Empowerment is an internationally recognised concept commonly incorporated in First Nations and in this instance Aboriginal and Torres Strait Islander health and wellbeing programs. The Family Wellbeing Program is an empowerment program developed in partnership with Aboriginal and Torres Strait Islander peoples that has been widely delivered to Aboriginal and Torres Strait Islander communities across Australia for close to 30 years. To date, there has been limited quantitative analysis of how this program is linked to health and empowerment outcomes.n = 9,843) recruited using multi-mode random sampling including mail out survey and in community convenience sampling. Logistic regression models were performed to calculate Prevalence Ratios (PRs) and 95% Confidence Intervals (CIs) to examine the association between personal control, life satisfaction, general health, family wellbeing and cultural wellbeing outcomes for Family Wellbeing participants (n = 718) versus non-participants .Cross sectional analysis of Mayi Kuwayu, the National Study of Aboriginal and Torres Strait Islander Wellbeing, baseline data , be aged 35–54 (41.8% versus 32.0%) and live in a remote area (17.7% versus 10.4%) and have educational attainment at the Year 12 level or above (57.8% versus 53.2%). Family Wellbeing participation was associated with a 13% higher reporting of family functioning, a 74% higher reporting of cultural participation and a 21% in higher reporting of local decision making in the local community compared to non-FWB participants. There were significant associations between FWB exposure compared to non-FWB exposure including reporting lower levels of health risk factors including quitting alcohol (26.4% versus 20.4%), regular exercise (67.7% versus 66.3%), quitting smoking (33.4% versus 31.9%). and e. FWB participants who had experienced both prison and youth detention were nearly double that of Non-FWB (3.5% versus 1.4%) and more reported being removed from their families as children (Stolen) (7.0% versus 4.1% Non-FWB).There are significant associations between Family Wellbeing exposure and organisation and community level empowerment outcomes, but only for some individual level empowerment outcomes. There is a lower reporting health risk factors including increased physical exercise, reduced alcohol use and smoking; and educational attainment among FWB participants compared to non-FWB participants. The results suggest individual, community and organisational empowerment needs to be explored further with more robust study designs that can attribute causality and direction of association.The online version contains supplementary material available at 10.1186/s12889-023-16450-9. Empowerment theory has become a common concept in public health since the World Health Organisations’ 1986 Ottawa Charter recognised ‘the process of enabling people to increase control over, and to improve, their health’ as vital to creating global health equity , 3. ObseEmpowerment is complimentary to Self-determination which comes from an international rights framework  and is commonly advocated for by Indigenous communities worldwide. Self-determination is a long standing legal and policy concept advocated by Aboriginal and Torres Strait Islander peoples since the early 1970’s and is a dynamic right to make decisions and to control their implementation. While there are differences, empowerment can be thought of in this instance as the processes of realising self-determination.Wallerstein’s work on empowerment as a health enhancing strategy asserts that understanding the role of social protective factors is key to developing effective theory of change models that can reduce ill health brought about by social inequity . This apCommunity empowerment theorists pointed out that consideration must be made to variations in protective factors by population, geography and time , 7. TheyThe Family Wellbeing program (FWB) was developed in 1993, conceived in consultation with a group of survivors of the stolen generations. The stolen generation were and are Aboriginal people forcibly removed from their families and were required by the state to undertake domestic or agricultural labour training, then be in the employ of non-Indigenous families from the early 1900’s through to the end of the 17,970’s. The Family Wellbeing program it is a group learning program that focuses on five stages of personal, family and community empowerment. Initiated as an employment and training program it has been adapted and delivered for a range of community identified needs and audiences; including suicide prevention, family violence, inmate education and parenting support . CommonlThe FWB has been delivered in 60 known sites across Australia with more than 5,400 participants , 14.Existing studies and evaluations of the FWB have analysed the applicability of empowerment used in the program to Aboriginal and Torres Strait Islander contexts –17, the This analysis examined the relationship between exposure to the FWB on health, wellbeing, cultural and empowerment outcomes.Mayi Kuwayu: The National Study of Aboriginal and Torres Strait Islander Wellbeing (the Mayi Kuwayu Study) is a longitudinal study that has been designed to quantify the links between social and cultural determinants, such as cultural expression, community decision making and family connection, to health and wellbeing , 9. It iN = 9,843), and includes all responses received between 2018-December 2020. FWB participants are identified by a question asking ‘Have you ever participated in: the Family Wellbeing Program?’, non-FWB were any survey participant who did not select the “Family Wellbeing Program” option. No information was collected on the date of participation in the program.This cross-sectional analysis is conducted using the third release (R.3) of the Mayi Kuwayu baseline data . The research was conducted with ethics approvals from relevant Aboriginal and Torres Strait Islander organisations, and from national, state and territory Human Research Ethics Committees (HRECs).‘Have you ever participated in: Family Wellbeing Program’.Family Wellbeing participants are identified through Mayi Kuwayu question. Outcome variables used in the study were selected based on analysis of the literature, including previous evaluations of the program , 17, 23 Personal empowerment was measured with responses to the question, “how much are you in control of your life?”’, with response options “A lot”, “A fair bit”, “A little bit” and “Not at all”. These were then dichotomised as a (0) not at all and (1) little—a lot. Life satisfaction was measured according to responses to the question, “How satisfied are you with your life?”, with response options “a lot”, “a fair bit”, “a little bit” and “not at all”. These were then categorized as a (0) not at all and (1) little to a lot. General health was measured according to the question “How would you rate your general health?”; response options were “poor”, “fair”, “good”, “very good” or “excellent”. Responses were categorised as (0) poor-fair and (1) good–excellent.Family functioning was measured according to a composite score of responses to a set of nine questions asking “In my family…”, “We get together and cope in the hard times”, “we celebrate special days/ events”, “we talk with each other about the things that matter”, “we are always there for each other”, “we manage money well”, “we have common interests”, “people are accepted for who they are”, “we have good support from mob”, “we have family knowledge and traditions that we pass on to our children”. With response options “not at all” (1), “a little bit” (2), “a fair bit” (3) to “a lot” (4), and “unsure” (0). Responses were summed (range: 0–36), and participants were categorized as having (0) low- moderate family functionality (score 0–29) or (1) high family functionality (score 30–36). Responses to the nine questions were summed for participants with complete data only; participants missing responses to any of the questions were coded as missing. All outcome measures above were coded as binary for use in the regression analysis.For organisational empowerment a composite score of the cultural knowledge and practice subset was created, that includes questions relating to contribution to community and participation in community-based events. The score was made up of responses to ten questions, summed together to generate a total cultural wellbeing score ranging from zero to 40. The questions asked “How much time do you spend…” 1. “With someone who has cultural knowledge (elder or knowledge holder)?” 2. “Learning and using knowledge from Aboriginal/Torres Strait Islander Law (or Lore)?” 3. “On country?” 4. “Getting or eating bush tucker ?” 5. “Learning culture, kinship and respect?” 6. “Making art, music or painting?” 7. “Passing on cultural knowledge?” 8. “Participating in social events related to Aboriginal/Torres Strait Islander people ?” 9. “Contributing to your community ?” 10. “Receiving Aboriginal/Torres Strait Islander healing methods ?”