians (GYRH and PL) to confirm current health status and study eligibility. Age in years and education level were assessed by self-report. Standing height was measured as stretch stature to the 0.1 cm per standard protocol. Weight was measured twice to the 0.1 kg on a calibrated digital scale. Waist-to-hip ratio was determined by measuring the widest part of the hip circumference and the waist just above the navel in centimeters. The Functional Comorbidity Index  assessedGlobal cognition was assessed using the MMSE  and the All MRI was conducted at the University of British Columbia (UBC) MRI Research Center located at the UBC Hospital on a 3.0 Tesla Intera Achieva MRI Scanner  using an 8-channel SENSE neurovascular coil. The fMRI consisted of two successive runs with 165 dynamic images of 36 slices (3 mm thick) with the following parameters: repetition time (TR) of 2000 ms, echo time (TE) of 30 ms, flip angle (FA) of 90 degrees, field of view (FoV) of 240 mm, acquisition matrix 80 × 80, voxel size of 3 mm × 3 mm × 3 mm. High resolution anatomical MRI T1 images were acquired using the following parameters: 170 slices (1 mm thick), TR of 7.7 ms, TE of 3.6 ms, FA of 8 degrees, FoV of 256 mm, acquisition matrix of 256 × 200.During each scanning session, the study participants were asked to perform a finger tapping motor task that had been previously administered and described . BrieflyRun A: Rest, Left Tap, Rest, Right Tap, Rest, Right Tap, Rest, Left Tap, RestRun B: Rest, Right Tap, Rest, Left Tap, Rest, Left Tap, Rest, Right Tap, Rest3) at baseline was quantified with structural MRI data acquired on the same MRI scanner  at the UBC MRI Research Centre. A T2-weighted scan and a proton-density-weighted (PD-weighted) scan were acquired for each subject. For the T2-weighted images, the repetition time (TR) was 5,431 ms and the echo time (TE) was 90 ms, and for the PD-weighted images, the TR was 2,000 ms, and the TE was 8 ms. T2- and PD-weighted scans had dimensions of 256 × 256 × 60 voxels and a voxel size of 0.937 mm × 0.937 mm × 3.000 mm. Briefly, WMLs were identified and digitally marked  by a radiologist on T2 and PD weighted images. Marked WMLs were automatically segmented by a customized Parzen windows classifier that estimated the intensity distribution of the lesions – which also included heuristics that optimized the accuracy of the estimated distributions  and the Short Physical Performance Battery (SPPB). The TUG required participants to rise from a standard chair, walk a distance of three meters, turn, walk back to the chair and sit down . We recoFor the SPPB, participants were assessed on performances of standing balance, walking, and sit-to-stand. Each component is rated out of four points, for a maximum of 12 points; a score < 9/12 predicts subsequent disability .Participant’s cardiovascular capacity was assessed using the 6-Minute Walk Test . The totMonthly total physical activity level was determined by the Physical Activities Scale for the Elderly (PASE) self-report questionnaire .Image preprocessing was carried out using tools from FSL (FMRIB’s Software Library) [78], MATLAB (Matrix Laboratory), and toolboxes from SPM . Excess unwanted structures  in high resolution T1 images were removed via Brain Extraction Tool (BET); rigid body motion correction was completed using MCFLIRT ; spatial smoothing was carried out using Gaussian kernel of Full-Width-Half-Maximum (FWHM) 6.0 mm; temporal filtering was applied with high pass frequency cut-off of 120 s. In addition, a low pass temporal filtering was also included to ensure the fMRI signal fluctuated between 0.008 < f < 0.080 Hz, the ideal bandwidth to examine functional connectivity. Furthermore, the application of a low pass filter eliminated high frequency signals that could be confounds. Participants’ low-resolution functional data were registered to personal high resolution T1 anatomical images, which were subsequently registered to standardized 152 T1 Montreal Neurological Institute (MNI) space.Noise generated from both physiological and non-physiological sources were removed through regression of the cerebral-spinal fluid (CSF) signal, white matter signal, and global brain signal. Global signal regression had been reported as both valid and useful step in functional connectivity analyses  that mayTable 1.Previous studies guided our choice of seeds in the whole brain analysis of the FPN . The FPNFrom each ROI, preprocessed time-series data were extracted with 14 mm spherical regions of interest drawn around their respective MNI coordinates in standard space. The different conditions  within each block of the motor task were extracted and compiled together. To concatenate the time-series data, the stimulus onset time for each task condition was acquired from the task program. Each volume of the data was then sorted according to their respective condition. Once the data were properly categorized, the task-specific volumes  were merged using a script provided in the FSL program. The first three volumes of any condition were discarded to account for delay of the hemodynamic response. Evidence in the literature has demonstrated that functional connectivity derived from temporally spliced/merged resting-state data from blocked fMRI design is not significantly different from connectivity derived from continuous data . Recent Z = 2.33, with cluster correction of p < 0.05.Region of interest time-series data were subsequently cross-correlated with every