To assess the shear bond strength (SBS) between metal orthodontic brackets and zirconia after receiving different mechanical and chemical surface treatments, and different types of resin adhesive. The failure mode of each treatment protocol was also evaluated.The present in vitro experimental study consisted of six surface treatment protocols with two different resin adhesives. One-hundred and forty-four rectangular-shaped 3 mol% yttrium-stabilized tetragonal zirconia polycrystal blocks were milled, sintered, and embedded in acrylic resin. They were randomly divided into three mechanical  and two chemical surface treatment conditions (no primer and Z-primer). The specimens were divided into two groups according to the resin adhesive received: self-cured (RelyX U200) and light-cured adhesives (Transbond XT). The SBS between the metal bracket and zirconia was tested using a universal testing machine (1-mm/min crosshead speed), and the failure mode was evaluated. Differences in SBS and failure mode were analyzed using Welch ANOVA followed by post-hoc comparison and Fisher’s Exact test, respectively.p < 0.001). Without primer application, RelyX U200 provided a higher SBS than Transbond XT (p < 0.001). After grinding, using Z-primer and RelyX U200 resulted in a higher SBS than no primer and using Transbond XT (p < 0.001). Adhesive failure at the zirconia–adhesive interface occurred only when Transbond XT was applied without bur grinding, and when using Transbond XT after grinding, but no Z-primer application.Bur grinding produced the highest SBS, followed by air abrasion. Z-primer application typically provided a higher SBS regardless of resin adhesive used (Bur grinding combined with applying an MDP-containing primer and resin adhesive enhances the SBS between zirconia and metal orthodontic brackets. All-ceramic materials become more popular in fixed dental prosthodontics because they provide a high esthetic appearance with optimal strength , 2. MonoBecause the demand for orthodontic treatment in adults has increased, the orthodontic brackets frequently need to be bonded to enamel and different types of ceramic restorations, including zirconia , 6. HoweThe factors affecting the adhesion of an orthodontic bracket to zirconia includes the bracket materials , 10 and Therefore, the objective of this study was to assess the shear bond strength (SBS) between metal orthodontic brackets and zirconia after receiving different mechanical and chemical surface treatments, as well as different types of resin adhesive. The failure mode of each condition was also evaluated. The null hypothesis was that neither mechanical and chemical surface treatment nor resin adhesive type would have an impact on the SBS between metal orthodontic brackets and zirconia.t-test and accounting for a 20% failure rate during specimen preparation, a minimum of 12 samples per group were required to achieve 90% power and a 5% type I error.The present in vitro experimental study consisted of six surface treatment protocols with two different resin adhesives. Table 3 blocks using a low-speed saw . The blocks were polished with #800 and #1200 grit sandpaper attached to a polishing machine  at a polishing speed of 250 rpm with a 50-N force for 1 min. After sintering in a high-temperature furnace  at 1,500 oC for 2 h, the sintered YTZP blocks were embedded in dental gypsum  in a polyvinyl chloride pipe. The specimen’s surface was re-polished with #800 and #1200 sandpaper attached to a polishing machine at a polishing speed of 250 rpm for 1 min. The surface roughness of the specimens was measured using a contact profilometer  to ensure that the baseline surface roughness of the specimens was similar.A total of 144 rectangular pre-sintered 3 mol% yttrium-stabilized tetragonal zirconia polycrystal (YTZP) blocks  were milled into 8 × 8 × 2 mmTM Plus) was applied onto the specimen’s surface using a microbrush, which was changed for each specimen, and dried with an air syringe for 5 sec [The prepared specimens were randomly divided into three mechanical  and two chemical surface treatment conditions (no primer and primer application). In the air abrasion protocol, the YTZP surface was sandblasted with 50-µm aluminum oxide particles at a 10-mm distance from the surface using a sandblasting machine  at 0.25-MPa pressure for 15 sec, perpendicular to the specimen’s surface. To grind the YTZP surface, a standard double cone-shaped diamond bur  was aligned with a flat surface parallel to the specimen surface  according to the adhesive type received; light-cured (TransbondTM XT) and self-cured  resin adhesives. The resin cement was mixed according to the manufacturer’s instructions and applied on the specimen’s surface. The metal bracket was adhered to the zirconia using a 1-kg static load with the metal bracket surface perpendicular to the applied force. After removing the excess cement with a microbrush, the bonded specimens were light-cured with a light-curing unit  on four sides of the specimens for 20 sec on each side. The specimens were stored in distilled water at 37oC in an incubator for 24 h prior to the SBS test.After the surface treatment, the specimens in each group were randomly divided into 2 groups (2).The SBS was determined using a universal testing machine  according to a protocol modified from ISO 29022:2013 [The bonded surfaces of the zirconia and bracket sides were examined using a stereomicroscope at 45x magnification to determine the mode of failure, which was categorized into 4 types:  adhesiveThe data were analyzed using SPSS version 28 statistical software  at