. With response options "Not at all" (1), "A little bit" (2), "A fair bit" (3) and "A lot" (4). Responses for the 10 questions were summed together to generate a total cultural wellbeing score ranging from zero to 40; participants missing any data had their score coded as missing. A binary category was created of (0) low cultural wellbeing  and (1) high cultural wellbeing (scores between 15 and 40).Decision making was measured by the question “in the Aboriginal and/ or Torres Strait Islander community where I live now local Aboriginal and Torres Strait Islander people make community decisions” with response options “Not at all”, “A little bit”, “A fair bit”, “A lot” and “Unsure”. Responses were coded and categorised to (0) not at all  and (1) A little – a lot; unsure was coded as missing.The presence of a drug and/ or alcohol problem was measured by response to the question that asked, “has a doctor ever told you that you have…drug or alcohol problem” with responses “no” and “yes”. Participants were also asked “do you drink alcohol?” with responses categorised as “current drinker”, “ex-drinker” and “never drinker”. Current smoker was measured with response to question “Do you smoke?”, with response categories created for “current smoker”, ex-smoker” and “never smoker”. Current smokers were also asked “do you want to quit smoking?” with five response options that were categorised as “not at all or unsure” and “a little to a lot”. For exercise in the past week where participants were asked to select the days that they did 30 or more minutes of exercise (Monday – Sunday) the responses (1 for each day selected) were summed into three categories “none”, “1–2 days” and “3 or more”.Eight demographic variables were used in the study including gender, age, location, and education. Following consultation with FWB providers we added measures for incarceration, low income, being removed as a child Stolen and housn) overall and by FWB exposure. A Chi-square statistic was calculated to test for significant differences between FWB exposure and the categories of each demographic variable  and 95% Confidence Intervals (CIs) are presented for each exposure/outcome association. Models are presented unadjusted and adjusted accounting for gender, age, location and being an ex-drinker as these were conceptually thought to be linked to both exposure and outcome variables. A sensitivity analysis was conducted to address possible contamination within the analysis with Family Wellbeing Services  run by tOf the total study population , 718 indicated participating in the FWB program and 9,125 did not indicate participation in FWB. Around 6 in 10 (59.5%) were female and the predominant age category was 55 + (40.9%). Close to half of the study respondents lived in a regional area (47.6%), with 41.1% residing in a major city and 10.9% residing in a remote area.p < 0.05) observed between FWB and non-FWB participants including by gender with around two-thirds (67.1%) of FWB participants being female (versus 58.4% Non-FWB), being in the 35-54 yr age category . Regional areas were the most common area of residence for both FWB (49.4%) and Non-FWB (47.4%), however more of the FWB group resided in remote areas (17.7%) compared to 10.4% of non-FWB participants. Less than one third of FWB participants (32.0%) lived in a major city (versus 41.8% non-FWB).There were a number of significant differences . There was no difference in family financial status between the two groups. A significantly higher level of overcrowding was reported by FWB participants (21.6% versus 16.3% Non-FWB).There were significant differences in reporting of incarceration and stolen generations between FWB and non-FWB participants with 7% of FWB participants reporting being stolen or removed as children compared to 4% of Non-FWB participants. Most FWB participants (83.3%) reported no experience of incarceration (versus 88.6% non-FWB). Overall FWB participants reported a significantly higher level of both prison and youth detention (3.5% versus 1.4% non-FWB).Overall, 6.3% of Mayi Kuwayu participants reported a diagnosis of a drug or alcohol problem by a doctor  of FWB participants had stopped drinking (versus 20.4% Non-FWB) and around a third 33.4% had quit smoking (31.9% Non-FWB). Of current smokers 79.8% in the FWB participant group reported wanting to quit smoking, compared to 76.6% of Non-FWB smokers. The majority of FWB participants (67.7%) had undertaken 3 or more days of exercise in the past week, (versus 66.3% non-FWB), less than one-fifth (18.3%) of the exposed group reported having done no exercise in the past week to 22% of the non-FWB.Against the personal empowerment outcomes, 94.2% of all participants reported a little to a lot of personal control, similarly 91.6% reported a little to a lot of life satisfaction (91.6%). Against the organisational empowerment outcome 41.2% of participants reported high levels cultural wellbeing. Around half of survey participants (47.9%) felt that local people made decisions in the community that they lived.The prevalence of high personal control and life satisfaction was similar across FWB and Non-FWB participants. There were significant differences on family and community empowerment including almost half of FWB participants (45.7%) reporting high-very high outcomes on the family functionality scale (versus 34% Non-FWB) and 70.9% reporting moderate-high cultural wellbeing (versus 38.9% Non-FWB). Almost three-quarters (74.5%) of FWB participants reported that local Aboriginal and/ or Torres Strait Islander people made decisions in their community, compared to less than half of the Non-FWB group (45.8%).There were no significant differences in life control PR 1.00 (CI. 1.00–1.00), life satisfaction PR 1.00 (0.98–1.02) or general health 0.95 (0.90–1.02) between FWB participants and non-Participants. There was a significant 13% increase in reporting high levels of family wellbeing PR 1.13 (CI 1.04–1.22); a 74% increase in reporting high levels of cultural wellbeing PR 1.74 (CI 1.66–1.83); and a 21% increase in reporting high levels of local decision making by Aboriginal and Torres Strait Islander people PR 1.21 (CI 1.16–1.25). After adjustment for gender, age, location and being an ex-drinker there was no difference in all but one exposure outcome relationship. Being a former drinker did reduce the prevalence ratio of cultural wellbeing to a 13% increase from a 74% increase in the unadjusted model.In this cohort of Aboriginal and Torres Strait Islander adults we observed significant associations between FWB participation and higher reporting of some outcomes including cultural wellbeing, community empowerment and for high-very high family functioning (13%). We did not observe a significant association between FWB participation, personal empowerment and general health. We did observe significant associations between FWB participation and increased prevalence of addressing health risk factors including quitting smoking, increased physical inactivity and quitting alcohol. We also observed a significant increase in year 12 or higher educational attainment amongst FWB participants compared to Non-FWB (57.8% v 52.8%) participants.These findings are in line with previous qualitative studies where participants reported wanting to connect with their spirituality. This accords with previous qualitative FWB studies where participants felt that colonisation had undermined Indigenous culture and spirituality . This maThe higher prevalence of participation in the FWB program by people who live remote areas, have been Stolen and experienced incarceration , accords with a history of program delivery for stolen generations and those experiencing incarceration and supports the flexibility of the program to address local needs identified by communities, including removal and incarceration . The outIt is unclear why there is no significant association between FWB program exposure and life control and life satisfaction in this study, given both the strong focus in the program on individual healing and wellbeing, and the associated improvements in the outcome measures of family functioning, cultural wellbeing and community decision making. However, the findings are still in line with definitions of empowerment processes as a dynamic continuum , 5; wherA limitation of this study is that it is cross-sectional and therefore no causality can be attributed and we are also unable to assess the direction of association. Another limitation is the high scores in life satisfaction and control across both the exposed and non-exposed group, contributing to a ‘ceiling effect’ which limits variation in outcomes between the two groups. To overcome these limitations, exploration of how these outcomes change over time for participants using more robust study designs would be desirable. The possible confusion of the program with other similarly named programs or services was also raised as a possible weakness of the study, as stated previously this was a particular concern in Queensland, however the sensitivity analysis identified no significant difference and therefore all Queensland participants were included in the final analysis. We note that mandating of the program may have occurred in some areas but we were unable to determine if participants were mandated, which may impact on the results. There is also a question of cultural nuance that requires adaption to the levels of analysis, or domains within them, to be more in line with Aboriginal and Torres Strait Islander cultural understandings of self. This would be in line with earlier qualitative evaluations of the Family Wellbeing program that found participants placed particular emphasis on relational and spiritual traits as attributes of empowerment , 18.Missing from the analysis is information on structural change, or evidence of how an empowerment intervention such as the Family Wellbeing program can influence changed conditions, different policies or impact experiences of racism. For example, the ability of participants to improve their housing conditions in remote areas where housing infrastructure is limited and over-crowding more common . There aThe FWB program was significantly associated with higher reporting of cultural wellbeing, family functioning and local decision-making, reporting included reduction of risk factors, such as drinking and smoking, implies a model whereby both immediate and intermediate effects when compared to non-FWB participants. These positive associations are encouraging, particularly in combination with past consistent qualitative evidence of program benefits. A fuller evaluation of this program is warranted, using more robust study designs to provide a prospective quantification of the benefits of participation.Additional file 1: Appendix 1. Framework for empowerment evaluation outcomes.


