At this stocking rate, yak weight gain per ha was 88% of that achieved at higher stocking rates typically used by farmers, but with little or no evidence of land degradation.The Qinghai–Tibetan Plateau is a very large land unit and an important terrestrial ecosystem within the Eurasian continent. Because of the harsh climate associated with the high altitude, alpine meadows on the plateau are susceptible to degradation from overgrazing. For this region, and for other alpine meadow pastures internationally, there is a need to define the sustainable stocking rate, to develop sound policy pertaining to future land use. Here we report biomass and liveweight gain per animal and per ha for pastures grazed by yaks at high, medium, or low stocking rates over 4 growing seasons from 2010 to 2013. Measures of herbage nutritive value are reported. The influence of inter-year variation in precipitation on standing herbage biomass was also evaluated. Higher precipitation increased standing herbage biomass and herbage nutritive value, indicating that vegetation suffered summer water deficit even in this environment. The sustainable stocking rate in this environment was determined to be approximately 1 yak ha Bos grunniens) and Tibetan sheep (Ovis aries). Global environmental changes are strongly affecting ecosystem functioning, and therefore, are affecting societies that rely on ecosystem services for subsistence [The Qinghai–Tibetan Plateau grassland is the largest geomorphological unit and one of the most important terrestrial ecosystems on the Eurasian continent . Historisistence .The increase in the size of the human population has led to a greater pressure on land resources and demand for milk products and meat for human consumption, and has resulted in increases in stocking rates to levels that exceed the carrying capacity of the ecosystem . Since tGrassland degradation not only affects the resilience of ecosystems, but also jeopardizes the basic subsistence of dependent herdsmen. Overgrazing reduces herbage production  and alteOvergrazing on the Qinghai–Tibetan Plateau is extremely common because the local grazing system aims to maximize animal liveweight gains per hectare (ha), which may produce higher economic returns in the short term. It has been reported that the local regulation of grazing activities of Tibetan Plateau yak herdsmen does not meet the targets for sustainable grassland-based animal production, from the view point of either ecology or economics . VariatiThe current management pattern of herdmen of continuously grazing the same grassland area each year with high stocking rates, combined with variation in precipitation, makes grasslands more susceptible to degradation . This poThe focus of this study was to examine how different stocking rates in the Tibetan Plateau grasslands affect yak performance and vegetation, as reflected by changes in the nutritive value and yield of herbage. Specifically, the aim of the present study was to determine the effects of stocking rate and precipitation on the biomass, nutritive value, and nutritive yield of herbage, as well as on yak performance measured as liveweight gain (LG) per yak and per ha. The study also aimed to determine the extent to which the ecosystem is either damaged or improved in terms of its productivity and quality under different stocking rates and precipitation regimes. The interaction between herbage and yak stocking rate was analyzed to determine how LG responds to increasing stocking rate, and whether the expected grazing-induced increase in herbage nutritive values compensate for the expected decrease in biomass. We conducted a restricted grazing experiment to analyze the effects of yak grazing at different intensities, from ungrazed to heavily grazed, over four consecutive years (2010–2013). Based on the results of this study, we recommend an optimal and sustainable level of grazing that avoids overgrazing and prevents further grassland degradation.Polygonum viviparum and Kobresia capillifolia contribute more than 50% to the herbage biomass yield. Stipa purpurea, Leontopodium nanum and Saussurea pumila are common accompanying species, and toxic plants include Stellera chamaejasme, Oxytropis coerulea, and Gentiana farreri. Soils are classified as Calcic Chernozems [-1 of the experimental paddock in July before grazing commenced.The study area is located in Nannigou Village, Zhuaxixiulong Town, Tianzhu County , Gansu Province, on the northeast edge of the Tibetan Plateau. This grassland area has an alpine climate with mean annual precipitation of 416 mm (1968–2013) and mean temperature of 0.7°C. Approximately 80% of the annual precipitation coincides with highest temperatures in warm season (May–September). The evaporation rate is 1521 mm and snow cover is 107 d in one year. Data for long-term mean climate variables as obtained from the Wushaoling meteorological station. The growing season lasts for approximately 150 d from May to September. The grazing season is slightly shorter than the growing season because early season grazing is prohibited to protect the grassland. Due to climate conditions in the Qinghai-Tibetan plateau, herdsmen divide alpine meadows into warm (from June to September) and cold (from October to May) paddocks respectively; this experiment was conducted in a warm paddock. The perennial dominant species ernozems . The exp-1), (2) medium stocking rate , (3) low stocking rate  and (4) no grazing (C). These were repeated three times for each stocking rate to give a total of 12 plots. Each plot was 4 ha. Yak management followed traditional practice, with animals grazing through the day and returning to a barn overnight; grazing time was about 10 h day-1. The paddock was owned by a local farmer, who agreed to its use for this experimental study. There was no endangered or protected species within the paddock.The effects of yak grazing on herbage biomass, herbage nutritive value, and herbage nutritive yield, as well as on yak LG, were analyzed using an experiment with a randomized complete block design. Experimental grazing periods were consistent with the typical local grazing season and lasted from June to September. Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, Ministry of Science and Technology of China , and werA quadrat survey was conducted each year in the middle of July, August, and September. Nine quadrats (1 m × 1 m) were randomly placed in each plot, replicated three times for each stocking rate, ensuring that the distance to the edge of the plot was greater than 20 m. This gave a total of 1296 quadrats in four years. Plant species, height, and coverage were recorded within each quadrat. The plant samples from each quadrat were dried to constant weight at 85°C and then weighed. The samples were grounded in a mill through 0.25 mm sieve to minimize variation in nutritive values and yield arising from the effects of selective feeding by livestock at different sampling locations.-1) was calculated as follows: ME = TDN × 0.82 × 0.04409 × 4.182.Samples were analyzed in the laboratory to determine the concentration of nitrogen (N) and crude fat (CF) using AOAC methods . The con-1 d-1 (1) and seasonal LG per ha (LG ha) in g ha-1 d-1 (2) were calculated as follows:i is the mean liveweight of yak in each treatment (g yak-1) at the beginning (B) and end (E) of the grazing season, d is the number of grazing days, and SR is the stocking rate (yak ha-1 grazing season-1).The grazing animals were 16-month-old non-pregnant female yaks. All animals had free access to water and mineral lick stones and were given anthelmintic treatments at the beginning of June and the end of July each year, and shelters were provided. The average yak liveweight was 78, 77, 76, and 78 kg at the beginning of the grazing season in 2010, 2011, 2012, and 2013, respectively. According to yaks liveweight and intake, a standard yak with 200 kg equals to 4 sheep equivalents. The yaks in each plot had similar initial live weights, which were determined after a 15-d adaptation period. Yaks were weighed on two consecutive days, and the liveweight for each animal was calculated as the average of these two measurements. Seasonal LG per yak (LG yak) in g yakData were analyzed by ANOVA using the Mixed Model option in SPSS version 16.0  with an autoregressive covariance structure. “Stocking rate”, “block”, “year” and their interactions were considered fixed effects with “year” treated as a repeated effect and “stocking rate × block” as the subject effect. Multiple comparisons of means were made by Tukey’s test . StatistAnnual precipitation was 295, 486, 526, and 252 mm in 2010, 2011, 2012, and 2013, respectively. During the experimental period (2010–2013), average annual precipitation was 390 mm, the variation in annual precipitation was 33% . The pre-1, in seasons with recorded precipitation rates of 295, 486, 526, and 252 mm in 2010, 2011, 2012, and 2013, respectively. Regression analysis defined a positive linear relationship between precipitation and standing herbage biomass   .2 = 0.8, P<0.001) (-1) than in HS (925 kg ha-1). Herbage biomass reduction associated with grazing by yaks was 0%, 2%, 15%, and 27% in C, LS, MS, and HS, respectively. There was a negative quadratic  more than a negative linear  relationship between herbage biomass and stocking rate  . Herbageing rate .2 = 0.9, P<0.001) more than a negative linear relationship  with precipitation .The nutritive value of herbage improved with increasing stocking rate , that is-1 d-1 and 425, 405, 469, and 433 g ha-1 d-1, respectively. Therefore, maximum animal performance and productivity was in the wet year, 2012. However, stocking rate was the most important factor in determining LG per yak  and per ha . The daily LG per yak was significantly higher in LS than in MS and HS (P = 0.016) (-1). The function among stocking rate, biomass and animal production ha-1 was as LGha = 162+ 259SR+ 0.0114HB , then LGyak = 599- 178SR+ 0.0184HB  and was used to describe the relationship among SR, biomass and animal production per head.Precipitation significantly affected yak LG . In 2010S and HS . In cont= 0.016) . Therefo= 0.016) . The intOur results indicate that inter-annual variability in precipitation can explain the inter-year differences in herbage biomass. The standing biomass of herbage increased linearly with increasing annual precipitation. Because of the high variability in precipitation, herbage biomass varied markedly among the four years. Such a situation makes it difficult to predict the best stocking rate in any single year, which has led to the excessive stocking rate in this region. A yak growing from 80 kg to 120 kg would need around 360 kg DM biomass  per yak for a 90 day grazing season, then, we could image the utilization ratio of standing biomass is 33.5% in dry year (2013) and 30.2% in wet year (2012).The results of this study also demonstrate that stocking rate adversely affected herbage biomass. The herbage biomass after grazing, which was a useful indicator of whether herbage biomass production was adequate to support the applied stocking rate, indicated that the gradations in stocking rate were appropriately chosen. Herbage biomass showed a negative linear relationship with stocking rate; at the end of the four years, there was 27% lower herbage biomass in the high stocking rate (HS) treatment than in the ungrazed treatment (C). This appears to be a common herbage response in the alpine meadow ecosystem, at least in the short term, because comparatively small biomass reductions in successive years have a cumulative effect. There is an ongoing argument as to whether grazing at current stocking rates will increase or decrease the primary productivity of grasslands , 29.