voxel within the brain to establish functional connectivity maps of their associated neural networks, in which pairwise correlation between time-series extracted from ROI listed above was calculated. Individual-level within-subject results were generated via ordinary least-squares (OLS) regression using FSL’s flameo  in FSL bp ≤ 0.05 for all analyses. Change in network connectivity strength was computed in SPSS as 6-month FPN connectivity minus baseline FPN connectivity. Linear mixed models with random intercepts and time-varying outcome measures were constructed to statistically test for significant between-group differences in change in network connectivity while adjusting for baseline total WML and age. A group by time interaction indicated group differences in changes in FPN connectivity from baseline to post-intervention. Similar analyses were conducted to determine whether there were group differences in changes in TUG, SPPB, 6MWT, and PASE scores. The primary analyses included the 21 participants with valid baseline and post-intervention fMRI data. Secondary analyses followed the intention-to-treat principle by including nine additional individuals with valid baseline fMRI but were lost to follow-up; maximum likelihood estimation allowed for these individuals to inform the treatment effects, despite having missing follow-up data and to determine whether loss to follow-up might bias the treatment effects estimated with only treatment completers.Statistical analysis was conducted using the IBM SPSS Statistic 23 for Windows . Statistical significance was set at n = 12) correlated with change in mobility, as measured by TUG and SPPB, or change in 6MWT across the 6-month study duration.Bivariate correlation analyses were performed to determine whether any significant changes in intra-network FPN connectivity  in the AT group , indicating that AT had a positive effect on cardiovascular capacity . Study demographics are reported in Table 2, pedometer information over the intervention period is reported in Table 3, mobility and cardiovascular capacity measures are reported in Table 4; these measures do not differ between groups at baseline nor differ significantly from the 70 eligible participants enrolled in the parent study (SD = 8.7 years), which is not significantly different from the mean age of the parent cohort at 74.3 years (SD = 8.3 years). Compared to the nine individuals with valid baseline data only, the study sample did not differ on baseline FPN connectivity  but did have higher average baseline MoCA scores . Neither of the mobility measures nor self-reported physical activity differed significantly between groups across the 6 months. However, we observed a trend-level group difference in the change in 6-Minute Walk Test performance , in which the AT group showed greater improvement (48.6 m) compared with the CON group (-0.3 m).Among the 70 randomized individuals in the parent study, we observed a significant effect of AT on 6-min walk performance, a well-established tool that accurately evaluates cardiovascular fitness   showed there were no significant between group differences in the mean network connectivity strength at baseline, regardless of task conditions (Table 5). At trial completion, compared with AT, CON exhibited significantly greater intra-network coupling of the FPN during right finger tapping (p < 0.02) after adjusting for baseline WML and age. No AT effects were observed for FPN connectivity during left finger tapping (p = 0.26) or during rest (p = 0.50). We conducted a secondary, intention-to-treat analysis using all 30 participants with usable baseline data, regardless of loss to follow-up, and observed similar, though weaker, between-group differences in FPN coupling during right finger tapping (p = 0.08). As with the primary analyses, CON showed an increase in intra-network coupling of the FPN , whereas AT showed no significant change over time .Results from the seed-based functional connectivity analysis on the FPN . Within the AT group (N = 12), the change in FPN connectivity during right tapping was significantly associated with change in TUG performance . Specifically, reduced FPN connectivity from baseline to post-intervention correlated with improved TUG performance over the same period of time .Bivariate correlation across the study sample showed that the change in FPN connectivity during right tapping was significantly associated with change in 6-Minute Walk Test performance . In assessing within-network connectivity, it may be that the effects of induced anti-correlation are less significant. However, as stated by Murphy and Fox  ‘there iOur results demonstrate that neural network functional connectivity may contribute to the effects of aerobic exercise on mobility among older adults with SIVCI. We observed that 6 months of AT maintains motor task-based connectivity within the FPN of older adults with SIVCI, and the degree of decoupling within this region correlates with improvements in mobility. As such, our current findings support emerging results from others that altered functional connectivity within certain neural networks might represent a beneficial change in older adults with mild SIVCI, especially vis-à-vis their mobility. More broadly, these results bring further support to the burgeoning notion that functional neural changes contribute to exercised-induced improvements to mobility among older adults. As extension of these findings, future studies should explore potential interaction between mobility and cognitive outcomes among this population.TL-A had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.TL-A and RH were involved in the study concept, design, acquisition of data, preparation and critical review of the manuscript. CH, MM, WC, and TL-A were involved in data collection. CH, TL-A, and JB were involved in writing of the manuscript. CH, TL-A, and JB were involved in statistical analyses. CH, TL-A, and JB were involved in interpretation of data. CH and SW were involved in fMRI data analyses. MV and TH were involved in critical review of the manuscript. All authors had full access to all of the data  in the study and can take responsibility for the integrity of the data and the accuracy of the data analysis.