The aim of this study was to evaluate the effect of cyclic mechanical loading on the fracture resistance of 3D-printed zirconia crowns in comparison to milled zirconia crowns.n = 30) were manufactured using subtractive milling (group M) and 3D additive printing (group P). Nine samples of each group were fractured under one-time loading while the other 6 samples were subjected to cyclic loading for 1.2 million cycles before being subjected to one-time loading until fracture. Scanning electron microscope (SEM) fractographic analysis was carried out on fractured fragments of representative samples.Monolithic zirconia crowns .The fabrication technique and cyclic loading affect the fracture resistance of zirconia crowns. Although the fracture resistance values for the 3D-printed crowns were lower than those of the milled, still they are higher than the masticatory forces and thus could be considered being clinically acceptable.Concerning fracture resistance, 3D-printed crowns can withstand the masticatory forces for the long term without any cracks or failure. Patients and dentists have been looking for metal-free tooth-colored restorations due to the increased esthetic demands, the more conservative preparations, and the high concerns of toxic and allergic reactions to certain alloys . Yttria The development of these materials and the CAD/CAM fabrication techniques has created a wide range of applications for dental restorations with conservative tooth preparation, such as partial coverage restorations, crowns, and bridges . MonolitThe fast expansion and the development of materials and techniques used in additive manufacturing allowed the possibility of the production of monolithic zirconia dental restorations from a variety of materials; additive manufacturing techniques include selective laser sintering (SLS), selective laser melting (SLM), stereolithography (SLA), ink-jet printing (IJP), and fused deposition modeling (FDM) . In thisThe fracture resistance of all-ceramic restorations is one of the major concerns in clinical applications of these materials, and it is influenced by many factors such as surface roughness, elastic modulus, crack resistance, and fabrication technique , 13. ZirIt has been well-established in the literature that fracture is the main cause of failure of dental ceramics after years of service inside the oral cavity, as they are subjected to repeated loads in the patient’s mouth, which can influence the long-term reliability of the crowns. Consequently, to estimate the mechanical performance of zirconia crowns, it is not sufficient to test them under one-time loading only, cyclic loading must be tested as well . SeveralAccordingly, the aim of our study was to evaluate the fracture resistance of 3D-printed zirconia crowns and the effect of cyclic loading in an aqueous solution on their fracture resistance, compared to milled zirconia crowns. The first null hypothesis was that there would be no significant difference in the fracture resistance between the crowns fabricated by milling or 3D-printed techniques. The second null hypothesis postulated that the cyclic loading would not affect the fracture resistance of zirconia crowns fabricated by both techniques.An upper premolar typodont tooth was prepared using tapered stone with a round end to create a deep chamfer finish line with 1 mm thickness, axial wall convergence of 6%, and 1.5 mm occlusal reduction , 20.Thirty impression replicas were taken for the prepared tooth using addition silicone impression material  and putty soft and light consistency to pour 30 epoxy stumps  that were allowed to set until full hardening .After scanning the epoxy stump with an extraoral scanner , the crowns were digitally designed using exocad software .A total of 30 zirconia crowns (3 mol % yttria-stabilized zirconia) were fabricated using 2 different fabrication techniques with 15 crowns in each group. As a control group (group M), IPS e.max ZirCAD LT  zirconia crowns were milled using a milling machine ; then, they were sintered in a zirconia furnace  up to 1530 °C, while in group P, Lithoz 210 3Y  zirconia crowns were 3D printed using a CeraFab7500 printer ; then, they were preconditioned up to 120 °C for 134 h, then debound at up to 1000 °C for 103 h, then sintered at up to 1450 °C for 17 h in 3 consecutive furnaces . The materials used for each group in the study and the sintering parameters are listed in Tables d) ranging from 1.46 to 1.82, with an actual power (1 − β error) of 0.8 (80%) and a significance level (α error) of 0.05 (5%) for a two-sided hypothesis test [A power analysis study was performed using the G power statistical power analysis program  to estimate the sample size. A total sample size of 30 samples  was found to be sufficient to detect a large effect size  were loaded using a metal stylus with a 5-mm-diameter spherical tip with a speed of 1 mm/min which was applied directly to the center of each crown and perpendicular to the occlusal surface until fracture using a universal testing machine  . Samples of each group  were subjected to dynamic mechanical loading using a commercial pneumatic setup . 1.2 million cycles were applied with a load between 20 and 200 N at 2 Hz in distilled water 37 ± 2 °C to simulate approximately 5 clinical years , 24, 25.Representative fractured crown samples from each group were selected, cleaned using alcohol in an ultrasonic cleaner, and coated with a thin gold/platinum layer using a sputter coater  for 60 s. After sputtering, the samples were ready to be examined under a scanning electron microscope  at an operating voltage of 10 kV and a spot size of 5 using secondary emission modes.p < 0.05 within all tests. Statistical analysis was performed with R statistical analysis software  version 4.1.3 for Windows (R Core Team 2022).Numerical data were represented as mean with a 95% confidence interval, standard deviation (SD), and minimum and maximum values. Shapiro–Wilk’s test was used to test for normality. The homogeneity of variances was tested using Levene’s test. Data showed parametric distribution and variance homogeneity and were analyzed using a two-way ANOVA test. The significance level was set at p = 0.002) and the mechanical cycling loading (p = 0.001) had a significant effect on fracture resistance while there was no significant interaction between tested variables (p = 0.325).Descriptive statistics for fracture resistance values are presented in Fig. SEM showed similar fracture patterns in the selected samples, the cracks in all the groups originated away from the point of load application, and the direction of crack propagation started from the tensile zone at the internal surface and propagated outwards to the external surface of the crown. Twist hackles and arrest lines are present as shown in Figs. The first null hypothesis of the present study that there would be no significant difference in the fracture resistance between the crowns fabricated by milling and 3D-printing techniques was rejected. Also, the second null hypothesis was rejected, as the cyclic loading decreased the fracture resistance of zirconia crowns fabricated by both techniques.In the present study, premolars were used as they require an esthetic restoration that can withstand high masticatory forces , 20. SimAccelerated aging tests provide essential information on lifetime predictions of ceramic restorations; the applied fatigue protocol with 1.2 million cycles is equivalent to 5 years of clinical service for dental restorations . The cycIn this study, the immediate fracture resistance of all crowns (1890 ± 191 N for the milled crowns and 1658 ± 333 for the 3D-printed crowns) exceeded 790 N, which is higher than the maximum biting forces (450–520 N) that increases to 790 N in bruxism , 37. AftMilled zirconia crowns showed significantly higher fracture resistance than the 3D-printed zirconia crowns. These results were consistent with the results of Zhai et al. .This may be contributed to the higher amount of porosity in the 3D-printed zirconia crowns. This effect has been widely studied by Branco et al. , who fouFractographic analysis showed similar fracture patterns for the examined specimens of all the tested crowns. The fracture origin was located away from the loading site, in the tensile zone (inner surface of the crowns), and the direction of crack propagation was outwards; this could be explained by the transverse expansion that occurs in the abutment material owing to the Poisson effect when the crown was subjected to occlusal loading. The Poisson ratio of epoxy (0.31 μ) is similar to that of dentin (0.3 μ) ; therefoAmong the limitations of this study is the use of abutments with no mobility; the dynamic load was vertical with no chewing cycles and in the clinical situation; the crown’s design may vary according to the patient. Additionally, the effects of other environmental factors, such as saliva, or the effects of different kinds of beverages and pH fluctuation were not taken into consideration in the present study.Zirconia crowns fabricated by the 3D-printing technique can withstand chewing forces for over 4 years (1.2 million cycles).The fracture resistance of these crowns will decrease during function in the oral cavity but is still higher than the masticatory forces (450–520 N).The fracture resistance of the printed crowns is lower than that of the milled crowns but is still higher than the masticatory forces (450–520 N).Among the limitations of this in-vitro study, it could be concluded that


Ankylopteryx in Ankylopterygini for the first time. Comparative analyses of the mitogenomes of Chrysopidae were conducted. The arrangements of the mitogenomes were consistent with the other Chrysopidae species. The phylogeny of Chrysopidae was reconstructed and the divergence time within Chrysopidae was estimated. Chrysopinae were recovered as the sister group to Apochrysinae + Nothochrysinae. Within the subfamily of Chrysopinae, Nothancylini were recovered as the sister group to (Leucochrysini + Belonopterygini) + (Ankylopterygini + Chrysopini). The three extant subfamilies of Chrysopidae diverged from each other during the Early Cretaceous. Nothancylini diverged from other Chrysopinae in the Early Cretaceous. Leucochrysini diverged from Belonopterygini in the Late Cretaceous. Ankylopterygini diverged from Chrysopini in the Middle Cretaceous.Chrysopidae are the second largest group of Neuroptera. Their larvae can prey on various agricultural and forestry pests. In this study, we sequenced two mitochondrial genomes (mitogenomes) of Ankylopteryx in the chrysopine tribe Ankylopterygini for the first time. The characteristics of these mitogenomes were analyzed in comparison with other green lacewing mitogenomes published to date. In the phylogeny herein reconstructed based on mitogenomes, Chrysopinae were recovered as the sister group to Apochrysinae + Nothochrysinae. Within the subfamily of Chrysopinae, Nothancylini were recovered as the sister group to (Leucochrysini + Belonopterygini) + (Ankylopterygini + Chrysopini). The divergence time estimation suggested an Early Cretaceous initial divergence within the extant Chrysopidae. Within Chrysopinae, the four tribes except Nothancylini diverged around mid-Cretaceous.Chrysopidae (green lacewings) are a cosmopolitan and species-rich family of Neuroptera, with remarkable significance of biological control against various agricultural and forestry pests. However, the phylogenetic position of Chrysopidae in Neuroptera and the internal relationships within the family remain equivocal among previous studies based on different types of data and sampling. Here we sequenced the mitochondrial genomes (mitogenomes) of two species of the genus  Chrysopidae are the second largest group of Neuroptera, with over 1400 species from 82 genera, widely distributed around the world ,4.cox1, 