-1, lower than high-productive grassland (more than 10 sheep ha-1).There is no intrinsic contradiction between the results of our study and those of other studies , 31. OurHigh inter-year variability in precipitation explained most of the inter-annual variation in herbage nutritive values. Thus, not only the grassland biomass but also the nutritive values of herbage depended on the amount of precipitation. Our results suggest that herbage nutritive values increased with higher rainfall. In alpine meadows, precipitation directly determines the amounts of plant-absorbable water and available water. Because of accelerated mineralization, an increase in precipitation is associated with an increase in the amount of plant-available nitrogen. However, while higher moisture levels increase nitrogen availability, they also increase the nitrogen assimilation rate of plants. There are several explanations for the increase in nitrogen assimilation with increasing precipitation, including the increased activities of enzymes involved in nitrogen anabolism , increasThe herbage contained more fiber in the drier year, 2013, than in the other years. Plant maturation was accelerated by the drier in this year. Thus, water stress resulted in more fibrous, less digestible herbage, whereas higher precipitation rates increased herbage nutritive values, likely because of delayed maturation. Plant maturation can be delayed under moderate water stress, and so herbage nutritive values remain at higher levels . The lowOur result that herbage nutritive values increased with increasing stocking rate is consistent with the results of other studies –41. The P. viviparum or K. capillifolia, to unpalatable species (e.g. O. coerulea or S. chamaejasme), annual species (e.g. Artemisia annua), and subshrubs (e.g. Potentilla fruticosa).The results of this study indicate that grazing positively affected herbage nutritive values over the four-year short-term trial. However, long-term heavy grazing may negatively affect herbage nutritive values in alpine meadows, because there can be a shift from high- to poor-quality herbage species after long-term grazing , 47. TheOur results show that variations in precipitation among years affected LG per yak and per ha, as reported in other studies , 49. In -1 grazing season-1. Grazing at this intensity avoided the reductions in herbage biomass typically associated with grazing, and the LG per ha was 88% of that obtained in HS  can reduce herbage biomass by 27% . We founed in HS . This re-1, which provides 88% of the highest LG per ha, satisfies the demands for sustainable grazing management. However, other more developed grazing management strategies, such as rotation grazing, supplementary feeding in winter and yak turn over period from 4 years to 2 years, may also increase the biomass and nutritive value of herbage and animal LG in the winter. Implementation of such strategies may improve the sustainability of grassland use on the other parts of Qinghai-Tibetan Plateau with the similar conditions.Herbage biomass and herbage nutritive values were dependent on annual precipitation. Grazing also affected herbage performance, in that herbage biomass decreased and herbage nutritive values increased with increasing stocking rate. The increase in herbage nutritive values by grazing could not compensate for the decrease in herbage biomass. Both precipitation and stocking rate were important factors in determining livestock LG. We conclude from our results that a moderate stocking rate of 1 yak ha


Vitexin induced apoptosis in a concentration-dependent manner, and caused up-regulations of Caspase-3, Cleave Caspase-3, and a down-regulation of Bcl-2. The expression of autophagy-related protein LC3 II was significantly decreased after vitexin treatment. Moreover, western blot analysis presented that vitexin markedly up-regulated the levels of p-JNK and down-regulated the levels of p-Erk1/2 in SK-Hep1 cells and Hepa1-6 cells. Cotreatment with JNK inhibitor SP600125, we demonstrated that apoptosis induced by vitexin was suppressed, while the inhibition of autophagy by vitexin was reversed. The results of colony formation assay and mouse model confirmed the growth inhibition role of vitexin on HCC in vitro and in vivo. In conclusion, vitexin inhibits HCC growth by way of apoptosis induction and autophagy suppression, both of which are through JNK MAPK pathway. Therefore, vitexin could be regarded as a potent therapeutic agent for the treatment of HCC.Vitexin, a flavonoids compound, is known to exhibit broad anti-oxidative, anti-inflammatory, analgesic, and antitumor activity in many cancer xenograft models and cell lines. The purpose of this study was to investigate the antitumor effects and underlying mechanisms of vitexin on hepatocellular carcinoma. In this study, we found that vitexin suppressed the viability of HCC cell lines (SK-Hep1 and Hepa1-6 cells) significantly. Vitexin showed cytotoxic effects against HCC cell lines  Hepatocellular carcinoma (HCC), the fifth most common cancer worldwide, is characterized as having a poor prognosis and high mortality . Liver tVitexin, (apigenin-8-C-D-glucopyranoside), a naturally-derived flavonoids compound, has been used as a traditional Chinese medicine for the treatment of a variety of diseases , 8. The Apoptosis, generally recognized as a form of programmed cell death, involves the suicide and disposal of cells in response to a number of stimuli, including growth factor deprivation, antitumor drugs and ionizing radiation . ApoptosAutophagy is a basic catabolic process through which damaged cytoplasmic constituents and organelles are delivered to lysosomes for proteolysis to maintain energy homeostasis . Increas2-terminal kinase (JNK), a stress-responsive kinase, plays a vital role in the regulation of cell growth, differentiation, apoptosis, tumorogenesis, and other signaling pathways [Recent findings have revealed that the c-Jun NHpathways . JNK is pathways , 25. In pathways . Of partpathways , 28. Bclpathways . In our P<0.001, Figure P<0.001, Figure P<0.001, Figure 50 values in SK-Hep1 and Hepa1-6 cells were approximately 100 μM, indicating that cell viability was significantly decreased at 100 μM concentration of vitexin treatment.The effects of vitexin on cell viability in human SK-Hep1 cells and mouse Hepa1-6 cells were examined in response to increasing concentrations of vitexin for 24 h. Vitexin exerted a dose-dependent inhibitory effect on the viability of SK-Hep1 and Hepa1-6 cells Figure . CompareP<0.001, Figure P<0.001, Figure To detect whether vitexin inhibited the viability by inducing apoptosis in liver cancer cells, apoptosis was determined by flow cytometry with Annexin V-FITC/PI Staining. Both SK-Hep1 and Hepa1-6 cells were treated with 100 or 150 μM vitexin for 24 h. As shown in Figure To further evaluate the molecular mechanisms of apoptosis induced by vitexin, we explored the expression of apoptosis-related proteins in response to different concentrations of vitexin for 24 h. As shown in Figure P<0.01, Figure P<0.001, Figure To determine whether the inhibition of autophagy was regulated by vitexin in SK-Hep1 and Hepa1-6 cells, levels of the proteins in autophagy pathway were measured by western blot. As shown in Figure P<0.05, Figure P<0.01, Figure In order to confirm the protective role of autophagy against vitexin-induced apoptosis, we investigated the effects of activating autophagy with use of rapamycin, a specific autophagy activator. The combination of rapamycin and vitexin significantly increased the survival rate of SK-Hep1 cells to 75.3% versus 56% with vitexin alone  was used to block JNK activity. We found that cotreatment with SP600125 (10 μM) reversed LC3II reduction caused by vitexin (100 μM) in both cell lines Figure . In SK-Hin vitro within liver cancer cells. In Figure in vivo within the established liver cancer C57BL/6 mouse model. As compared with tumor formation obtained in the non-vitexin treated group, vitexin pretreatment resulted in a significant suppression in tumor size . Vitexin, obtained from SelleckInc (USA), was dissolved in dimethylsulfoxide  at a concentration of 300 mmol/L stock solution and stored at -20°C. MTT , DAPI (6-diamidino-2-phenylindole dihydrochloride), and the Annexin V-FITC/PI Staining Apoptosis Detection Kit were purchased from Sigma-Aldrich. Rapamycin and SP600125 (JNK inhibitor) were purchased from Selleck Inc (USA). Antibodies for Beclin1, LC3, JNK, p-JNK, Erk1/2, p-Erk1/2, Bcl-2, Caspase-3, Cleave Caspase-3, Ki-67, MMP-2, GAPDH and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology Inc (USA). All other reagents were purchased from Sigma-Aldrich or as indicated.2.SK-Hep1 cells (a human liver cancer cell line) and Hepa1-6 cells (a mouse liver cancer cell line) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences . Cells were incubated in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (100 U/mL) at 37°C under 5% CO5 cells/mL in 6-well plates and divided into four groups: (1) Control group: Cells without treatment were served as the control condition. (2) Vitexin group: Cells were treated with different concentrations of vitexin for 24 h. (3) Rapamycin group: Cells were treated with 100 μM vitexin and 40 nM rapamycin for 24 h. (4) SP600125 group: Cells were treated with vitexin (100 μM) combined with SP600125 (10 μM) for 24 h.SK-Hep1 and Hepa1-6 cells cultured in DMEM were plated at a density of 2 × 104 cells/well) and after 24 h of incubation at 37°C, then treated with 0, 10, 20, 30, 40, 50, 75, 100, 150, 200, or 300 μM vitexin for 24 h. Fresh medium and 20 μL MTT solution (5 mg/mL) was added to each well and cultured for 4 h at 37°C and 5% CO2. After incubation, the medium was removed and 200 μL DMSO was added to each well to detect the absorbance. The OD values were determined at a wavelength of 570 nm. IC50 value represented the concentration of vitexin required to inhibit cell viability by 50% relative to untreated cells and were calculated by the logit method.The effects of vitexin on SK-Hep1 and Hepa1-6 cells viability were assessed with use of a MTT kit (Sigma-Aldrich). Cells were plated into 96-well plates (5 × 10The cells were placed in a 6-well plate and incubated with vitexin (100 or 150 μM) for 24 h. Cells without any deal served as control. Cells were harvested, washed in PBS and resuspended in annexin-binding buffer. Then 5 μl Annexin V-FITC and 10 μl PI Staining Solution were added and cells were cultured at room temperature in the dark for 20 min. Apoptotic cell death was analyzed after DAPI staining by flow cytometry. For quantification and distribution, the rate of cell apoptosis was determined under fluorescent microscope using 100× magnification with an average from five different fields used to calculate this quantified value.Protein samples extracted from HCC cell lines were lysed with SDS-PAGE sample buffer. Protein samples were separated and transferred onto nitrocellulose membranes by 10% or 12% SDS-PAGE. The membranes were blocked with TBS containing 5% nonfat milk and then incubated with antibodies  at 4°C overnight. After washing, the membranes were incubated with HRP-conjugated antiIgG at room temperature for 1 h. Signal detection was performed with an ECL system .SK-Hep1 and Hepa1-6 cells were transfected with the GFP-LC3 plasmid using Lipofectamine 3000™ (Invitrogen) according to the manufacturer's guidelines. After cells had been treated with or without 100 μM vitexin, the puncta formation of GFP-LC3 was determined under fluorescent microscopy (Olympus BX5). Cells were considered to have accumulated autophagosomes when five or more puncta were counted. This criteria was established as the small number of mock-treated cells rarely displayed more than one to four puncta.The single-cell suspensions of SK-Hep1 and Hepa1-6 cells were placed in a fresh 6-well plate in triplicate for each group in medium containing 10% FBS at 37°C. After 24 h, the cell lines were replaced with new medium together with 100 μM vitexin. Drugs were washed out at 48 hours after treatment, and fresh 10% FBS