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. The reviewers KM, GA and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Tetrapyrroles make critical contributions to a number of important processes in diverse organisms. In plants, tetrapyrroles are essential for light signaling, the detoxification of reactive oxygen species, the assimilation of nitrate and sulfate, respiration, photosynthesis, and programed cell death. The misregulation of tetrapyrrole metabolism can produce toxic reactive oxygen species. Thus, it is not surprising that tetrapyrrole metabolism is strictly regulated and that tetrapyrrole metabolism affects signaling mechanisms that regulate gene expression. In plants and algae, tetrapyrroles are synthesized in plastids and were some of the first plastid signals demonstrated to regulate nuclear gene expression. In plants, the mechanism of tetrapyrrole-dependent plastid-to-nucleus signaling remains poorly understood. Additionally, some of experiments that tested ideas for possible signaling mechanisms appeared to produce conflicting data. In some instances, these conflicts are potentially explained by different experimental conditions. Although the biological function of tetrapyrrole signaling is poorly understood, there is compelling evidence that this signaling is significant. Specifically, this signaling appears to affect the accumulation of starch and may promote abiotic stress tolerance. Tetrapyrrole-dependent plastid-to-nucleus signaling interacts with a distinct plastid-to-nucleus signaling mechanism that depends on GENOMES UNCUOPLED1 (GUN1). GUN1 contributes to a variety of processes, such as chloroplast biogenesis, the circadian rhythm, abiotic stress tolerance, and development. Thus, the contribution of tetrapyrrole signaling to plant function is potentially broader than we currently appreciate. In this review, I discuss these aspects of tetrapyrrole signaling. Figure 1).Tetrapyrroles contribute a number of functions to processes that are critical for the viability of diverse organisms including essential functions to the light reactions photosynthesis, which underpins life on earth. Tetrapyrroles contain four pyrroles— aromatic rings containing four carbon atoms and one nitrogen atom—often linked together with methine groups, in linear  or cyclic arrangements . Porphyrins chelate metal ions, such as cobalt, iron, or magnesium ions. These structural features along with a variety of ring substituents contribute to the diverse functions of tetrapyrroles . These tetrapyrroles provide critical functions to important processes, such as light signaling, the detoxification of reactive oxygen species, the assimilation of nitrate and sulfate, respiration, programmed cell death, and the light-harvesting reactions of photosynthesis. In vivo, many tetrapyrroles, such as porphyrins, are sequestered to prevent them from generating singlet oxygen (1O2), a toxic reactive oxygen species that causes stress and cell death. Electronically excited versions of porphyrins produce 1O2 when they transfer energy to molecular oxygen  to plants. ALA is the 5-carbon universal precursor of tetrapyrroles. ALA is hydrophilic and is readily taken up by plants. Exogenous ALA was demonstrated to flood the tetrapyrrole biosynthetic pathway with intermediates, induce increases in the levels of chlorophyll precursors , and dowthe dark . These dthe dark . More rethe dark genomes uncoupled (gun) mutant screen . GUN2 encodes heme oxygenase. GUN3 encodes phytochromobilin synthase , which synthesizes heme from protoporphyrin IX  to test for gun phenotypes. However, norflurazon appeared to block chloroplast biogenesis when Arabidopsis seedlings were grown in 10 μmol m-2 s-1 white light   mutants  . Additio mutants . Thus, tgun phenotypes  Attempts to show that norflurazon-treated gun4-1 and gun5 accumulate less reactive oxygen species (ROS) than norflurazon-treated wild type by quantifying ROS-responsive gene expression, staining with ROS responsive dyes, and by treating plants with ROS scavengers were not successful   reduced chlorophyll levels to 20% of the chlorophyll found in wild type. Thus, in contrast to norflurazon treatments, sig2 attenuates but does not block chloroplast biogenesis. sig2 mutants expressed lower levels of PhANGs than wild type. Etiolated gun5 sig2 expressed higher levels Lhcb2.2 and other PhANGs than etiolated sig2 (Arabidopsis seedlings that were not treated with norflurazon. (7) Tetrapyrrole metabolism regulated starch-related nuclear gene expression in Bright Yellow-2 (BY2) cells derived from tobacco, which lack photosynthetic plastids  No mutants) . (3) gun mutants . (4) Bloncreased . (5) Higinifolia . (6) Nulted sig2 . Thus, pplastids . (8) In flurazon . (9) Phy mutants .gun phenotypes  chloroplast biogenesis in Arabidopsis seedlings would need to exhibit such extreme sensitivity to this photooxidative stress that the perturbations in the tetrapyrrole biosynthetic pathway exhibited by gun2, gun3, gun4-1, gun5, and gun6-1D would not affect the photobleaching activity of norflurazon and (2) the perturbations in tetrapyrrole metabolism induced by gun5 would need to affect a distinct mechanism to affect the expression of PhANGs in the dark.The idea that no evidence of partial resistance to norflurazon was obtained in so many independent experiments because during chloroplast biogenesis, the norflurazon-induced increase in ROS is transient or localized  is diffienotypes , etiolatted sig2 , and nei mutants . If norfGUN2/HY1, GUN3/HY2, GUN4, and GUN5 that were isolated from gun mutant screens are loss-of-function alleles , aba-insentive4 (abi4), and phytochrome-associated protein phosphatase 5 (papp5)  . These fin vitro , 2011. Fin vitro . In appaArabidopsis when chloroplast biogenesis was blocked with norflurazon treatments  to eliminate the possibility of the circadian regulation of PhANG expression obscuring the plastid regulation of PhANG expression , Mg-Proto methyltransferase (ChlM), and Mg-ProtoME cyclase (Chl27) in 10-day-old Arabidopsis seedlings with an inducible RNAi system. Within 24 h, Mg-ProtoME accumulated—to higher levels than observed by Chl27 RNAi lines. It is not clear whether the approach of Chl27 RNAi lines—24 h after suppression was induced—there were no macroscopic changes in the plants and no significant differences in the expression of PhANGs or ROS-regulated genes. Four days of downregulated Chl27expression induced chlorophyll deficiencies, necrosis, and downregulated PhANG expression. Plastid-to-nucleus signaling was proposed to not cause the changes in gene expression that were observed 24 h after the RNAi-induced suppression of ChlH, ChlM, and Chl27  and 60-fold (tobacco). Mg-ProtoME accumulated 100 (tobacco) and 200-fold (barley). This accumulation occurred within 30 min (tobacco) and 60 min (barley) and declined within another hour . An indeic light . In tobater dawn , severalter dawn . Thus, iter dawn  and the hcb mRNA .An attempt to identify the cytosolic receptor for Mg-Proto in plants revealed that a large number of different cytosolic proteins bound to an affinity column constructed by linking Mg-Proto to Affi-Gel 102 . SeveralLhcb expression, Lhcb genes. RNAi lines that reduced the expression of all HSP90.1–HSP90.4 genes exhibited lower levels of PhANG expression relative to wild type, but only norflurazon- or methyl viologen-treated lines were tested , which binds to promoters of genes encoding HAP and stimulates their transcription. HAP and CBP induced transcription in general and thus, were proposed to promote the transient increases in the levels of cellular RNA that porphyrin signaling is proposed to induce  that overexpressing catalytically active FC1 within the physiologically relevant range and feeding ALA to norflurazon-treated seedlings induced an increase in the expression of PhANGs  and (2) flurazon . SIG2 isplastids . The synynthesis . The levignaling . Consist nucleus .Arabidopsis. The expression of FC1 is observed in non-photosynthetic tissues and is induced by stress. FC2 is coexpressed with PhANGs . The current data are also consistent with changes in the flux through the tetrapyrrole biosynthetic pathway activating a plastid-to-nucleus signaling mechanism  induces increases in the levels of Mg-Proto and Mg-ProtoME and (2) is correlated with downregulated expression of PhANGs was observed under distinct developmental and environmental conditions relative to the conditions used to screen for gun mutants. The evidence consistent with oxidative stress inducing an increase in the levels of Mg-Proto or Mg-ProtoME that affects gene expression in the nucleus is also consistent with oxidative stress attenuating heme signaling or at least in some instances, ROS signalingThe plastid-to-nucleus signaling mechanism that is specifically interrogated by the At this point, it is perhaps most important to understand the mechanism of tetrapyrrole signaling in plants. Understanding this mechanism may help us to understand why perturbations in tetrapyrrole metabolism sometimes upregulate, downregulate, or have no effect on the expression of nuclear genes. Understanding this mechanism may help us to understand the interactions between tetrapyrrole signaling and GUN1-dependent plastid-to-nucleus signaling, interactions between tetrapyrrole signaling and light-regulated development, and the biological function of tetrapyrrole signaling. Identifying a protein that specifically contributes to tetrapyrrole signaling  is perhaps the highest priority. Mutants with defects in such a gene are essential for learning the biological function of tetrapyrrole signaling in plants. The evidence for tetrapyrrole signaling contributing to abiotic stress tolerance and for the interactions between tetrapyrrole metabolism and GUN1-dependent plastid-to-to-nucleus signaling are consistent with an important role for tetrapyrrole signaling in plants. Thus, future research in this area is expected to advance our understanding of plant biology and possibly contribute to the development of new germplasm that will help to meet the needs of agriculture in our changing environment.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.