16s rDNA, cad and mitogenomes) [Chrysopidae are composed of three extant subfamilies  and an extinct subfamily (Limaiinae) . Howevergenomes) ,8,9. Howgenomes) ,11.cad, cox1 and 16s rDNA) [Among the five tribes, i.e., Nothancylini, Leucochrysini, Belonopterygini, Ankylopterygini and Chrysopini of Chrysopinae, Nothancylini were recovered as the sister group to (Belonopterygini + Leucochrysini) + (Chrysopini + Ankylopterygini) based on morphological data and mitogenomes ,12,13. C6s rDNA) ,11.16s rDNA, cox1, CPSase, cad, wg, pepck, atpase, 18s and mitogenomes), the Bayesian inference (BI) suggested Apochrysinae was the sister group to Nothochrysinae + Chrysopinae based on the molecular supermatrix, while the tree of maximum likelihood (ML) supported Chrysopinae was the sister group to Nothochrysinae + Apochrysinae [16s rDNA, cox1, CPSase, cad, wg, pepck, atpase, 18s and mitogenomes) [In recent years, many new data and methods have been used to resolve the phylogenetic relationships within Chrysopidae. In a recent study based on molecular data  . In addigenomes) . Herein,http://www.ncbi.nlm.nih.gov (accessed on 11 June 2023)), but there is only an incomplete representative mitogenome of Ankylopterygini. In this study, we newly sequenced the mitogenomes of two species of the genus Ankylopteryx  in Ankylopterygini. These two mitogenomes were annotated and uploaded to GenBank with accession numbers OQ269716 and OM510943. Comparative analyses of the mitogenomes of Chrysopidae were conducted here, such as codon usages, the rates of nucleotide substitution and secondary structure predictions of RNAs. The phylogeny of Chrysopidae was reconstructed based on mitogenomes with addition of the newly sequenced Ankylopteryx data, and the divergence time among major lineages within Chrysopidae was estimated.There are eleven mitogenomes of Chrysopidae published in GenBank (Ankylopteryx octopunctata (Fabricius) was collected by Hongyu Li on 30 July 2018, in Jiangxin, Medog, Xizang, China and that of A. gracilis (Nakahara) was collected by Qicheng Yang on 10 June 2016, in Baisha River, Hekou, Yunnan, China. Both the specimens were preserved in 95% alcohol at −20 °C before the DNA extraction. Genomic DNA was extracted using the DNeasy Blood and Tissue kit  from the thoracic muscle tissue. The DNA concentration was measured using a Nucleic acid protein analyzer .The specimen of Drosophila” as genetic code. The secondary structures of tRNAs were predicted by the MITOS Web Server  and checked through manual proofreading [rrnS and rrnL were predicted using RNA Structure (http://rna.urmc.rochester.edu/RNAstructureWeb/ (accessed on 27 May 2023)) [Whole genomes were sequenced on the Illumina HiSeq 2500 Platform  by Majorbio . Raw reads were checked by FastQC 0.11.9  and low-freading , and they 2023)) .Hemerobiidae, two species of Mantispidae as well as two species of Myrmeleontidae were used as outgroups to reconstruct the phylogeny in this study  prisca [Chrysoperla was calibrated with a minimum age of 21 Ma based on the fossil species Chrysopa glaesaria [The divergence time was estimated under the uncorrelated lognormal relaxed clock using MCMCtree in BEAST v.2.5.0  with 1,0f 165 Ma ; (2) thesochrysa ; (3) the) prisca ; (4) thelaesaria . The analaesaria .A. gracilis was 18,284 bp in length, while that of A. octopunctata is incomplete with a length of 13,881 bp. The mitogenome sequence of A. octopunctata was not complete since the high AT content of the control region (AT-rich region). The complete mitogenome was composed of 13 PCGS, 22 tRNAs, 2 rRNAs and a control region  and the longest was An. gracilis . In the mitogenomes of Chrysopidae, the content of C was higher than G and the content of A was higher than T except C. pallens. The A + T content was 78.76% to 81.75%  and the longest were those of Ch. externa and Ch. nipponensis . The highest AT content was observed in Abachrysa eureka (79.39%) and the lowest AT content was detected in Ch. externa (77.0%)  .C. pallens (0.00), and the weakest AT-skew was in Apochrysa matsumurae (−0.04). The highest GC-skew was in Ch. externa (−0.12), and the weakest GC-skew was in Ap. matsumurae (−0.18) (Nothancyla sinica (−0.14) and the weakest AT-skew was in Ch. externa (−0.18). The strongest GC-skew was in Ch. externa (0.09), and the weakest GC-skew was in Nothochrysa sp. (0.04)  . In the . (0.04) . Comparef base G . Analysif base G . This phnad2 of Italochrysa insignis, L. pretiosa and Parankylopteryx sp. The use of start codons in Chrysopidae was a typical form with ATN in all PCGs  used TAA as stop codons in all species, while other PCGs used TAA/TAG or TA-tRNA/T-tRNA as stop codons. The stop codons of nad3 were TAA/TAG or T-tRNA in different species, and nad5 was terminated with T-tRNA in all species. The stop codons of cox1, cox2 and nad4 were T-tRNA in most species, and the stop codons of cob, nad2 and nad6 were TAA in most species.Start codons and stop codons of most PCGs were identified except the incomplete all PCGs . Most PCAn. gracilis and An. octopunctata. The most common codons were UUA (Leu1), AUU (Ile), UUU (Phe) and AUA (Met), indicating the preference of nucleotide composition for A/T.The relative synonymous codon usages (RSCUs) of PCGs in the two newly sequenced mitogenomes were shown in cox2) to 0.205 (atp8). The gene atp8 (Pi = 0.205) showed the highest nucleotide diversity. There was also relatively high nucleotide diversity in nad6 (Pi = 0.200) and nad2 (Pi = 0.173), while cox2 (Pi = 0.098) and cox1 (Pi = 0.102) showed relatively low nucleotide diversity, which suggested they were conserved genes. In order to better understand the evolution of PCGs, the rate of nucleotide substitution (Ka/Ks) of PCGs among Chrysopidae was analyzed (cox1) to 0.552 (atp8), indicating that cox1 had the slowest evolution rate, while atp8 had the fastest evolving rate.The nucleotide diversity (Pi) of 13 PCGs among Chrysopidae was shown in analyzed . The Ka/An. gracilis between rrnS and trnI (3416 bp) is much longer than the other URs (1–136 bp). Some predicted secondary structures contain T-rich loops   . In addiatp8-atp6 and nad4-nad4L, which had been reported in many other insect mitogenomes [nad4-nad4L, while the sequences were ATGATAA between atp8-atp6 in most Chrysopiadae and ATGGTAA in Ch. externa. In addition, there was an overlapped nucleotide ‘A’ between atp6 and cox3 found in the 13 Chrysopidae species.There were overlapping regions of seven nucleotides between the gene pairs ogenomes . All theAn. gracilis were predicted and compared with those of An. octopunctata  arm formed a simple loop. This characteristic occurred frequently in the sequenced mitogenomes of metazoan [The secondary structures of tRNAs of punctata . There wmetazoan .rrnL was located between trnL1 and trnV. The length of rrnL was 1301–1319 bp. The A + T content ranged from 81.64% to 83.85%  and LG + C60 + F (dataset PCGAA), respectively . The topParankylopteryx diverged from Ankylopteryx during the Late Cretaceous . The divergence between An. gracilis and An. octopunctata occurred during the early Paleogene .The divergence time was estimated based on the phylogeny of dataset PCG123RNA and the chronogram was shown in An. gracilis and An. octopunctata were newly sequenced and analyzed in this study. The arrangements of these two mitogenomes were consistent with other Chrysopidae species. The use of start codons of Chrysopidae was relatively simple with the form of ATN. Most PCGs used TAA as stop codons, while some PCGs used TA-tRNA or T-tRNA as stop codons. Incomplete stop codons TA-tRNA and T-tRNA would transform into complete stop codons after transcription in Chrysopidae [The mitogenomes of ysopidae .Concerning the phylogeny of Chrysopidae, Chrysopinae were recovered as the sister group to Apochrysinae + Nothochrysinae. This result had also been confirmed by many studies using molecular evidence, such as nuclear genes, mitogenomes and low-coverage whole genomes ,12,13,16


The results showed that a single-phase Sr2CeZrO6 refractory could be fabricated at 1400 °C for 12 h, and its space group was Pnma with a = 5.9742(3) Å, b = 8.3910(5) Å and c = 5.9069(5) Å. An interaction layer with a 40μm thickness and dense structure could be observed in Sr2CeZrO6 crucible after melting TiAl alloy. Additionally, the interaction mechanism showed that the Sr2CeZrO6 refractory dissolved in the alloy melt, resulting in the generation of Sr3Zr2O7, SrAl2O4 and CeO2−x, which attached to the surface of the crucible.Vacuum induction melting in a refractory crucible is an economical method to produce TiAl-based alloys, aiming to reduce the preparation cost. In this paper, a Sr However2O3 [2 [2O3 [2O3, ZrO2, and Al2O3 [2O3 crucible. In contrast, the absorption of oxygen from the ZrO2 and Al2O3 crucibles occurred at a notably higher level . Koichi’s work presented that the oxygen concentration could be controlled at 0.1–0.13 wt.% with a holding smelting time ranging from 5 to 20 min in the CaO crucible [2O3 and CaO had higher stability than that of Al2O3 and ZrO2. Thus, they were more suitable for melting TiAl alloys. However, there are significant challenges that need to be addressed before industrial-scale manufacture. For instance, Y2O3 has an inherent drawback of poor thermal shock resistance, and CaO exhibits a hygroscopic nature [Until now, researchers have explored various oxide materials as refractory crucibles for melting TiAl alloys, such as Y2O3 , CaO [122O3 [2  and Al2O [2 [2O3 . However [2 [2O3 . Toshimind Al2O3 . Notablycrucible . From a c nature ,18. Give3 was an attractive candidate material due to its exceptional resistance to corrosion, especially in alkaline melts and vapors [3 structure are very well-known inorganic materials. The A and B sites in perovskite materials possess the capability to accommodate diverse metal cations, providing an avenue to regulate both the chemical compositions and the properties of perovskite materials. Substituting elements on the B sites can lead to the formation of double perovskite A2B′B″O6 compounds, where A represents an alkaline-earth metal, and B′ and B″ represent two different transition metal elements. The positioning of B′ and B″ cations within the crystal structure can vary, either occupying indistinguishable sites or distinct sites, contingent upon their charge and ionic radii. Double perovskites exhibit a wide array of intriguing properties owing to their diverse compositions and structures, prompting extensive investigations into their structural characteristics [2 has a high melting point (nearly 1950 °C). It is typically