medium was added. After 14 days, cells were fixed with methanol and stained with 0.1% crystal violet. Dishes were washed with distilled water, and visible colonies were manually counted.7) were re-suspended in 200 μl serum-free culture medium and were subcutaneously injected into the left liver lobes of C57BL/6 mice [Male C57BL/6 mice were purchased at 6-7 weeks of age from the experimental animal center of the PLA Military Medical Science Academy. Animal experiments were performed according to the guidelines of the National Institute of Health (NIH). A mouse xenograft model of HCC was established as described previously [L/6 mice . The micSamples from the liver tumor tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated and embedded in paraffin. Five micrometer-thick sections were cut from each paraffin embedded tissue and stained with hematoxylin and eosin (H&E) for histopathologic examination under a light microscope.2O2 for 15 min, sections were blocked with 5% goat serum, and incubated with antibodies (Ki-67 and MMP-2) at 4°C overnight. Sections were incubated at room temperature for 60 min with the HRP-conjugated secondary antibody, then DAB solution was used. Counterstaining was performed with hematoxylin.Tumor tissues samples were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, and sectioned at a 5-μm thickness. Tissue sections were deparaffinized and rehydrated with xylene and alcohol of gradient concentrations, then cultured in 5 mmol/L citrate buffer (pH 6.0) for 15 minutes to retrieve internal antigens by microwave. After blocking endogenous peroxidase activity by incubating in 3% HAfter retrieving internal antigens and blocking endogenous peroxidase activity as described above, tumor sections were treated with 5% goat serum and incubated with LC3 antibody at 4°C overnight. Sections were incubated with 20μl fluorophore-conjugated secondary antibody for 1 h at room temperature before imaging. After washing with PBS, sections were counterstained with DAPI (10 ng/mL) for 10 min. A fluorescence microscope was used for LC3 co-localization analysis.P<0.05. All statistical analysis were performed with SPSS 19.0 and GraphPad Prism Version 5.0 softwares.The results were expressed as the mean ± standard deviation (SD) of independent experiments. The data were analyzed using one-way analysis of variance (ANOVA) to determine statistically significant differences among groups. Differences were considered statistically significant for values of


The status of “trying to become pregnant” is complex and may last for years. Many female global health profession-als of reproductive age, if they heed the advice of the CDC, would avoid work-related travel to Central and South America. This could mean that for the current cohort of global health projects, women's input would be significantly reduced. Lack of gender balance is known to hamper effective implementation of global health initiatives.Women may be hesitant to express their concern about Zika. Citing Zika travel policies as a reason to avoid work-related international travel may signal to employers a woman's intention to have children. Many women worry about revealing their plans to have children to their employers. Women are aware that revealing an intention to become pregnant might be detrimental to their career advancement. For example, employers may prefer to hire and promote those they suspect less likely to have children, to avoid having to cover maternity leaves. While the practice is technically illegal in the United States, women are often discriminated against in hiring, such as by being asked in job interviews if they plan to have children.Women are aware that revealing an intention to become pregnant might be detrimental to their career advancement.Monitor and report on whether women's participation in global health programs and policies is decreasing. Compare measures of participation before and after Zika travel policies were in place.Mitigate the impact. Seek more flexible ways to involve women in global health projects. For example, I acted as gender consultant for a maternal health initiative in Guatemala by combining a shorter in-country trip with later teleconferencing for interviews with maternal health project staff based in Northern Guatemala.Consider increasing the participation of local as opposed to expatriate women. The reality is that most global health projects are funded and managed by international institutions headquartered in the North that employ many expatriates. If instead local professional women were brought to the fore, this could be a silver lining. Involving more local women would improve adaptation of the project to the local context and mitigate the overall loss of women's perspectives resulting from the Zika travel policies.Involving more local professional women in global health projects could be a silver lining.Know you are not alone. For women of reproductive age who are involved in global health, navigating adherence to Zika travel policies is complex. I hope this letter will stir reflection and push these issues from private dilemmas to public debate.Suggest to global health employers alternative ways for women to be involved. If your intention to become pregnant means you cannot risk travel to Zika-endemic areas, suggest to your employer alternatives such as teleconferencing or partnering with a local counterpart who is a woman.Zika travel policies may have the unintended consequence of reducing women's participation in global health programs. Addressing gender equity in global health projects is complex. Recognizing and dealing with gender imbalances in global health leadership is perhaps more subtle, but no less important.


Influenza A viruses are important human and animal pathogens. Seasonal influenza viruses cause infections every year, and occasionally zoonotic viruses emerge to cause pandemics with significantly higher morbidity and mortality rates. Three cases of laboratory confirmed human infection with avian influenza A (H7N9) virus were reported in 2013, and there have been several cases reported across South East Asia, and recently in North America. Most patients experience severe respiratory illness, with mortality rates approaching 40%. No vaccine is currently available and the use of antivirals is complicated due to the emergence of drug resistant strains. Thus, there is a need to identify new drugs for therapeutic intervention and disease control. In humans, following H7N9 infection, there is excessive expression of pro-inflammatory factors CCL2, IL-6, IL-8, IFNα, interferon-γ, IP-10, MIG and macrophage inflammatory protein-1β, which has been shown to contribute to fatal disease outcomes in mouse models of infection. In the current study, the potent inhibitor of CCL2 synthesis, Bindarit, was examined as a countermeasure for H7N9-induced inflammation in a mouse model. Bindarit treatment of mice did not have any substantial therapeutic efficacy in H7N9 infection. Consequently, the results suggest that Bindarit may be ill-advised in the treatment of influenza H7N9 infection. Infected children typically suffer only mild disease,2 whereas elderly patients are generally more severely afflicted.3 This suggests that there is minimal heterologous immunity from previous influenza infections. Most patients infected with H7N9 experienced severe respiratory illness, such as pneumonia (97.3%) and acute respiratory distress syndrome (71.2%), leading to high rates of intensive care unit admissions.4 The mortality rate attributed to influenza H7N9 infection in human is >38%, with 175 deaths from 450 confirmed cases reported within a 20-month period.5 No evidence of sustained human-to-human transmission of H7N9 has been recorded. However, there is some evidence of limited person-to-person spread under rare circumstances.6 After human H7N9 infections were first reported in China, the virus spread rapidly to other countries.7 Recently, the first case of human H7N9 infected in North America was documented in Canada.8Despite vaccines and therapeutics for circulating strains, influenza A virus (IAV) remains a serious global health threat affecting humans, wildlife and agricultural species. Avian IAV H7N9 virus (H7N9) infections in humans were first reported in China in March 2013.9 There are several antivirals available for the treatment of IAV infections including the M2 ion channel inhibitors amantadine and rimantadine, and the neuraminidase inhibitors zanamivir and oseltamivir.11 Early treatment with these antiviral drugs reduces the duration of symptoms and recovery time. However, the use of antiviral drugs is complicated by the emergence of drug resistant viruses,13 and oseltamivir-resistant H7N9 strains have recently been described in Taiwan.14 In addition, the use of antiviral drugs may affect population vulnerability due to lack of seroconversion, as well as driving drug resistance among circulating strains.15 The development of new drugs and vaccines against H7N9 may take many years. Drug repurposing, or the use of clinical drug candidates, may help to overcome this lengthy process of drug development and reduce the impact of H7N9-induced disease. H7N9 disease results in a potent immune response believed to contribute to tissue destruction and pathology.16 The host's immune response towards H7N9 infection has not been fully characterized, which is an issue for the discovery of novel therapeutics and target identification for drug repurposing.No specific vaccine is currently available for H7N9.16 Proliferation of H7N9 in the lower respiratory tract causes excessive activation of the innate immune response.16 This leads to production of inflammatory mediators, many expressed by intrapulmonary macrophages and alveolar cells.16 Some of the major histological characteristics of H7N9 infection include diffuse alveolar damage, hyaline membrane and fibroproliferation in the lung, and spotty necrosis in the liver.16 Histological observations have shown a depletion of T cells, a fluctuating numbers of neutrophils, and highly abundant and activated macrophages which are characteristic of H7N9 infection in the alveoli.16 In addition, high levels of intrapulmonary inflammatory mediators, such as the interferon gamma-induced protein 10 (IP-10) and interleukin-6 (IL-6) have been detected.16 Plasma levels of IL-6 and IL-8 are sharply upregulated, whereas IL-10, macrophage inflammatory protein-1β, and interferon-γ are increased to intermediate levels.4 In contrast, IL-1β, tumor necrosis factor α (TNFα), and MIP-1α are found only at minimal concentrations. Whether the elevated cytokine and chemokine levels cause or contribute to the severity of H7N9 disease has yet to be determined.4 However, the upregulation of pro-inflammatory cytokines and chemokines, such as the monocyte chemoattractant protein-1 (MCP-1/CCL2), IL-6, IL-8, IFN-α, IP-10, MIG and macrophage inflammatory protein-1β was described in H7N9-infected patients with lung injury and severe pneumonia.2 The cytokine levels in C57BL/6 and BALB/c mice infected with H7N9 (A/Anhui/A/2013 strain) were compared. C57BL/6 mice exhibited more severe lung injury, slower recovery from lung damage, less effective viral clearance, higher levels of CCL2, IL-6 and IL1β, and lower levels of TNFα and interferon-γ than BALB/c mice. These data suggest that TNFα and IFNγ may help to suppress viral gene expression and increase viral clearance, while CCL2 and IL-6 may contribute to lung injury during H7N9 disease.17In studies of fatal H7N9 infections in humans, there is evidence of immune pathological changes caused by a heightened innate immune response.18 Bindarit has been successfully used to alleviate virus-induced inflammation in several animal models of disease. For example, Bindarit was efficacious in mouse models of Chikungunya and Ross River virus infections, where it was shown to ameliorate infections and disease.20 