a 5% significance level. Descriptive statistics were performed to determine the mean and standard deviation (SD) values. The Kolmogorov-Smirnov test was used to determine the normality of the data distribution. Welch-analysis of variance (ANOVA) and the Games-Howell post-hoc comparison test were used to analyze the differences in the SBS, and Fisher’s Exacts test was used to evaluate the differences in the mode of failure between the tested groups.p < 0.001). Mechanical surface treatment by grinding produced the highest SBS, followed by air abrasion. Z-primer application typically resulted in a higher SBS regardless of the resin adhesive used (p < 0.001). When the primer was not applied, RelyX U200 resin adhesive resulted in a significantly higher SBS than Transbond XT (p < 0.001). In contrast, there was no significant difference in the SBS between the two resin adhesives when the Z-primer was applied , except for the higher SBS of RelyX U200 when the specimens were subjected to grinding (p < 0.001). After grinding, using Z-primer and RelyX U200 resulted in a higher SBS compared with no primer application and using Transbond XT (p < 0.001).The SBS of the surface treatment groups is presented in Table After loading, 100% adhesive failure at the zirconia–adhesive interface occurred when Transbond XT was applied without bur grinding, and when Transbond XT was used after grinding but without Z-primer application Fig. . In contThe present study evaluated the combined effects of chemical and mechanical surface treatment, as well as resin adhesive types on the adhesion between metal brackets and zirconia restoration. Our results indicated that zirconia surface grinding, applying primer, and using RelyX U200 resin adhesive significantly improved the SBS between metal orthodontic bracket and zirconia. Therefore, the null hypothesis was rejected. The failure mode results also supported the quantitative findings, indicating that the groups with a relatively high bond strength demonstrated a combination of adhesive and cohesive failures within the resin adhesive.Our study found that mechanical grinding provided the highest bond strength, followed by air abrasion, which is consistent with a previous study . HoweverThe chemical surface treatment using zirconia primer application demonstrated a positive effect on improving the adhesion of metal bracket to zirconia, as seen in previous in vitro studies , 18. ThiWithout primer application, RelyX U200 resin adhesive achieved better adhesion between the metal brackets and Zirconia compared with Transbond XT. Transbond XT has been specifically recommended as a two-step adhesive for bonding metal or ceramic bracket with tooth structure , whereasBased on our findings, a synergistic effect was observed when combining mechanical and chemical surface treatment with a specific type of resin adhesive. When performing zirconia surface grinding in conjunction with primer application and using RelyX U200, improved adhesion between the metal brackets and zirconia was observed, as supported by the highest bond strength and ARI score. This can be attributed to the increased flow and adaptation between the two layers due to the micromechanical interlocking created through the ground surface. In addition, the MDP in the primer and resin adhesive may create chemical bonds with zirconia restorations. Grinding zirconia can result in increased surface roughness, potentially causing color instability due to color infiltration . This isThe mode of failure results revealed that the cohesive failure within the resin adhesive was the most common failure type when using the primer and RelyX U200, particularly in combination with grinding. Therefore, the adhesion between metal brackets and zirconia can be improved through a combination of mechanical interlocking and chemical bonding created by surface roughening and using an MDP-containing primer and adhesive. Despite a high SBS between the metal bracket and zirconia, the force required for bracket removal is unlikely to result in zirconia breakage. This is because debonding primarily occurs within the resin cement or at the interface between the metal bracket and resin cement.Our findings suggest that selecting the appropriate surface treatment protocol and resin adhesive materials can improve the adhesion of orthodontic brackets to zirconia restorations. However, some limitations are noted in this study. The findings from this in vitro study may provide different results in clinical performance because the oral environment may affect the adhesion. Further clinical studies using a long-term follow-up are suggested to verify the clinical significance of the present findings. Moreover, it is imperative to explore the impact of optimizing bur types for grinding, grinding speed, and the use of water coolant on the bond strength between metal brackets and zirconia restorations.Our findings indicated that mechanical surface treatment using bur grinding, combined with applying an MDP-containing primer and resin adhesive, enhances the SBS between zirconia and metal orthodontic brackets. These combinations also result in a more cohesive failure within resin adhesive, rather than adhesive failure between the two layers.Table 1, Table 2, Table 3