stable at high temperatures, making it potentially useful in high-temperature applications, such as in high-temperature lubricants or coating materials [2 was introduced into the preparation of the Sr-Zr oxide refractory. It could be considered that the Ce dopant could replace the position of Zr elements in the Sr-Zr oxides. Thus, a double perovskite structure Sr2CeZrO6 refractory was fabricated, and then its interaction with melting TiAl alloys was investigated. Until now, there have not been any prior exploration into utilizing Sr2CeZrO6 refractory for preparing TiAl alloys.Novel alkaline earth zirconate materials, such as Sr-Zr oxides, exhibit the essential properties for melting titanium alloys. For example, SrZrOd vapors . Generaleristics . In ordeeristics . The raraterials . In this2CeZrO6 refractory through a solid-state approach utilizing industrial-grade SrCO3, CeO2 and ZrO2 raw materials. Subsequently, the formation of the Sr2CeZrO6 crucible was achieved via shaping and sintering. Utilizing XRD and SEM, the synthesized powders were analyzed to ascertain the phase composition and microstructure. The structure of Sr2CeZrO6 refractory was investigated through the Rietveld method. Subsequently, TiAl alloy was melted in the Sr2CeZrO6 crucible, facilitating an exploration into the interaction between the TiAl alloy and Sr2CeZrO6 crucible, along with an investigation into the corresponding interaction mechanism.In this paper, firstly, the manufacturing process involved the synthesis of a Sr3 (99.9%), ZrO2 (99.9%) and CeO2 (99.9%) raw materials were employed for the preparation of a SrCeZrO6 refractory according to the solid-state reaction. The XRD  patterns for the raw materials are shown in 3): n(ZrO2): n(CeO2) = 2:1:1. Subsequently, they were subjected to ball milling , followed by drying at 120 °C for 12 h. Then, the discs with φ20 mm × 3 mm were fabricated at 120 MPa, holding for 2 min. Finally, the unfired discs were heat-treated at 1400 °C for 12 h.SrCOhttps://subversion.xray.aps.anl.gov/trac/pyGSAS). Microstructure analysis was carried out using scanning electron microscopy .Prior to the phase analysis, the sintered discs were ground into powders and then sieved through a 400-mesh sieve. XRD was used to analyze the phase structure of the powders through Rietveld refinement via GSAS-II software  and sponge Ti (>99.9%) in a water-cooled copper crucible. In the VIM furnace, the space surrounding the crucible was filled with Al2O3 ramming mass. Then, the master alloy was inserted into the crucible. The furnace chamber was evacuated to 10−3 mbar. The high-purity argon gas was backfilled into the chamber at least three times. This effectively prevented oxygen contamination during the melting. As the molten alloy became visible, high-purity argon gas was reintroduced, and the temperature was slowly raised to 1600 °C and maintained for 5 min. Then, the alloy melt was allowed to cool in the crucible. X-ray diffraction was used to analyze the crucible surface in order to examine the interaction products between them. The interaction behavior was investigated after the fabrication of the samples using a buzz saw. Selected sections underwent examination using a digital microscope (VHX-1000). Interaction analyses for the microstructure were conducted using a scanning electron microscope with an energy dispersive spectrometer (EDS).Our goal was to assess the interface stability between the Sr3, CeO2 and ZrO2 after sintering at 1400 °C for 12 h. The sintered powder exhibited a significant sintering phenomenon, as shown in 2CeZrO6, the XRD pattern of the sintered powder was refined by the Rietveld method. The Rietveld refinement of the XRD data of the powder in the 2θ° angle 10~120° is shown in 2CeZrO6 had a similar peak pattern to SrCeO3 (Pnma 62). Therefore, the XRD data of the Sr2CeZrO6 sample were refined using the orthogonal structure Pnma space group. In this structural model, Sr atoms occupied the 4c position at coordinates , while both Ce and Zr atoms occupied the 4a position  with an equal occupancy of 0.5:0.5. O atoms were positioned at 4c  and 8d . 2CeZrO6. The experimental intensity (depicted by the green dotted line) of the Sr2CeZrO6 coincided with the simulated intensity (shown as the red continuous line). The lattice constants were a = 5.9742(3) Å, b = 8.3910(5) Å, c = 5.9069(5) Å, respectively, with a unit cell volume of 296.11(6) Å3 using the least-squares method. The reliability factors Rwp and GOF stood at 8.94% and 1.57, respectively. This indicated that the Rietveld refinements were conducted at a reasonably high standard. 2CeZrO6.To further confirm the crystal structure of Sr2CeZrO6 crucible surface after sintering at 1700 °C and holding for 4 h is shown in 2CeZrO6 phase could be detected on the crucible surface, confirming the analysis in The SEM picture of the SrThe macroscopic picture of the cooled alloy and the crucible is shown in The macroscopic pictures of the cross-sections of the crucibles before and after melting are shown in 3Zr2O7, CeO2−x and SrAl2O4 phases. This can be elucidated in conjunction with the suggested interaction model outlined below.2CeZrO6 phase. After melting, an interaction layer (~40 μm thickness) and dense structure could be observed in the crucible, as shown in 2O4, resulting in the enrichment of Al elements in the interaction layer. Additionally, there were essentially no Ti elements, which were residual along the crucible sidewall. This indicated that this refractory exhibited a good non-wettability during the alloy melts, consistent with the analysis in The SEM pictures of the cross-section of the crucibles before and after melting are shown in 3 and ZrO2, respectively. However, the coexistence of Ce4+ and Ce3+ was observed in this layer. This indicates that there was non-stoichiometric CeO2 in the interaction layer . Evidently, the interaction between the refractory and the alloy melt caused the phase change in the interaction layer. The detailed interaction mechanism is described next.The detailed electronic states of the constituent elements in the interaction layer were characterized by X-ray photoelectron spectroscopy (XPS) measurements, as shown in 2, TiO, Al2O3, CeO, ZrO2, SrO and Sr3Zr2O7, according to results obtained from the HSC software (Version 6.1). The Gibbs free energy of Sr2CeZrO6 was not confirmed. Because no interaction products, such as TiO2 and TiO, could be detected  and (3) were showed as follows.From 2O3 products. Then, Al2O3 reacted with SrO, resulting in the generation of SrAl2O4. The reaction Equations (4) and (5) are presented below.As the O elements dispersed along the interface between the crucible and the alloy melt, it further combined with the Al elements to generate Al6 refractory was synthesized using the solid-state method. The interaction of the SrCeZrO6 refractory with a TiAl alloy melt was investigated. Conclusions were obtained as follows: (1)2CeZrO6 was successfully synthesized by a solid-state reaction after sintering at 1400 °C for 12 h. The Sr2CeZrO6 phase showed Pnma space group symmetry with a = 5.9742(3) Å, b = 8.3910(5) Å, c = 5.9069(5) Å.The pure compound Sr(2)2CeZrO6 refractory crucible maintained an intact shape after contacting with the TiAl alloy melt, and the thickness of the interaction layer was about 40 μm.The Sr(3)2CeZrO6 refractory decomposed into Sr3Zr2O7, SrAl2O4 and CeO2−x. The products were generated by the dissolution of the Sr2CeZrO6 refractory in the alloy melt, which was the main factor responsible for the interaction mechanism between them. This study provides some theoretical guidance for the development of new refractory materials for melting titanium alloys in the future.The SrIn this study, a novel perovskite SrCeZrO


Solanum tuberosum L. cv. Desiree) tubers under variable circumstances. Starch-interacting proteins were extracted from developing tubers of wild type and transgenic lines containing antisense inhibition of glucan phosphorylases. Further, proteins were separated by SDS-PAGE and characterized through mass spectrometry. Additionally, starch-interacting proteins were analyzed in potato tubers stored at different temperatures. Most of the proteins strongly interacting with the potato starch granules corresponded to proteins involved in starch metabolism. GWD and PWD, two dikinases associated with starch degradation, were consistently found bound to the starch granules. This indicates that their activity is not only restricted to degradation but is also essential during storage starch synthesis. We confirmed the presence of protease inhibitors interacting with the potato starch surface as previously revealed by other authors. Starch interacting protein profiles of transgenic tubers appeared differently from wild type when tubers were stored under different temperatures, indicating a differential expression in response to changing environmental conditions.The complete mechanism behind starch regulation has not been fully characterized. However, significant progress can be achieved through proteomic approaches. In this work, we aimed to characterize the starch-interacting proteins in potato ( Starch is a carbohydrate polymer synthesized by plants and most algae for energy storage. It is naturally produced as a highly ordered and dense packaging of glucan chains, leading to the formation of insoluble granules located mostly in plastids (chloroplasts and amyloplasts) . The mec32 that phosphorylation occurs in de novo synthesis of starch. On the other side, in cassava (Manihot esculenta) GWD repression had a significant effect on both leaf and root starch, and the starch excess generated in leaves led to retarded plant and storage root growth [In this regard, to facilitate starch degradation, the external structure of the starch granules must be shifted from a water-insoluble to a soluble state. This is achieved by the action of Glucan, Water Dikinase (GWD) and Phosphoglucan, Water Dikinase (PWD), two plastidial enzymes that mediate these transitions through cycles of phosphorylation of glucans ,14. Gluct growth . OverallStarch dephosphorylation on native starch granules is achieved by the glucan phosphatases SEX4 (Starch Excess 4) and LSF2 (Like Sex Four 2) ,20. ArabOther proteins that have been found associated with the starch granules in Arabidopsis and potato tubers are Early Starvation Protein 1 (ESV1) and its homologue Like Early Starvation Protein (LESV) ,23,24. BSeveral proteins, with so far unrevealed catalytic activity, are crucial in the different phases of the starch formation and degradation process. Arabidopsis SS5, for