Furthermore, Bindarit was able to reduce arthritic inflammation without showing any detrimental effect on virus clearance in these animal models of alphavirus infection.21 Therefore, the activity of Bindarit activity was determined to reduce pulmonary and serum CCL2 levels in a mouse model of H7N9 disease. Bindarit selectively inhibits CCL2. CCL2 is a critical mediator of neuroinflammation in myriad disease states, including multiple sclerosis,22 human immunodeficiency virus (HIV)-1-induced encephalitis,23 Guillain-Barré syndrome,24 Alzheimer's disease,25 ischemia,26 neurotrauma,27 epilepsy,28 neurogenic hypertension29 and alcoholism.30 Bindarit has also been studied for therapeutic intervention for these diseases. The clinical tolerability data for Bindarit in different CCL2-dependent illnesses demonstrated Bindarit safety up to a maximum dose of 2400 mg per day for as long as 6 months, and suggests the potential of Bindarit to be beneficial for a range of diseases.31The focus of this study was to assess the effects of drug inhibition of the pro-inflammatory factor CCL2, which may have a pathogenic role during H7N9 disease. Bindarit represents a novel class of inhibitor that reduces CCL2 synthesis.This study aimed to explore the role of CCL2 in H7N9 disease and the potential of Bindarit to act as a countermeasure against H7N9-induced pathology in a mouse model. Intriguingly, the survival rate of Bindarit-treated mice was comparable to that of non-treated mice, while weight loss, cellular infiltration and viral titers were considerably increased with Bindarit treatment. Thus, the use of Bindarit as a therapeutic to treat H7N9 disease seems ill-advised.CCL2 gene expression during infection with A/Ca (H1N1), a representative, currently circulating IAV subtype, in a human epithelial cell (A549) line. CCL2 was considerably upregulated by 3.21-fold (P<0.01) in A/Ca (H1N1)-infected A549 cells compared to mock-infected controls at 24 h pi (m), CCL2 gene expression was significantly (P<0.01) reduced to a level comparable to mock-infected controls.To evaluate the effectiveness of Bindarit in reducing CCL2 production, lung epithelial cells were infected (MOI=0.1) with A/California/04/09  virus and simultaneously treated with Bindarit. Quantitative reverse transcription PCR (RT-qPCR) was used to investigate the effect of Bindarit on  24 h pi . When A55 PFU) and treated with Bindarit (70 mg kg−1) twice daily starting at day 1 post-infection (pi). Mice were monitored for weight loss and survival. Bindarit treatment had no detectable impact on weight loss or survival of mice. Mice in both groups lost body weight to a similar extent, reaching a 25% reduction in body weight by day 5 (P<0.1) difference was noted in the survival rate between the two groups.The ability of Bindarit to protect mice from lethal H7N9 infection was investigated. Mice were intranasally (i.n.) infected with a lethal dose of H7N9  and orally treated with Bindarit (70 mg kg−1) twice daily starting at day 1 pi. Mice were killed at day 4 pi and lungs were collected for histopathology. Lungs in both groups, mock- and Bindarit-treated, showed moderate to severe necrotic bronchitis and bronchiolitis. The peribronchiolar and perivascular infiltration was mild to moderate for both groups and animals showed mild to severe alveolitis (−1). All mice showed a lung score of 3  increase in total number of leukocytes was observed in mice orally treated with Bindarit (70 mg kg−1) after lethal infection with H7N9 at day 4 pi  fluid from infected mice. A significant . The lethal challenge may have been overwhelming and therefore the effect of Bindarit insufficient. Therefore, a study using a sub-lethal dose of virus was performed. Mice were infected 102.7 PFU with a sub-lethal dose of H7N9 and orally treated with Bindarit (70 mg kg−1) twice daily starting at day 1 pi. Mice in both groups lost body weight until day 4 pi. However, on days 5, 6 and 8, Bindarit-treated mice showed a considerable increase in weight loss compared to mock-treated control mice  had higher lung virus titers compared to mock-treated control mice at day 8 pi  increased when H7N9-infected mice were treated with Bindarit. Bindarit treatment did not appreciably change the level of other pro-inflammatory cytokines. Bindarit oral treatment also had minimal effect on cytokine expression levels in the lungs. Interestingly, CCL2 protein level was not reduced, but appeared enhanced after treatment with Bindarit.To corroborate the RT-qPCR results, a Luminex enzyme-linked immunosorbent assay platform was used to measure the pro-inflammatory cytokines IL-6, IL-15, CCL2, RANTES and TNF in the BAL at day 8 pi . The IL-P<0.01) increased after Bindarit treatment, but were slightly higher than those in mock-treated mice  knockout mice showed increased virus titers and disease.35 In a different study on CHIKV a similar importance for the CCR2 was found. CCR2 deficiency promoted exacerbated chronic erosive neutrophil dominated CHIKV-induced arthritis in mice.40CCL2 is upregulated in several viral diseases in humans, such as HIV, hepatitis C virus, several herpes viruses, Japanes encephalitis virus and respiratory syncytial virus, and has been considered as a biomarker linked to disease severity in HIV.2 However, in this study, when H7N9-infected mice were treated with Bindarit, mice exhibited heightened disease signs as demonstrated by an increase in weight loss, pro-inflammatory factors, cellular infiltration and virus titers. Thus, blocking CCL2 dampened viral clearance and was associated with upregulation of pro-inflammatory cytokines and cellular infiltration. In earlier studies examining the effects of anti-CCL2 antibodies on IAV disease, mice exhibited enhanced pneumonitis compared to non-treated animals, despite reduced numbers of cellular infiltrates such as leukocytes, macrophages and neutrophils in the lungs.41 Furthermore, infection of CCL2 knockout mice with a non-lethal dose of a mouse-adapted strain of IAV resulted in a profound increase in weight loss, elevated viral loads and pro-inflammatory cytokines, and enhanced leukocyte recruitment into the infected lungs compared to wild-type mice.42 Interestingly, in that study, pro-inflammatory cytokines such as TNFα, IL-6 and IFNγ were enhanced, but cellular infiltrates into the lungs were reduced.42 However, one limitation of that study was the analysis of the cellular infiltrate in full lung homogenate as opposed to BAL, which may have influenced the outcome of the study. In the current study, we observed an increase in pro-inflammatory cytokine expression and cellular infiltration in the BAL.CCL2 is highly upregulated in patients suffering from H7N9 influenza infection, and has been linked to lung injury in mouse models.Ccl2 gene expression was not reduced in the lungs after oral treatment with Bindarit, despite its known capability in reducing CCL2 synthesis in vitro and in vivo from earlier studies by other groups.18 Oral administration of Bindarit may be a limitation in the treatment of pneumonia, as there are difficulties in reaching therapeutic concentrations of drugs in the lungs when administered by this route.43 It remains a possibility that oral delivery of Bindarit was not completely effective or led to a systemic reduction of CCL2 production that contributed to disease enhancement. In future studies this limitation will be addressed by i.n. administration of Bindarit, which may increase the concentration of the drug in the lungs. In addition, IL-15 was highly upregulated in the BAL of Bindarit-treated H7N9-infected mice. IL-15 has recently been described as a critical factor in the pathogenesis of IAV in mice with virus-induced acute lung injury.44 Whether there is a link between increased IL-15 production in the lung and a systemic inhibition of CCL2 synthesis remains a subject of further studies.Interestingly, 45 Due to increased drug resistance among circulating and novel IAV strains and the lack of specific vaccines against H7N9, there remains an imminent need for drug repurposing because the development of novel antivirals and specific vaccines will require many years of preclinical and clinical studies before their availability for clinical use.Various approaches have been investigated for the treatment of H7N9 infection in the recent years. Treatment with corticosteroids was evaluated, but it led to increased mortality in patients suffering from acute H7N9 infection.in vivo experiments were performed under the guidance of the Institutional Animal Care and Use Committee (IACUC) and Animal Resources at the Animal Health Research Center (AHRC), which has approved biosafety level 3 laboratories. Avian IAV A/Anhui/1/2013  was propagated in embryonated chicken eggs. Influenza A/California/04/09  was propagated in Madin-Darby canine kidney cells (ATCC CCL-34). Viruses were titrated on Madin-Darby canine kidney cells as described previously.46 The human type II respiratory epithelial cell line A549 (ATCC CCL-185) was maintained in Dulbecco's modified eagle's medium  supplemented with 5% heat-inactivated fetal bovine serum (HyClone) in a 37 °C incubator with 5% CO2. Cell lines were regularly tested for mycoplasma contamination. Virus propagation in embryonated eggs was carried out in strict accordance with the recommendations by the University of Georgia IACUC. The protocol was approved by the University of Georgia IACUC.All m. After 24 h, cells were collected in TRIzol (Thermo Fisher) for total RNA purification. Gene expression was assessed using RT-qPCR. All infections were performed in the presence of 1 μg ml−1 (l-tosylamido-2-phenyl) ethyl chloro-methyl ketone (TPCK)-treated trypsin  in modified Eagle's medium (MEM) supplemented with 0.3% bovine serum albumin (Thermo Fisher Scientific).Bindarit (2-Methyl-2-[[1-(phenylmethyl)-1H-indazol-3-yl]methoxy]propanoic acid) was synthesized by Chemlin . Bindarit was dissolved in ultrapure water . A549 cells were grown to 80% confluency in a 48-well plate and infected with A/Ca (H1N1) at multiplicity of infection (MOI) of 0.1. After infection, Bindarit was added to the wells at a concentration of 100 μ−1 (maximum solubility). To evaluate the effect of Bindarit on disease burden, Bindarit was administered using 0.2 ml  oral gavage twice daily starting at day 1 pi. Mice were inoculated i.n. with influenza virus strain A/Anhui/1/13 (H7N9) with either a lethal dose (105 PFU) or a sub-lethal dose (102.7 PFU) of H7N9. BAL fluid was collected in phosphate-buffered saline (PBS) to determine cell number with flow cytometry and cytokine levels using multiplex enzyme-linked immunosorbent assay. Lungs were collected in 10% buffered formalin for histopathological analyses or homogenized in serum-free Dulbecco's modified eagle's medium and processed with TRIzol for RNA extraction and subsequent RT-qPCR.BALB/c female mice (6-to-8 weeks old) were obtained from the National Cancer Institute. All experiments and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia. All experiments were performed with 5–7 mice per group and repeated independently at least twice. Bindarit was dissolved in 0.5% methylcellulose (Thermo Fisher Scientific) at 7 mg ml47 In brief, the concentration of total RNA was measured using a microplate spectrophotometer . RT-qPCR was used to validate mRNA expression changes and virus load using the Stratagene Mx3005P real-time PCR system . The reverse transcription reactions were performed using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) and 1000 ng total RNA for each reaction. qPCR was performed using the GoTaq Green Master Mix  to determine mRNA levels, and data were normalized to 18S expression using the 2−ΔΔCt method.48 Primer sequences for CCL2 were: 5′-GAACACACTCAGCGCAGTTA-3′ (forward primer) and 5′-CACCCACCCTCTCTTTGATTAC-3′ (reverse primer). The virus load was determined using 5 ng of total RNA with a TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific). The standard curve was produced using an M gene plasmid.49Total RNA was isolated using TRIzol as previously described.50 The slides were evaluated by a pathologist without prior knowledge of the infection and treatment status.Lungs from infected mice were harvested at 4 days (pi), perfused with 10% buffered formalin, and fixed in 10% buffered formalin. The sections were embedded in paraffin, cut into 5 μm sections, and stained with hematoxylin and eosin. The sections were evaluated using light microscopy. A histological score for each lung was determined according to the following criteria: 0=no lung abnormality; 1=<10% of airways inflamed; 2=10–30% of airways inflamed; 3=30–50% of airways inflamed and 4=>50% of airways inflamed.g for 5 min at 4 °C. The recovered supernatants were collected and stored at −80 °C until assessed for cytokine concentration, and the cell pellet were resuspended in 200 μl of 10% buffered formalin. Total cell numbers were counted using a hemocytometer. Cytokines in BAL supernatants were quantified with the Luminex xMAP system using a MILLIPLEX MAP mouse cytokine immunoassay  according to the manufacturer protocol. In brief, beads coupled with anti-CCL2, anti-IL-6, anti-interferon-γ, anti-RANTES, anti-IL-15 and anti-TNF monoclonal antibodies were sonicated, mixed and diluted 1:50 in assay buffer. For the assay, 25 μl of beads were mixed with 25 μl of PBS, 25 μl of assay buffer, and 25 μl of BAL supernatant, and incubated overnight at 4 °C. After washing, beads were incubated with biotinylated detection antibodies for 1 h and the reaction mixture was then incubated with streptavidin-phycoerythrin (PE) conjugate for 30 min at room temperature, washed and resuspended in PBS. The assay was analyzed on a Luminex 200 instrument  using the Luminex xPONENT 3.1 software.Eight days pi, mice were killed and tracheotomy was performed. The mouse lungs were flushed with 1 ml of PBS, and the retained BAL was centrifuged at 400 + , CD45+SiglecF+CD11clow (eosinophils), CD45+SiglecF+CD11chigh , CD45+CD3+ , CD4 T cells (CD45+CD3+CD4+) and CD8 T cells (CD45+CD3+CD8+).For flow cytometry analysis, cell suspensions were incubated in FACS staining buffer (PBS containing 1% BSA) and subsequently stained for 30 min at 4 °C with an optimized concentration of antibodies : PE-conjugated anti-CD3, PerCPCy5.5-conjugated anti-CD8, PE Cy7-conjugated anti-CD4, PerCPCy5.5-conjugated anti-CD45, APC-conjugated anti-CD11c and PE-conjugated anti-SiglecF to determine cell types in the BAL. Cells were acquired on an LSRII flow cytometer (BD Bioscience) and the data were analyzed using the FlowJo software . Based on surface marker expression, six different cell types were identified: CD45t-test (two tailed). P-values of <0.05 were considered significant. All analysis was performed using Graphpad Prism Software .All experiments were performed in minimum in triplicate to ensure adequate power and the experiment independently repeated at least twice. Using power analysis, 12 animals in each group are required to give a 90% probability of detecting a treatment difference at a 5% significance level if the true difference between the treatments is one s.d. of the variation with each treatment group. However, decreasing the number of mice to five per group, gives a 90% probability of detecting a treatment difference if the true difference between the treatments is 1.63 s.d.'s of the variation with each treatment group. Further decreasing the treatment group size to three mice per group, gives a 90% probability of detecting a treatment difference only if the true difference between the treatments is 2.36 s.d.'s of the variation with each treatment group. Data are expressed as mean±s.e.m. Differences between groups were determined by one-way analysis of variance. Individual differences between groups were tested by multiple comparison and analysis using the Tukey post-test. Pairs of groups were compared by Student's


CYC-B) could catalyze cyclization of lycopene to β-carotene. In this study, the differential accumulation patterns of loquat with different colors were analyzed and virus-induced gene silencing (VIGS) was utilized in order to verify CYC-B gene function. Using a cloning strategy of homologous genes, a CYC-B gene orthologue was successfully identified from the loquat. At a later stage of maturation, CYC-B gene expression and carotenoids concentrations in the ‘Dawuxing’ variety were higher than in ‘Chuannong 1-5-9′, possibly leading to the difference in pulp coloration of loquat. Interference of CYC-B gene expression in the loquat demonstrated clear visual changes. The green color in negative control fruits became yellow, while TRV2-CYC-B silenced fruits remained green. CYC-B gene expression and total carotenoid content in the pulp decreased by 32.5% and 44.1%, respectively. Furthermore, multiple key genes in the carotenoid metabolic pathway synergistically responded to downregulation of CYC-B gene expression. In summary, we provide direct evidences that CYC-B gene is involved in carotenoid accumulation and coloration in the loquat.Carotenoids are the principal pigments in the loquat. Although the metabolic pathway of plant carotenoids has been extensively investigated, few studies have been explored the regulatory mechanisms of loquat carotenoids because knowledge of the loquat genome is incomplete. The chromoplast-specific lycopene β-cyclase gene ( Eriobotrya japonica Lindl) is a member of the Rosaceae family and is native to the southeast of China [Loquat  is an effective tool for analyzing plant gene function over a short period of time , as succLoquat fruits with different colors were sampled from the loquat germplasm resource preservation garden during 2016–2017 . ‘Dawuxing’  and ‘Chuannong 1-5-9′  fruits of different developmental stages were harvested for experiments, including immature green (IG), breaker (B), degreening (D), yellow mature (YM), and fully mature (FM) . ‘ZaozhocCYC-B_F: 5′-CCACTTYCCTTTGACCTTCA-3′ and cCYC-B_R: 5′-GTGTTRCATTATTATGCAGCT-3′) were designed by this laboratory. A high-fidelity enzyme was used to amplify the CYC-B gene from ‘Dawuxing’, with homology alignment verified after sequencing. A phylogenetic analysis of CYC-B proteins from different plants was constructed using MEGA software  with neighbor-joining method (testing of 1000 bootstrap replications).Loquat fruits were selected to extract total RNA, and first-strand cDNA was synthesized using a PrimeScript™ RT reagent Kit with gDNA Eraser , used as the template. A homology-based cloning method was selected because genome information of the loquat is incomplete. Degenerate primers containing the complete coding region (TRV2-CYC-B plasmids were shown in EcoR I and BamH I restriction enzyme cutting sites (underlined) were designed in the conserved region of CYC-B gene sequence (pCYC-B_F: 5′-CGGGATCCATGGAGTTTGGGTTGATGAA-3′ and pCYC-B_R: 5′-CGGAATTCGCCTACTAACAAGCGAAGTT-3′). The fragment of CYC-B gene from ‘Zaozhong 6′ was inserted in an antisense orientation before the nopaline synthase (NOS) terminator and after the cauliflower mosaic virus (CaMV) 35S promoter. CYC-B fragment was purified using a SanPrep Column DNA gel extraction kit . The purified product and TRV2 plasmid were both digested by EcoR I and BamH I restriction enzymes. After ligating with T4 ligase overnight, it was transferred into DH5α Escherichia coli (E. coli) competent cells. E. coli was spread using selective media containing kanamycin . After expansion, the quality of E. coli was ascertained by running PCR and agarose gel electrophoresis. A pair of detection primer for TRV2-CYC-B (S1_F: 5′-CGGACGAGTGGACTTAGATTCTG-3′ and S1_R: 5′-GCCTATGGCTTCTGTTCATGTG-3′) were synthesized. TRV2-CYC-B plasmid DNA was extracted using a SanPrep Column Plasmid Mini-Preps kit , as identified by EcoR I and BamH I digestion then verified by sequencing.Construct maps of the TRV1, TRV2, and TRV2-CYC-B and TRV1 and TRV2 plasmids were transfected into Agrobacterium tumefaciens GV3101 competent cells by freeze-thaw. The method of Chen et al. [Agrobacterium cells were spread in selective Luria Bertani (LB) solid medium containing Gentamicin , Kan (50 mg/L), and Rifampicin , then cultivated at 28 °C for 48 h–72 h. The growth of the colonies was observed. Recombinant, TRV1, and TRV2 colonies were inoculated with 50 mL of LB liquid medium containing 40 mg/L Gen, 50 mg/L Kan, 20 mg/L Rif, 10 mmol/L 2-morpholinoethanesulfonic acid (MES), and 20 μmol/L acetosyringone (AS), shaken at 28 °C until the OD 600 had risen to 1.2. The presence of the inserts was verified using 1 μL of bacterial solution as a template for PCR. TRV1 detection primers were created as described by Chen et al. [TRV2-CYC-B detection primers were the same as S1 primers. TRV2 detection primers were as follows: TRV2_F: 5′-GCCATTAGCGACATCTAAAT-3′ and TRV2_R: 5′-CTAAGTCCACTCGTCCGTAA-3′. The verified cells were collected by centrifugation at 4000 g for 15 min and resuspended in infection buffer  twice until the final OD-600 was 1.2. Finally, resuspended cells were gently shaken at 28 °C for 4 h.n et al.  and Zhain et al. . TRV2-CYTRV2-CYC-B and TRV2 were mixed with TRV1 bacterial solution at a ratio of 1:1, respectively, using the empty TRV2 plasmid as a negative control. In addition, groups in which sterile water was injected, or not, were used. Twelve fruits of ‘Zaozhong 6′ in each group were selected, using a single fruit as a repeat. A loquat fruit was injected with 300 μL of mixed bacterial solution using sterile syringes. After a week, the pulp of the injection area (no necrosis) was cut into small pieces and immediately frozen in liquid nitrogen, then stored at −80 °C. The primers for viral molecular detection (the TRV1 and TRV2 primers described above) were used to amplify samples, in order to verify whether the TRV virus had successfully invaded the loquat fruits.Disease-free fruits of uniform size and maturity at the breaker stage were selected as injection materials. In this study, recombinant TRV2-CYCB silenced) were selected to determine total carotenoid content, respectively. Total carotenoid content in loquat samples was conducted using acetone extraction. After extraction using 80% acetone for 24 h, the OD at 470 nm of the supernatant was measured, with 80% acetone used as the control. Every fruit sample was performed in three replicates.Twelve fruits of two groups (negative control or TRV2-CYCB silenced). Appropriate gene-specific primers of carotenoid biosynthesis were designed by Fu et al. [®Premix Ex Taq™ II  kit. The 2ct−ΔΔ values were computed to quantify relative gene expression levels [Real-time quantitative PCR (qRT-PCR) was performed to quantify gene expression levels in fruits of different developmental stages and the VIGS samples  was used to perform Duncan’s pairwise comparison with a significance level of 5%  and Pearson bivariate correlation analysis.In this study, IBM SPSS statistical software  gene. Full-length cDNA of the CYC-B gene was successfully isolated from the loquat, nominated as EjCYC-B.In this study, the 1534 bp-length sequence of analysis . The loqCYC-B gene demonstrated a rising trend before the YM stage, which was downregulated at the FM stage except for the pulp of ‘Dawuxing’ (CYC-B gene were significantly different between loquats with different colors both in the pulp and peel after the B stage (p < 0.05). In the pulp, CYC-B gene expression of ‘Dawuxing’ and ‘Chuannong 1-5-9′ reached their maximum at the FM and YM stages, respectively. In the peel, CYC-B gene expression both reached its maximum at the YM stage. At the FM stage, the CYC-B gene expression level of ‘Dawuxing’ was approximately 3-fold higher than that of ‘Chuannong 1-5-9′ in the pulp, and approximately twice that of ‘Chuannong 1-5-9′ in the peel.The relative expression levels of the awuxing’ . ExpressCYC-B gene expression levels in the pulp of ‘Dawuxing’ at different developmental stages (p < 0.05). This result indicated that the change in CYC-B gene expression level may affect differential accumulation of carotenoids and coloration in the loquat.The total carotenoid content of loquats with different colors at different developmental stages was detected in our previous study (doi: 10.13417/j.gab.037.004407). The content of total carotenoids of ‘Dawuxing’ and ‘Chuannong 1-5-9′ varied greatly at different maturity stages. Pearson correlation analysis found that there was a significant and positive correlation between carotenoid content and CCS gene sequence of the pear (Pyrus bretschneideri Rehd.) (GenBank accession number: XM_009379544.2) demonstrated that sequence similarity reached 98.34%, indicating that the CYC-B gene fragment from loquat was successfully cloned and linked next to the TRV2 plasmid.After PCR amplification of the conserved region, a 543-length sequence was obtained. Comparison of homology with the CYC-B gene obtained. The target gene region of the recombinant plasmid was sequenced, the result identical to that of the previous CYC-B gene cloned fragment. Therefore, one can conclude that the TRV2-CYC-B silencing vector had been successfully constructed.After double digestion of the recombinant plasmid, two distinct target bands appeared , and a tAgrobacterium, obvious phenotypic changes of the loquat fruits were observed. It was found that groups that were not injected or had been injected with sterile water and the negative control changed as normal from green to yellow, while the TRV2-CYC-B silenced fruits remained green . The results demonstrated that TRV-mediated CYC-B gene silencing resulted in a significant decrease in carotenoid accumulation in loquat pulp and interfered loquat coloration. The results also indicated that the VIGS system had been successfully constructed in the loquat.ng 35.2% C. Total ng 35.2% D. PearsoTRV2-CYC-B silenced and negative control fruits  mRNA levels also decreased significantly. To summarize, the expression of multiple structural genes involved in carotenoid accumulation changed after silencing the CYC-B gene.mRNA levels of 12 genes connected to carotenoid metabolism were analyzed in the pulp of l fruits . In linechromoplast-specific lycopene β-cyclase (CYC-B) gene by Ronen et al. [CYC-B gene become more widespread, finding that over-expression of CYC-B promoted formation of β-carotene in the tomato. Therefore, it is clear that the CYC-B gene plays an important role in the regulation of the accumulation of carotenoids. In this study, a CYC-B gene orthologue was successfully identified from the loquat using a homology-based cloning method, a simple, fast, and efficient method that uses highly conserved sequences to design degenerate primers to clone target genes. This has been used for carrots [Phalaenopsis Hybrida [Carotenoids are the principal pigments in loquat, regulated by multiple factors. Since the discovery of the n et al. , studies carrots , Phalaen Hybrida , and tob Hybrida , etc.CYC-B gene exhibited significant upregulation at the majority of developmental stages of yellow and white-fleshed loquats, as previously reported by Hadjipieri et al. [CYC-B gene was downregulated except in the pulp of ‘Dawuxing’. However, total carotenoid content increased at the FM stage in our previous study. We speculate that in addition to the CYC-B gene, other genes cooperate to regulate total carotenoid biosynthesis at the FM stage. Previous research has found that PSY, CYCB, and BCH genes have a synergistic effect on carotenoid accumulation [In this study, the transcription levels of the i et al. . At the mulation . In our mulation  have vermulation  also fouAgrobacterium, the in vitro loquat fruits changed color from the breaker stage, fruit phenotype demonstrating obvious changes. It was found that there was shrinkage in the infected region of the silenced fruits, which we speculate is caused by the pressure difference of the infected region caused by enlarged interstitial spaces after excessive bacterial liquid injection. Following CYC-B gene silencing, the infected part of the silenced fruits retained a green phenotype, suggesting a delay in fruit maturity and interfering loquat coloration. In this study, decreased expression levels of the CYC-B gene were found in the pulp, and as a result of VIGS, led to additional reduction in carotenoid content, indicating that the CYC-B gene could positively regulate carotenoid accumulation in the loquat. So, we further confirmed that the CYC-B gene was closely related to loquat coloration, as found in other studies [CYC-B gene silencing efficiency was 35.2%, while total carotenoid content reduced by 44.1%, indicating that the CYC-B gene played a key role in the regulation of carotenoid synthesis. Furthermore, this laboratory constructed the TRV-PSY silencing vector and found the silencing efficiency was 55.6%, total carotenoid content reducing by 46.8%. Additional experiments were conducted to explore why a lower silencing efficiency of CYC-B gene also caused a large drop in carotenoid concentration. After interfering with CYC-B expression, apart from the PDS, ZDS, and BCH genes, eight genes in the carotenoid metabolism pathway synergistically responded to downregulation of CYC-B gene expression, and the expression levels of seven genes  were downregulated, except for the ECH gene. We speculate that suppression of multiple key structural genes may lead to a large reduction in carotenoids. Although ECH gene expression levels were upregulated, metabolized substrate synthesis of ECH gene was downregulated, which would reduce the synthesis and accumulation of carotenoids from the source. Moreover, downregulation of PAP gene expression might affect the development of plasmid structure and disturb the accumulation of carotenoids. Leitner-Dagan et al. [CHRC (a cucumber PAP) expression by RNAi resulted in a 30% decrease in carotenoid content in tomato flowers.After injection with  studies ,24. In an et al.  also fouCYC-B gene after silencing was not complete. These factors influencing this are likely to be a selection of silencing vector [Nicotiana benthamiana, with a modified tobacco mosaic virus (TMV) vector used to silence the PDS gene of N. benthamiana [In this study, we found that the downregulation of the g vector , temperag vector ,42, lengg vector . VIGS wathamiana . A variethamiana ,42. Studthamiana . VIGS isOR transgene regulates the accumulation of carotenoids by controlling differentiation and formation of chromoplasts [OR gene regulates carotenoid biosynthesis by controlling PSY gene expression, revealing a novel regulatory mechanism of carotenoids. Phytochrome interacting factors (PIFs) are a central factor in light-mediated reactions [PSY gene expression and carotenoid accumulation by binding to G-Box element on PSY gene promoter, and transducing light signals [The biosynthesis of plant carotenoids is a complex process, regulated not only by structural genes . Moreovemoplasts . Zhou etmoplasts  discovereactions , which r signals . EnvironCYC-B gene, finding that the CYC-B gene positively regulated carotenoid accumulation and affects coloration in the loquat. Moreover, there was a multiple-gene synergistic response to the downregulation of CYC-B gene expression in the carotenoid metabolic pathway. These results directly indicate that the CYC-B gene plays a vital role in carotenoid accumulation and coloration in the loquat. Clarifying CYC-B gene function is of great significance for breeding new loquat varieties.TRV-mediated VIGS was used to cause effective silencing of the


Most of real-world image distortions are multiply distortion rather than single distortion. To address this issue, in this paper we propose a quaternion wavelet transform (QWT) based full reference image quality assessment (FR IQA) metric for multiply distorted images, which jointly considers the local similarity of phase and magnitude of each subband via QWT. Firstly, the reference images and distorted images are decomposed by QWT, and then the similarity of amplitude and phase are calculated on each subband, thirdly the IQA metric is constructed by the weighting method considering human visual system (HVS) characteristics, and lastly the scores of each subband are averaged to get the quality score of test image. Experimental results show that the proposed method outperforms the state of art in multiply distorted IQA. With the large-scale use of intelligent mobile phone and computer in our modern society, evaluating the images after compression and transmission has become an increasingly important issue, and image quality assessment (IQA) has the great practical significance.IQA can be divided into three types: full-reference IQA, reduced-reference IQA, and no-reference IQA. The full reference IQA is developed earliest, which uses the original image as a reference. Full reference IQA can be roughly divided into error visibility and structural similarity methods. Peak signal-to-nosie (PSNR) is the simplest full reference IQA method. The structural similarity (SSIM)  index waIn recent years, quaternion wavelet transform (QWT) has been widely used in image processing. For example, Chen et al.  used hybMotivated by recent progress in IQA and QWT, in this paper we propose a QWT based full-reference IQA metric called QWT-IQA, which jointly takes into account the local similarity of phase and magnitude of each subband via quaternionic wavelet transform. In our QWT-IQA, we make use of a weighting method to compute the image quality score, inspired by human visual characteristics. A lot of experimental results demonstrate that our proposed QWT-IQA outperforms existing full-reference IQA method.The remaining part of the paper is organized as follows. In the section Background, we introduce the quaternion and QWT. In the section QWT-based full reference IQA metric, we propose a full-reference IQA metric based on QWT. Experiments on the LIVEMD and MDID2013 databases are carried out in the section Experimental results and analysis. At last, we give the conclusion in the section Conclusion.q is a quaternion, it can be expressed as , , and  respectively.A quaternion can also be expressed by amplitude and phase, i.e., φh and φg are scale functions, ψh and ψg are wavelet functions. According to δwhere thMagm(x) = max to make the IQA metric more consistent with human visual characteristics. Therefore, the SImilarity Metric in LL subband (SIMLL) considering HVS is calculated as following.After getting the similarity of each pixel, the similarity of the LL subband can be calculated. However, the human visual system has different perceptual effects on different regions of the picture. The more obvious the pixel amplitude is, the more prominent its corresponding phase is. The larger the amplitude is, the more important the corresponding pixel is, and the more likely it is to be in the texture changing structure. The smaller the amplitude is, the more likely it is to be in the smooth image region, and the value of the corresponding phase tends to be more unstable . In otheSIMLH, SIMHL, SIMHH can also been calculated in a similar form. Once the foregoing four similarity metrics are obtained, the QWT-based IQA metric (QWT-IQA) can be calculated by a weight sum of similarity metrics in all subbands as follows.a, b, c and d are used to adjust the importance of each subband.Following the same computation way in (10), the SImilarity Metrics in LH, HL and HH subbands, i.e., From a, b, c and d to 0.25, and the final IQA formula is defined as follows:So here we set all LIVE multiply distorted (LIVEMD) image database  consistsThere are several measures to evaluate the correlation between the quality scores and DMOS, such as Spearman rank order correlation coefficient (SROCC), Pearson linear correlation coefficient (PLCC), Kendall’s rank order correlation coefficient (KROCC), and Root mean squared error (RMSE). Note that, the closer correlation coefficient is to 1 and the lower RMSE is, the better the algorithm performs.Experiments are carried out on two image subsets and the entire database, respectively. We compare our proposed QWT-IQA with the FR PSNR, SSIM, FSIM, MS-SSIM and NR SISBLIM, DIIVINE, BLIINDS-II, NIQE methods. The results are listed in Tables We also give the scatter plot of several FR IQA scores on the LIVEMD image database against DMOS in Multiply distorted image database (MDID2013)  consistsThe comparison results of FR QWT-IQA, PSNR, SSIM, FSIM and MS-SSIM are given in We also compare the QWT-SIM metric with some NR metrics such as SISBLIM, DIIVINE, BLIINDS-II and NIQE, listed in On the MDID2013 image database, we also plot the scatter of QWT-IQA scores against DMOS, as shown in LIVE IQA database  is singlFor further test our proposed QWT-IQA metric, we also compare several IQA algorithms on the LIVE single distortion IQA database, and the results are listed in time consuming than FR PSNR, SSIM and MS-SSIM, but also efficient for real time application.Time efficiency is another important index for algorithm. We give the time of compared algorithm listed in In this paper, we have proposed a QWT-based full reference IQA metric called QWT-IQA for multiply distorted images. It first calculates the local similarity of phase and magnitude of each subband via QWT, and then uses a weighting method to gain image quality score through considering human visual characteristics. Many experimental results have demonstrated that our QWT-IQA has a higher consistency with the subjective measurement on multiply distortion images, compared with the state-of-the-art full reference IQA methods.