instance, participates in the regulation of the morphology and starch granule number in the chloroplasts, although lacking the glycosyltransferase activity associated with the SSs . SimilarPlastidial glucan phoshorylase (PHO1), an enzyme with both synthesis and degradation capabilities, has a strong implication on maltooligosaccharide metabolism and, consequently, on the starch metabolic pathway. It catalyzes the addition of glucosyl units to the non-reducing end of α-1,4 linked glucans using glucose-1-phosphate as substrate. In the reverse reaction, PHO1 generates glucose-1-phosphate using α-1,4 linked glucans and orthophosphate as substrates. Transgenic potato tubers with strong repression of PHO1 notably accumulate maltooligosaccharides with a high degree of polymerization, probably due to partial degradation of starch granules during synthesis  without being degraded by PHO1 . A distiIn this work, we investigated the proteins physically interacting with the potato tuber starch under different conditions. First, to analyze if the phosphorylating enzymes GWD and PWD are present during the synthesis of starch, we isolated and characterized the starch proteome in developing tubers, where the synthesis of starch takes place. In addition, as it was demonstrated that PHO1 has a strong implication on the maltodextrin and starch metabolism in potato tubers, the starch proteome of transgenic potato lines repressing the plastidial or cytosolic glucan phosphorylase was characterized and compared with the proteins interacting with the starch of wild type tubers. Moreover, to evaluate the effect of different environmental conditions on the starch proteome, starch-interacting proteins were analyzed in potato tubers stored under different temperatures, revealing that that the expression of specific starch-related proteins are stimulated.First, we identified all proteins bound to the starch granules isolated from potato tubers. Proteins interacting with the starch granules were isolated from 500 mg of potato tuber starch, concentrated, and separated by SDS-PAGE. To elucidate the nature of the interaction within the starch granules, protein isolation was conducted in two steps. Weakly interacting proteins to starch (WIP), more likely located on the starch surface, were first removed from the starch through successive washes with SDS. Later, strong interacting proteins (SIP) were recovered by mixing the starch samples with protein extraction buffer while disrupting the starch structure by heating at 99 °C. In Interestingly, one peptide was identified by MS/MS corresponding to the plastidial glucan phosphorylase PHO1 and it was the least abundant of the SIP.In To evaluate if changes in the profile of the starch-interacting proteins could be observed, we selected two transgenic potato lines for starch isolation, both sustaining a deficiency in the same catalytic activity but in different cell compartments, and we compared it with the starch-interacting proteins in wild-type tubers. Thus, a transgenic line repressing the glucan phosphorylase PHO1 isozyme, which is expressed in the amyloplast/chloroplast, and a second line repressing the cytosolic phosphorylase PHO2 was used. PHO1 is directly involved in starch and maltodextrin metabolism, whereas PHO2 is involved in starch degradation related maltose metabolism in the cytosol . RepressIn In addition, we detected starch-bound proteins with antibodies raised against specific enzymes involved in starch degradation (GWD and PWD) or initiation (PTST2) in the three different genotypes. GWD and PWD were not removable from starch by SDS washes and were exclusively found as SIP . PTST2, As shown, PHO1 was detected as SIP, but not as WIP. However, having as antecedent that PHO1 plays an active role on the starch metabolism in potato and rice plants growing at low temperatures, we assayed the isolation of the starch-interacting proteins after storage of wild type and PHO1-repressed tubers under different temperatures. Right after teir harvest, tubers derived from the same plant were stored at room temperature, warm (37 °C), or cold (4 °C) conditions for 15 days in the dark and starch proteins were later extracted. In the protein fraction corresponding to the SIP, no differences in the protein band patterns were observed. However, as it can be observed in the SDS-PAGE gel image of WIP , dispariIn this work, we explored the starch-interacting proteins in potato starch under different conditions. We classified the starch proteins in two different fractions based on the nature of the interaction considering the way we isolated them from starch. One fraction contained the proteins that were removable from starch by SDS and thus, more likely found loosely interacting with the starch surface, defined in this work as weak interacting proteins (WIP). Strong interacting protein fraction (SIP) was designated to the proteins which were able to recover only after breaking down the granular structure of the starch by heat-induced gelatinization. All identified proteins from the SIP were described as involved in starch metabolism. The protein profile of the SIP observed in the separation gel mainly resembles that obtained by . GBSS waFinding these phosphorylating dikinases strongly interacting with potato starch is not surprising, as it was previously demonstrated that phosphorylation occurs along the whole storage starch biosynthesis and accumulation in potato tubers . HoweverRegarding the WIP proteins, the protein amount that was possible to isolate from starch was considerably lower than that recovered from the SIP fraction (around 8 μg against ~30 μg of SIP), and less protein bands were detected after gel electrophoresis and staining . Of the A. thaliana. Proteins were isolated from potato wild-type starch and transgenic PHO1-repressed and PHO2-repressed potato starch samples. A 100 kDa protein band was detected in the starch surface protein fraction , a chloroplastic protein involved in starch granule initiation , was alsIn a second approach seeking for differences on the starch-interacting proteins, we stored PHO1-repressed and wild-type tubers under different temperature conditions. By these means, different patterns of WIP protein bands were visualized between the samples stored at room temperature, 4 °C, or 37 °C, either in wild-type or PHO1-repressed tubers. Differences were basically observed in the loss/detection of four bands having an apparent mass of ~ 130, ~100, ~50, and ~45 kDa. Unfortunately, due to the low protein concentration of these bands, attempts to identify them were unsuccessful so far. Noteworthy, a nearly 130 kDa protein was detected in the PHO1-repressed protein sample obtained from potato tubers stored at 4 °C, which is close to the molecular weight of PWD. Under such cold storing conditions, the storage starch is more likely under the so called cold-induced sweetening phenomenon, thus the degradation of starch must occur to self-supply reducing sugars. In any case, this band was not detected in wild type tubers stored at 4 °C and a relationship to be attributed to the missing PHO1 activity in the transgenic tuber triggering the expression of PWD is hard to establish.A. thaliana and N. sylvestris, and [Considering the findings presented by  who reporis, and , who recAttempts to further investigate the starch proteome under variable environments or simulated stress conditions would be supportive in revealing inducing mechanisms for specific starch-related enzymes and understanding their implication under specific conditions. Like we observed in this assay, expression of different starch-interacting proteins was stimulated by storage of tubers in different temperatures.The proteins identified in this study that were strongly interacting with the potato starch granules corresponded to previously described proteins having a specific role in the starch metabolism. Remarkably, GWD and PWD, despite being enzymes commonly associated with the starch degradation, were found to interact strongly with the starch granules in developing tubers. We assume that the activity of GWD and PWD is not exclusive to the starch breakdown but is also required during the synthesis of storage starch. More likely, these two dikinases play a significant role in the regulation of the starch granule architecture by facilitating the cleavage of glucosyl residues by other enzymes. However, the specific role and relevance of these enzymes during starch synthesis must still be investigated.Furthermore, we demonstrated that the starch proteome profile can be altered during the storage of potato tubers under different temperature conditions. This assay can serve as a milestone to further investigate the starch proteome under different scenarios. This might be helpful, for instance, in the identification of new proteins or revealing which specific starch-related enzymes are preferentially expressed under particular environmental or stress conditions.Solanum tuberosum L. cv. Desiree), and transgenic lines containing antisense constructs against the plastidial phosphorylase isozymes (PHO1a + b), and the cytosolic phosphorylase isozyme (PHO2) were grown under controlled conditions  in a grow chamber. Potato tubers were harvested after 3 months and immediately used for starch isolation, except where otherwise stated.Potato wild-type plants  SDS per each 100 mg native starch and recovering the supernatant following vortex and centrifugation . The SDS extraction was repeated twice, and the collected supernatants were pooled in a single tube. Later, starch samples were washed twice with 1 mL distilled water to remove the remaining SDS. Then, proteins which interacted strongly with the starch granules (SIP) were extracted by adding 1 mL of protein extraction buffer  SDS, 20% (w/v) glycerol, 50 mM DTE, pH 6.8) [g for 10 min and the supernatant was collected.For protein extraction 500 mg starch was used. Proteins weakly interacting to the starch granule (WIP) were extracted by adding 200 µL 2%  for protein concentration following the supplier manual instructions. Following centrifugation, the volume retained in the filter (nearly 200 μL) was recovered and transferred into a 2 mL Eppendorf tube for acetone precipitation. Protein samples were mixed with four volumes of −20 °C cold acetone, vortexed, and kept in −20 °C for at least 1 h. Then, samples were centrifuged at 20,000× g for 5 min. The protein samples were pelleted, and the acetone discarded. Tubes were left uncovered for 10 min for drying.The two protein fractions containing the WIP and SIP were separately transferred to 10 kDa filter  following the supplier protocol. The concentration of protein samples was estimated by comparing with a standard curve generated with known concentrations of Bovine Serum Albumin (BSA). Absorbance was measured at 562 nm as indicated in the supplier manual.