Background: The May 2016 wildfire in Fort McMurray, Alberta, Canada forced evacuation of the population of 88,000 individuals and destroyed 10% of the homes. Youth are particularly impacted by disaster.Methods: Eighteen months after the wildfire, Fort McMurray Public and Catholic Schools surveyed 3,252 of the 4,407 students in Grades 7–12 to determine possible long-term psychological impacts. The survey included validated measurement scales for post-traumatic stress disorder (PTSD), depression, anxiety, use of drugs, alcohol, and tobacco, quality of life, self-esteem, and resilience. Data analysis was possible for only 3,070 students, i.e., 70% of the total student population. Anonymized data were analyzed to compare students who directly experienced lesser or greater impact from the wildfire, with greater impact defined as personally seeing the fire or having one’s home destroyed. Also, students with greater or lesser scores on the Child and Youth Resilience Measure (CYRM-12) were compared.Results: Of the 3,070 students, 37% met criteria for probable PTSD; 31% met criteria for probable depression, and 17% for probable depression of at least moderate severity; 27% of students met criteria for probable anxiety, and 15% for probable alcohol or substance use disorder; 46% of all students met criteria for one or more probable diagnosis of PTSD, depression, anxiety, or alcohol/substance abuse, and this included students who were both present and not present in Fort McMurray at the time of the wildfire. Students with greater impact from the wildfire exhibited significantly higher scores on measures of PTSD, depression, anxiety, and alcohol/substance use. They also had lower self-esteem and quality of life scores. Students with lower resilience scores exhibited a similar pattern.Conclusions: These findings highlight first the negative impact of disasters on youth mental health, particularly for those who directly experience wildfire, and second the role of resilience on youth mental health, with lower resilience associated with substantially lower mental health outcomes. These results emphasize the need for long-term mental health supports for youth post-disaster, with specific focus on increasing youth resilience, which may serve as a protective factor against effects of disaster on mental health. On May 3, 2016, a large wildfire, called “The Beast” in the popular media   and incrIn a broader context, non-wildfire disasters, including floods, earthquakes, and tsunamis, also have an adverse effect on mental health [see review in Goldmann and Galea  and Kar Despite mental health and well-being challenges resulting from disasters, resilience plays an important role in the lives of children and youth by serving as a protective factor that mitigates the effects of disaster on mental health. Resilience refers to an individual’s capacity to handle adverse life experiences based on their mindset, resourcefulness, and support from family, friends, and community , 19. HigTo our knowledge, only one previous study prior to 2019 examined the impact of wildfires on youth mental health at a population level, focusing on PTSD and depression . Our goaBy analyzing school mental health survey data collected by Fort McMurray Public and Catholic School boards in November 2017 (18 months after the 2016 wildfire), we were able to help determine the overall population mental health effects in youth. This analysis allowed specific insights into measures of symptoms indicative of PTSD, depression, anxiety, alcohol and substance misuse, tobacco use, levels of self-esteem, quality of life, and resilience. In addition to capturing information on the mental health effects using standardized scales, the Fort McMurray schools were interested in understanding how other factors may influence these measurements. Therefore, in the survey, youth were also asked specific questions about their personal exposure to the wildfire and about the direct impact the wildfire had on their lives, including if they were present and witnessed the wildfire when it occurred, as well as if their own home was destroyed by the wildfire.We published an initial analysis  of the NThe current study presents an extended analysis of the Fort McMurray dataset, including an investigation of PTSD as well as examining the effects of severity of impacts from the 2016 wildfire experienced by different individuals on PTSD, depression, anxiety, alcohol/substance misuse, self-esteem, quality of life, and resilience.Based on previous research, our hypotheses were that more serious exposure to the wildfire and more direct impacts from the wildfire would have greater negative effects on youth’s mental health. More specifically, we hypothesized that these youth would have higher risk of having clinically significant scores on measures of PTSD, depression, anxiety, and alcohol/substance misuse, while having lower scores on measures of self-esteem, quality of life, and resilience.th, 2017 (ethics protocol number Pro00072669).The two school boards in Fort McMurray—Fort McMurray Public Schools and Fort McMurray Catholic Schools—asked all students in Grade 7–Grade 12 to complete a comprehensive survey of their mental health, in November 2017, 18 months after the 2016 wildfire. The survey was conducted to enable the school boards to evaluate the effectiveness of the programs that they put in place immediately following the wildfire  out of the survey if they desired. Students, themselves, had the option to participate or not in the survey, which was explained at the start of each survey data collection session  was a custom questionnaire assessing age, gender, the student’s grade, and school.The Impact of Fire Questionnaire  was a custom questionnaire assessing the impact of the 2016 wildfire on the student, including whether they were present in Fort McMurray during the fire, whether they were evacuated, whether they personally saw the fire, and whether their home was destroyed.Child PTSD Symptom Scale  assesses symptoms of PTSD  and geneThe Patient Health Questionnaire, Adolescent version  assesses symptoms of depression and suicidality , 25 and The Hospital Anxiety and Depression Scale  assesses symptoms of anxiety  and geneThe CRAFFT Questionnaire  assesses symptoms of alcohol and substance misuse , 28 and Tobacco Use Questionnaire (2 questions) includes two questions on tobacco use: “Over the past month: Do you smoke tobacco products? Do you use smokeless tobacco products?”The Rosenberg Self-Esteem Scale  assesses self-esteem  and geneThe Kidscreen Questionnaire  assesses quality of life  and geneThe Child and Youth Resilience Measure  assesses resilience to adverse experience or trauma  and genehttp://clojure.org).  Students either came to a computer laboratory or used laptops brought to their classroom, depending on their school. A survey description script was read to each class at the beginning of each survey session  Each participant used a laptop or desktop computer to fill in the survey. The survey website used an HTML/CSS front-end and a back-end server written in the Clojure programming language , depression (from PHQ-A), anxiety (from HADS), and alcohol/substance use disorder (from CRAFFT). We use the term “probable diagnosis” because scores were based on self-report scales, not psychiatric clinical interviews, and scores on a specific scale are not clinically diagnostic. Nonetheless, existing literature reports good correspondence between psychiatric clinical diagnoses of PTSD, depression, anxiety, and alcohol/substance use disorder with probable diagnoses based on widely published cutoff scores for the above four scales , 32–35. We defined five pairs of subgroups from participants, namely: 1) no impact of fire vs. any impact of fire; 2) present during the fire vs. not present; 3) saw the fire in person vs. did not see it; 4) home destroyed by the fire vs. home not destroyed; and 5) high resilience vs. low resilience.No impact of fire vs. any impact of fire: The no impact of fire group was defined as those participants answering “no” to the following four questions: “Were you in or near Fort McMurray during any part of the 2016 wildfire?,” “Did you evacuate because of the fire?,” “Did you see the fire in person?,” “Was your home destroyed by the fire?” The any impact of fire group was defined as those participants who answered “yes” to any one or more of those four questions. Participants who did not provide an answer to all four questions were excluded from both the no impact of fire group and the any impact of fire group.Present during the fire vs. not present: The present and not present during the fire groups were defined as participants answering “yes” and “no,” respectively, to the question “Were you in or near Fort McMurray during any part of the 2016 wildfire?” Participants who did not answer that question were excluded from both groups.Personally witnessed the fire vs. did not see it: The personally witnessed the fire vs. did not personally witness the fire were defined as participants answering “yes” and “no,” respectively, to the question “Did you see the fire in person?” Participants who did not answer that question were excluded from both groups.Home destroyed by the fire vs. home not destroyed: The home destroyed vs. not destroyed by the fire groups were defined as participants answering “yes” and “no,” respectively, to the question “Was your home destroyed by the fire?” Participants who did not answer that question were excluded from both groups.High resilience vs. low resilience: Resilience was measured with the CYRM-12 questionnaire. High and low resilience groups were defined as participants whose CYRM-12 scores were, respectively, equal/above the median or below the median CYRM-12 score.For each of these five pairs of groups, we compared the following 15 measures: 1) mean CPSS score, 2) mean PHQ-A score, 3) mean HADS score, 4) mean CRAFFT score, 5) mean Rosenberg score, 6) mean Kidscreen score, 7) mean CYRM-12 score, 8) percent probable PTSD, 9) percent probable depression, 10) percent probable moderately severe depression, 11) percent suicidal thinking, 12) percent probable anxiety, 13) percent probable alcohol/substance use disorder, 14) percent tobacco use, and 15) percent any of four probable diagnoses. Details of groups are given above. Details of questionnaire scores and probable diagnoses are given above.  