®®-Roth, Karlsruhe, Germany).Starch bound proteins were separated through sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) using polyacrylamide concentrations between 7.5 and 10% in the separation gel. The polyacrylamide concentration used in the stacking gel was always 3%. Electrophoresis was performed at 180 V and 40 mA. Following electrophoresis, gels were stained overnight with a commercial Coomassie-blue staining solution (Roti BlueThe relative intensity of the protein bands detected by Coomassie staining of SDS-PAGE gels was quantified using the image processing program ImageJ 1.51d.4HCO3/acetonitrile (1:1) for 15 min with a change of distaining solution in between. Later, the gel pieces were dehydrated in 50 µL acetonitrile for 5 min. The acetonitrile was discarded, and gel pieces were dried in vacuum. The gel pieces were rehydrated with 15 µL of pre-cooled trypsin solution (20–30 ng/µL trypsin sequencing grade reconstituted in 50 mM NH4HCO3) and incubated on ice for 15 min. Trypsin digestion was performed overnight at 37 °C. For peptide recovering, gel samples were rehydrated with 20 µL distilled water. Later, 20 µL acetonitrile was added and recovered after 5 min. Later, 20 µL of 5% (v/v) formic acid was added to the gel pieces, incubated for 15 min, and recovered. Finally, a second acetonitrile extraction was performed. Peptide samples were completely dried in a vacuum concentrator. The peptide pellet was resuspended in 10 µL of 20% (v/v) acetonitrile dissolved in 0.1% (v/v) trifluoracetic acid (TFA).SDS-PAGE protein bands were prepared for peptide mass fingerprint (PMF) analysis following the protocol described by  with somv/v) acetonitrile, 13% (v/v) EtOH, and 3% (v/v) trifluoroacetic acid 0.1% (v/v). Peptide mass spectra (m/z 500–4000 Da) were acquired as positive ions using the reflector mode. The peptide mass values were matched against NCBI and Swissprot databases listed in the MASCOT Peptide Mass Fingerprint and MASCOT MS/MS search platforms . Trypsin was selected as a digesting enzyme and the peptide tolerance set to 0.6 Da. One or no missed cleavage was allowed for the search and no fixed or variable modifications selected. For MS/MS ion search, the peptide charge was set to +1 and the MS/MS tolerance to 0.6 Da. Results with a MASCOT score above the threshold (p < 0.05) were considered significant.Peptides were analyzed through mass spectrometry using a MALDI-TOF system  and MS/MS MALDI-LTQ XL . A 0.35 µL peptide sample was spotted on the target plate and covered with the same volume of α-cyano-4-hydroxycinnamic acid matrix (HCCA) prepared as follows: 3.5 mg HCCA dissolved in 1 mL 84% (v/v) MeOH). Blotting was performed at 12 V for 12 h at room temperature.The transfer of proteins from SDS-PAGE gels to nitrocellulose membranes was carried out by placing two Whatman filter papers, the membrane, the protein gel, and two Whatman filter papers in a blotting device  following the manual instructions. All components were previously wetted with transfer buffer  Tween 20) containing 3% (w/v) milk powder and then with primary antibodies  diluted 1:1000 (v/v) in blocking solution. After one hour, the primary antibody solution was removed and the membranes were washed for five minutes with TBST buffer  Tween 20). This washing step was repeated six times. Later, blot membranes were incubated with secondary antibody ) for one hour. The membranes were washed again with TBST buffer as mentioned. To visualize alkaline phosphatase activity, BCIP®®/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) reagent (Sigma-Aldrich) was used following the manual instructions. For HRP conjugated antibodies, signal was detected using the Supersignal West Pico PLUS Chemiluminescent Substrate kit  following the manual instructions.Blots were incubated for one hour in blocking TBST solution (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (


The aim of this study was to analyze real-world practice patterns and graft survival after corneal transplantation for infectious keratitis in the Netherlands.All consecutive keratoplasties for infectious keratitis registered in the Netherlands Organ Transplant Registry were included. Graft survival was analyzed using Kaplan–Meier survival curves with Cox regression to compare the 3 most common pathogens with subgroup analysis for type and reason of transplantation, sex, and graft size. Multivariable analysis was performed using the same explanatory factors.Acanthamoeba (n = 121). Human leukocyte antigen (HLA) matching did not provide a significant survival benefit, whereas emergency procedures showed worse graft survival . Graft size >8.5 mm was significantly worse than graft size 8.5 mm . In therapeutic keratoplasty, graft survival was significantly worse for Acanthamoeba than viral keratitis . In the multivariable model, adjusting for graft size, type, and reason for transplantation, viral and bacterial keratitis did not differ significantly in graft survival, and Acanthamoeba showed a significantly worse prognosis .Between 2007 and 2017, 1111 keratoplasties for infectious keratitis were registered in the Netherlands Organ Transplant Registry. The most common pathogens were viruses (n = 437), bacteria (n = 271), and Acanthamoeba keratitis. HLA matching did not offer protection over elective non-HLA–matched procedures, whereas emergency procedures and grafts sized >8.5 mm showed poor survival. In optical keratoplasty, survival is high for all pathogens, whereas in therapeutic keratoplasty Acanthamoeba shows poor outcome.Viral keratitis was the most common indication for transplantation, followed by bacterial and  Donor corneas are centrally allocated and registered in the NOTR in the Netherlands. Using the NOTR, the NTS prospectively captures data related to the recipient, donor, eye bank processing, and surgical procedure of all corneal transplantation performed in the Netherlands. Corneal surgeons completed relevant follow-up data at predefined time points using a standardized electronic data capture system. Data collection was continued until graft failure or loss to follow-up. For this study, the NOTR Steering Group provided institutional review board approval for data extraction and analysis. Informed consent was obtained from all patients to participate in the registry and to use the data for research. The study adhered to the tenets of the Declaration of Helsinki and Dutch legislation.Data for this multicenter prospective registry study were obtained from the NOTR, a prospective national database founded by the Netherlands Transplantation Foundation , type of surgery , reason for surgery , and graft size . Cox regression analysis was performed over 5 years or 2 years postoperatively, depending on the number of patients still in the study at that time. The proportional hazard assumption was verified using a log(-log) survival function plot. Two-sided P values ≤0.05 were considered statistically significant.Statistical analyses were performed using the IBM SPSS Statistics for Windows . To avoid dependency between the 2 eyes, repeat transplants were excluded, and for patients who underwent corneal transplantation in both eyes, only the first eye was included in the graft survival analysis. Baseline characteristics are reported as frequencies with percentages or mean ± SD. The number of transplants over time was tested using the χnd, 2007, and December 24th, 2017. Although the volume of corneal transplant procedures has increased in the last decade, the overall number of transplants for infectious keratitis has not changed significantly over time (P = 0.070)  . In this cohort of primary transplants, patients were on average 61 (±18) years, 51% (n = 420) were male, 74% (n = 613) were phakic, and 13% (n = 105) were pseudophakic at the time of surgery . Of the surgeries, 65% (n = 540) were optical keratoplasties, 28% (n = 235) were therapeutic keratoplasties, and 7% (n = 54) was missing. HLA matching was performed in 6% (n = 53) of all cases, 15% of all cases (n = 126) were emergency (non-HLA matched) procedures, and 78% (n = 650) were elective non-HLA–matched procedures. The mean graft size was 8.3 mm, 16% of the grafts was 8.5 mm (n = 131), 58% was smaller than 8.5 mm (n = 481), 21% was larger than 8.5 mm, and 5% (n = 44) was missing. For viral, bacterial, and Acanthamoeba keratitis, 9% (n = 38), 3% (n = 9), and 5% (n = 6) were HLA-matched; 9% (n = 41), 23% (n = 62), and 15% (n = 18) were emergency (non-HLA matched) procedures; and 77% (n = 335), 69% (n = 184), and 76% (n = 92) were elective non-HLA–matched procedures, respectively. A total of 46 repeated transplants were identified. The causative pathogens included viruses , bacteria , Acanthamoeba , and fungi . Compared with the incidence of the causative agents of the first transplant, fungal keratitis had the highest risk of repeat transplantation (15%), followed by Acanthamoeba keratitis (8%), viral keratitis (5%), and bacterial keratitis (2%), P < 0.001. Of repeated transplants, 50% (n = 23) were therapeutic keratoplasties and 50% (n = 23) optical keratoplasties. HLA matching was performed in 11% (n = 5) of repeated transplants, 15% of repeated transplants (n = 7) were emergency procedures, and 74% (n = 34) were elective non-HLA–matched procedures.In total, 1111 corneal transplant procedures for infectious keratitis were registered in the NOTR between January 2Acanthamoeba keratitis   was 93%, 94%, and 76% at 1 year; 89%, 89%, and 67% at 2 years; 86%, 85%, and 56% at 3 years; and 83%, 79%, and 55% at 5 years, respectively. The outcomes of corneal transplantation for fungal keratitis were analyzed separately due to the low number of cases in our cohort (n = 47) and the high censoring rate (45% of cases available at 1 year). The graft survival for fungal keratitis was 90% (n = 26) at 1 year, 83% (n = 18) at 2 years, 83% (n = 13) at 3 years, and 71% (n = 4) at 5 years.A total of 784 procedures were available for graft survival analysis. Loss to follow-up was registered in 45 cases (5%). The overall graft survival rates for primary transplantation and retransplantation were 97% and 88% at 3 months, 95% and 88% at 6 months, 91% and 84% at 1 year, 86% and 75% at 2 years, and 82% and 70% at 3 years, respectively . Emergency procedures showed significantly worse 5-year graft survival compared with elective non-HLA–matched procedures . The graft survival rates for optical (n = 511) and therapeutic keratoplasty (n = 224) were 96% and 78% at 1 year; 92% and 72% at 2 years; 82% and 65% at 3 years; and 88% and 59% at 5 years, respectively , emergency procedures (n = 121), and elective non-HLA–matched procedures (n = 611) was 100%, 79%, and 92% at 1 year; 98%, 70%, and 88% at 2 years; 91%, 62%, and 85% at 3 years; and 90%, 59%, and 81% at 5 years, respectively  and donor sex . Graft survival rates for sex-matched (n = 381) and sex-mismatched (n = 403) cases were 90% and 91% at 1 year; 88% and 86% at 2 years; 83% and 84% at 3 years; and 82% and 83% at 5 years, respectively .No significant differences in 5-year graft survival were observed for both recipient sex . However, grafts sized >8.5 mm showed significantly worse 5-year graft survival compared with grafts sized 8.5 mm . In 44 cases, the graft size was missing.Graft survival for transplants with a graft size of 8.5 mm n = 123), <8.5 mm (n = 450), and >8.5 mm (n = 167) was 90%, 94%, and 81% at 1 year; 87%, 90%, and 75% at 2 years; 84%, 88%, and 69% at 3 years; and 83%, 87%, and 65% at 5 years, respectively  and bacterial keratitis   and bacterial keratitis    and bacterial keratitis   Fig. C. For thAcanthamoeba keratitis were the most common indications. Most transplants were performed for visual rehabilitation , and a minority for debulking and preserving globe integrity (therapeutic keratoplasty). Two-year graft survival was 72% for therapeutic keratoplasty and was comparable between viral and bacterial keratitis but substantially lower in Acanthamoeba keratitis, even after correction for type, reason for surgery, and graft size.This registry study demonstrated the practice patterns and outcomes of corneal transplantation for infectious keratitis in the Netherlands. Although the volume of corneal transplantation has increased substantially in the last decade due to endothelial keratoplasty, the number of corneal transplants for infectious keratitis has remained stable. Viral, bacterial, and 8 However, we do not know what the outcomes would have been, should these high-risk patients have received non-HLA–matched grafts. Nonetheless, it is unlikely that a randomized controlled trial would be conducted in high-risk patients with infectious keratitis, and the registry data may be as close as possible to answer this question. HLA matching in the Netherlands is performed at the discretion of the physician, taking into consideration the characteristics of the recipient and the delay associated with finding a matching donor. Because the use of HLA matching has become rare (<3% according to the EEBA 2008 report), our national registry provides a unique opportunity to explore this topic. A comparison of causative agents within emergency procedures revealed that viral and bacterial keratitis showed promising results, even in so-called high-risk procedures, whereas Acanthamoeba showed significantly worse outcomes.Infectious keratitis may rapidly deteriorate, necessitating corneal transplantation in an emergency setting, making it a high-risk therapeutic procedure. In our cohort, emergency procedures resulted in significantly worse graft survival rates. Although information on donor parameters for emergency transplantation is available in the registry, because of the relatively small number of cases compared with nonemergent cases, a comparison can be misleading and was therefore not included. The value of matching HLA subtypes for corneal transplantation remains controversial, and there are little data in the context of infectious keratitis. In the current study, HLA-matched grafts did not provide a significant survival benefit compared with elective non-HLA–matched procedures. Similarly, a recent randomized controlled trial on penetrating keratoplasty for any indication did not find a survival benefit of HLA matching.Acanthamoeba keratitis carried a higher risk of repeat transplantation than viral and bacterial keratitis. Half of the cases involved therapeutic procedures, indicating that the primary graft was insufficient for disease management or did not yield the desired outcome. The Australian Graft Registry reported that a consecutive graft has a lower survival rate than the previous graft.9 This trend is confirmed in this study for infectious keratitis and shows the burden on patients and health care.Fungal and 13 Against the background of penetrating keratoplasty for infectious keratitis, we failed to detect a significant effect for donor–recipient matching.A much-debated question is the impact of donor–recipient sex matching on graft survival. Previous studies reporting on sex matching in the context of both endothelial keratoplasty and penetrating keratoplasty have shown inconsistent results.16 but not all studies found a significant association.17 Lower graft survival of larger grafts in our cohort may be related to a higher antigenic load in the proximity of the limbus increasing the risk of rejection and a more severe infection requiring debulking.Large-diameter corneal grafts have been previously described as a risk factor for graft failure,18 in particular herpes simplex keratitis which affects 1.5 million people globally.20 In our study, 2-year graft survival for therapeutic viral keratitis measured 77% which is high compared with studies reporting 55%–75% at 2 years.23 The cause of failure is manifold, for instance, in herpes simplex keratitis, stromal keratitis is associated with deep vascularization which increases rejection risk,24 and disease recurrence is common after nerves regenerate in the graft. Moreover, herpes keratitis is accompanied by hypoesthesia, which facilitates recurrent trauma and exposure keratopathy.25 The relatively high graft survival in our cohort may be related to standard use of oral prophylaxis to prevent herpes recurrence in the Netherlands.26In most parts of the developed world, viral keratitis is the most common cause of unilateral infectious corneal blindness,27 Although promising, the US Center for Disease Control estimated that annually 2.8 million people are infected with drug-resistant microbes and resistance is increasing.28 In our cohort, the number of keratoplasties varies over time but does not follow a directional trend. Two-year graft survival of therapeutic keratoplasty for bacterial keratitis was 81%, slightly lower than that reported in previous studies (over 90%).30In bacterial keratitis, the introduction of newer and more potent antibacterial agents led to a decrease in the number of therapeutic keratoplasties in some centers.Acanthamoeba keratitis accounted for 14% of all indications and increased from a handful of cases before 2009 to double digits since 2015. This increase may be due to the use of silicone hydrogel contact lenses,31 multipurpose solutions,32 or local environmental factors.33 In line with the literature, corneal grafting for Acanthamoeba keratitis showed poor survival outcomes.36 Importantly, Acanthamoeba keratitis showed the worst outcomes of all causative agents even when outcomes were adjusted for type of procedure , reason for grafting , and graft size. Age was omitted from the multivariable analysis because Acanthamoeba predominantly affects younger individuals. Since the introduction of biguanides for medical therapy, most cases are treated medically and therapeutic keratoplasty is traditionally reserved for cases of insufficient response to medical therapy or severe complications.37 A much debated question is the value of early therapeutic deep anterior lamellar keratoplasty in Acanthamoeba keratitis.38 However, at the moment, this is not standard of care in the Netherlands, as a result of which there were too few cases available for separate analyses.In our cohort, the volume of corneal transplantation for 39 The disease accounts for a significant burden of blindness,39 causing nearly 5 times as many corneal perforations as bacterial keratitis.40 In our cohort, graft survival for fungal keratitis was 83% 2 years postoperatively, mirroring the outcomes of transplants performed for viral and bacterial keratitis, and comparable to reports in the literature.41Fungal keratitis has a global incidence of more than 1 million cases each year.Central graft allocation by the NTS ensured the robustness of our primary outcome measure: graft survival. Nonetheless, our study has the following limitations. The NOTR database is not designed to specifically capture differences in virulence, predisposing factors, extensiveness of keratitis, and ocular surface inflammation. Moreover, to reduce the registration burden, not all parameters are mandatory, for example, corneal neovascularization, sutures, and use of topical and systemic immunosuppressive therapy. The time between disease onset and transplantation is also an interesting factor that was unavailable in this study. However, therapeutic and emergency procedures may be used as surrogates.Acanthamoeba. Viral keratitis was the most common indication, followed by bacterial and Acanthamoeba keratitis. Graft survival was promising, except in Acanthamoeba keratitis and grafts larger than 8.5 mm. The benefit of HLA matching is unclear, but our data suggest that it may not provide additional survival benefits. Timely therapeutic keratoplasty should be considered as needed given the relatively successful outcomes in our study, especially for viral and bacterial keratitis.In conclusion, this prospective Dutch registry study shows that the volume of corneal transplantation for infectious keratitis has remained stable over the last decade, with the exception of The Netherlands Corneal Transplantation Network (excluding writing committee): Lies Remeijer, MD, PhD; Jeroen van Rooij, MD; Robert H.J. Wijdh, MD; Annette J.M. Geerards, MD; Cathrien A. Eggink, MD, PhD; Michel J.W. Zaal, MD, PhD; Carla P. Nieuwendaal, MD; Remco Stoutenbeek, MD, PhD; Tom A. van Goor, MD; Marjolijn C. Bartels, MD, PhD; Bart T.H. van Dooren, MD, PhD; Ype P. Henry, MD; Siamak Nobacht, MD; Mei L. Tang, MD; Robert P.L. Wisse, MD, PhD; Ivanka J. van der Meulen, MD, PhD; Ruth Lapid-Gortzak, MD, PhD; Chantal M. van Luijk, MD; Nathalie T.Y. Santana, MD, PhD; Isabelle Saelens, MD, PhD; Yanny Y. Cheng, MD, PhD; Sacha Gast, MD; Annemiek Rijneveld, MD, PhD; Allegonda van der Lelij, MD, PhD; Michel Zaal, MD, PhD; Nayyirih de Koning Tahzib, MD, PhD; Cesar Sterk, MD; Martine Jager, MD, PhD; Gabriel van Rij, MD, PhD; Mario Dhooge, MD; Hugo van Cleynenbreugel, MD, PhD; Isabel Bleyen, MD, PhD; and Benedicte Putting MD, PhD.