In analyzing data for a given measure , we included only those participants who provided answers for all questions in the relevant questionnaire or scale.5). Permutation testing is a nonparametric method, chosen for its robustness against non-normality. All tests were two-sided, two-sample tests, with a null hypothesis of no difference between the means of the two groups for the given comparison. In total, our analysis included 74 individual statistical tests. We addressed multiple comparisons using the Benjamini–Hochberg method for false discovery rate (FDR) correction. This method computed a threshold of p = 0.027 for FDR correction. Effect sizes reported in tables are Cohen’s d (mean difference divided by pooled standard deviation). We performed all analyses using in-house computer code in the Clojure programming language (http://clojure.org). The server for the questionnaire website was written in Clojure, and the server saved participants’  questionnaire answers in Clojure data structures. It was therefore simplest to analyze the data in Clojure as well. In-house analysis code included data collating, sorting, filtering, and questionnaire scoring functions as well as the permutation testing and Benjamini–Hochberg FDR algorithms, which are straightforward to implement. The code for statistical testing and FDR correction is available at http://github.com/mbrown/mrgbstats.Permutation testing was used for all statistical comparisons  or Catholic schools (52% of students) and who were attending their schools on the day the survey was conducted. Five public schools and two Catholics schools took part in the survey. In total, 3,252 students participated out of 4,407 students enrolled in both the public and Catholic systems in Grades 7–12. That is, a total of 72% of enrolled students participated in the survey. The results presented below are organized by comparison groups. For convenience, Of the 3,252 students who participated in the survey, data from 182 students were excluded due to one or more of the following exclusion criteria:Age less than or equal to 10 years.Age greater than or equal to 20 years.Inconsistent answers among the positive and negative questions from the Rosenberg questionnaire . There were 2,970 students for whom depression scores were available, and of these, 31% met criteria for probable depression (PHQ-A score ≥ 11), and 17% met criteria for probable depression of at least moderate severity (PHQ-A score ≥ 15). Of these 2,970 students, 16% exhibited suicidal thinking. Anxiety scores were available for 2,990 students, of whom 27% met criteria for probable anxiety (HADS score ≥ 11). Alcohol/substance use scores were available from 3,001 students, of whom 15% met criteria for probable alcohol/substance use (CRAFFT score ≥ 2). Tobacco use data were available from 3,011 students, and 13% exhibited tobacco use. Of the 3,070 students, 46% satisfied the “any of 4 probable diagnoses” criterion. .There were no statistically significant differences between the “no impact of fire” group and the “any impact of fire” group in 14 of the 15 measures compared Table 3.There were no statistically significant differences between the “present in Fort McMurray during the fire” group vs. the “not present” group in 13 of the 15 measures compared Table 4.Comparison of participants who personally witnessed the fire vs. those who did not see the fire revealed significant differences in 13 of the 15 measures tested see Table 5.Participants whose homes were destroyed vs. not destroyed by the fire exhibited significant differences in 12 of the 15 measures tested see Table 6.The median resilience score (CYRM-12 score) across the 3,070 participants included in the statistical analysis was 48. High resilience was defined as having a CYRM-12 score of 48 or more, while low resilience was defined as having a CYRM-12 score of less than 48. There were significant differences between participants with high vs. low resilience scores on all 15 measures tested see Table 7.This study examined the impact of the wildfire on the mental health of youth in Fort McMurray, Alberta. The findings indicate rates of probable PTSD 37%), probable depression (31%), probable anxiety (27%), and probable alcohol/substance use disorder (15%) in the population of Grades 7–12 students in Fort McMurray, Alberta 18 months after the 2016 wildfire. By way of comparison with previous Canadian studies, the prevalence of probable diagnosis of PTSD in children has been reported as 15.5% ; prevale%, probabIt was anticipated that those students who had the greatest level of impact from the 2016 wildfire, in the form of personally seeing the fire (vs. not seeing it) or having their homes destroyed (vs. not destroyed), would have more frequent mental health symptomatology. The findings indicate that students directly impacted by the fire had significantly higher scores on scales measuring symptoms related to PTSD, depression, anxiety, and substance misuse. These students also had significantly lower scores for self-esteem, quality of life, and resilience. The findings indicate that students directly impacted by the fire had higher rates of probable PTSD, depression, anxiety, and substance use disorder and higher rates of suicidal thinking and tobacco use. These results suggest that mental health impacts are more severe with increased severity of impact from wildfire on the individual, in the form of personally seeing the fire or having one’s home destroyed.Importantly, the findings also revealed an unexpected pattern in students who had no direct impact from the fire; that is, they were out of town during the fire for a variety of reasons or were not living in Fort McMurray during the fire. The scores on almost all scales and questionnaires for these students were very similar to those who had direct experience with any impact of the fire  in terms of mental health symptom scores, self-esteem, quality of life scores, and rates of probable diagnoses. These findings suggest that youth not directly impacted by the fire nonetheless experienced vicarious trauma as a result of the fire’s large impact on the community, similar to secondary trauma experienced by first responders and researchers in post-disaster situations , 42. TheFinally, resilience also played an important role in the mental health of youth. Low resilience was linked with substantially more severe mental health impacts from the wildfire. Though we do not have survey data from Fort McMurray prior to the 2016 wildfire, we believe that having a higher baseline level of individual resilience prior to a disaster would have beneficial effects on youth’s mental health. This is supported by previous research showing that higher resilience is associated with better mental health outcomes following disaster , 20, 21.In terms of interventions, there is often active discussion about whether to intervene in complete populations , even when only a subset of the population has issues, or whether is it better to be more focused  for those with the greatest need. The issue with the latter approach, however, is that it misses large numbers of individuals who have only mild or moderate symptoms. The approach we have previously advocated is a combined approach, and we have shown this to be successful , 44. NonThe results presented here are based on a large sample of 3,070 participants. One limitation of the study is that clinical measures were based on self-report questionnaires as opposed to clinical interviews. Conducting a full clinical interview with each participant would not have been feasible given the large sample size. It would have been useful to have data using the same questionnaire battery from the Fort McMurray student population from before the 2016 Wildfire. This would have allowed a before and after wildfire comparison with identical instruments. Instead, we have previously presented a comparison of the Fort McMurray mental health data with control data previously collected using a very similar battery in Red Deer, Alberta .In conclusion, the present results support existing findings that both youth and communities impacted by a wildfire, or similar major disaster, experience long-term mental health impacts. The present data extend this by examining all youth in grades 7–12 attending schools in Fort McMurray following the 2016 wildfire and identifying those groups who are most vulnerable and have the greatest risk. It is likely that providing additional assistance for all individuals to increase resilience, and also focusing on those with particularly severe wildfire impact, would be useful in terms of improving population mental health after a wildfire and other major disasters. Fort McMurray Public and Catholic Schools have put significant and ongoing work into establishing and maintaining mental health support programs for students in the aftermath of the 2016 wildfire. The findings presented here are consistent with the need for long-term mental health supports in disaster-affected communities such as Fort McMurray.The dataset analyzed in this study is available from the corresponding author on reasonable request.In November 2017, 18 months after the 2016 wildfire, the two school boards in Fort McMurray—Fort McMurray Public Schools and Fort McMurray Catholic Schools—asked all students in Grade 7–Grade 12 to complete a comprehensive survey of their mental health. The survey was conducted to enable the school boards to evaluate the effectiveness of the programs they had put in place immediately following the wildfire. Researchers from the University of Alberta were asked to collaborate and provide assistance in designing the survey and analyzing the survey data. After the data were collected, the anonymized data were made available for analysis by the researchers from the University of Alberta. The analysis of the survey data was approved by the University of Alberta’s Health Research Ethics Board on June 26th, 2017 (ethics protocol number Pro00072669).The survey included questions to determine demographics, mental health, resilience, personal exposure to, and direct impacts of the wildfire. All survey data were collected under the auspices and ethical guidelines of the two Fort McMurray school systems and were administered as part of their standard curriculum and as an ongoing assessment of the educational and support programs they had put in place after the wildfire. The selection of measurement instruments was determined by the school systems and was informed by the existing scholarly literature and findings. Parents and guardians were notified of the process by written letter 2 weeks prior to the administration of the survey in the schools, and they had the option to opt their child(ren) out of the survey. Students had the option to participate or not in the survey, and this was explained at the start of each survey data collection session. Survey participation was anonymous; participants were not asked for their names. This paper provides an analysis of the survey data that were collected from both school boards.Materials and Methods section, are shared with a separate manuscript by the authors .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.CPSS, Child PTSD Symptom Scale; CRAFFT, CRAFFT Questionnaire (proper name of the questionnaire is CRAFFT); CYRM-12, Child and Youth Resilience Measure; EMPATHY, Empowering a Multimodal Pathway Towards Healthy Youth project; FDR, false discovery rate; HADS, Hospital Anxiety and Depression Scale; Kidscreen-10, Kidscreen Questionnaire; PHQ-A, The Patient Health Questionnaire, Adolescent version; PTSD, post-traumatic stress disorder; Rosenberg, Rosenberg Self-Esteem Scale.