Chronic pressure overload triggers pathological cardiac hypertrophy that eventually leads to heart failure. Effective biomarkers and therapeutic targets for heart failure remain to be defined. The aim of this study is to identify key genes associated with pathological cardiac hypertrophy by combining bioinformatics analyses with molecular biology experiments.Comprehensive bioinformatics tools were used to screen genes related to pressure overload-induced cardiac hypertrophy. We identified differentially expressed genes (DEGs) by overlapping three Gene Expression Omnibus (GEO) datasets . Correlation analysis and BioGPS online tool were used to detect the genes of interest. A mouse model of cardiac remodeling induced by transverse aortic constriction (TAC) was established to verify the expression of the interest gene during cardiac remodeling by RT-PCR and western blot. By using RNA interference technology, the effect of transcription elongation factor A3 (Tcea3) silencing on PE-induced hypertrophy of neonatal rat ventricular myocytes (NRVMs) was detected. Next, gene set enrichment analysis (GSEA) and the online tool ARCHS4 were used to predict the possible signaling pathways, and the fatty acid oxidation relevant pathways were enriched and then verified in NRVMs. Furthermore, the changes of long-chain fatty acid respiration in NRVMs were detected using the Seahorse XFe24 Analyzer. Finally, MitoSOX staining was used to detect the effect of Tcea3 on mitochondrial oxidative stress, and the contents of NADP(H) and GSH/GSSG were detected by relevant kits.in vivo and in vitro. Knockdown of Tcea3 aggravated cardiomyocyte hypertrophy induced by PE in NRVMs. GSEA and online tool ARCHS4 predict Tcea3 involved in fatty acid oxidation (FAO). Subsequently, RT-PCR results showed that knockdown of Tcea3 up-regulated Ces1d and Pla2g5 mRNA expression levels. In PE induced cardiomyocyte hypertrophy, Tcea3 silencing results in decreased fatty acid utilization, decreased ATP synthesis and increased mitochondrial oxidative stress.A total of 95 DEGs were identified and Tcea3 was negatively correlated with Nppa, Nppb and Myh7. The expression level of Tcea3 was downregulated during cardiac remodeling both Our study identifies Tcea3 as a novel anti-cardiac remodeling target by regulating FAO and governing mitochondrial oxidative stress. Heart failure is a rapidly growing public health concern and a leading cause of mortality. Initially, cardiac hypertrophy develops as an adaptive response to persistent pathological stimuli, but it often progresses to fibrosis, dysfunction, and ultimately heart failure. Despite numerous studies on the mechanisms of heart failure, clinical outcomes remain suboptimal and the mortality rate is still high. Therefore, identification of new therapeutic targets is urgently required.Transcription factors are an important type of gene transcription regulatory element. In addition to the translation and transcriptional initiation steps, transcriptional prolongation involving RNA polymerase (Pol) II is now considered to be a key step in controlling full-length mRNA production . It was β-oxidation are shown to possess a vital role within the pathological progression of cardiac remodeling. Inhibition of fatty acid oxidation by CPT1B deficiency had been shown leading to myocardial lipid accumulation and exacerbate cardiac pathological progress , and differentially expressed genes were identified. The volcano plots and heatmaps were created by R software “pheatmap” package . The basic information of the datasets is shown in Supplementary Excel file.Gene expression profiles with series number GSE5500, GSE1621, and GSE36074 were downloaded from the Expression Omnibus Gene (GEO) database. Limma of R/bio-conductor was used for screening of the DEGs  < 0.25 were considered statistically significant.GSEA software V2.1.0 was used to identify the pathway associated with Tcea3 expression, and the expression matrix was 32,619(gene) × 14(sample). Predefined gene set “c2.all.v4.0.symbols” was selected. Enrichment results satisfying a nominal The hearts were obtained from the left ventricles of patients with DCM and non-cardiac deceased donors, which were all not suitable for transplantation. All procedures involving human samples were in accordance with the declaration of Helsinki, and informed consent was obtained from the families of all donors. The review committee of the People's Hospital of Wuhan University approved this study. The source of these donors has previously been identified , 16. TheWe obtained all of our male C57BL/6 mice from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, at 8–10 weeks of age and 23.5–27.5 g of body weight. All animal care and experimental procedures are approved by the Animal Care and Use Committee of Wuhan University People's Hospital . Transverse aortic constriction (TAC) was conducted to achieve overload-induced cardiac hypertrophy and heart failure in mice as previously described . BrieflyBriefly, ventricular cardiomyocytes were isolated from the left ventricles of 1–2-day-old Sprague Dawley rats and digested with Type 2 collagenase (0.5 mg/ml)  and pancreatin (0.6 mg/ml) (Sigma). The Isolated cardiomyocytes were re-suspended in DMEM containing 10%FBS and precultured for 24 h. Then Cells were incubated with 12%FBS containing Phenylephrine  for 48 h to stimulate cardiomyocyte hypertrophy.The sequences for rTcea3 siRNA were as follows: Sense sequence: 5′ GUGCAAGAAGAAGAAUUGUACCUAT 3′, antisense sequence:5′ AUAGGUACAAUUCUUCUUCUUGCACUU 3′. The scrambled and Tcea3 siRNAs were transfected into NRVMs with the RNAiMAX  according to the manual. RNAi duplex-LipofectamineTM RNAiMAX complexes (Contains 5 µl of RNAiMAX) should be added to each well of a 6-well culture vessel. This gives a final volume of 3 ml and a final RNA concentration of 20 nM. Twelve hours after transfection, PE was added to induce cardiomyocyte hypertrophy. Cells were harvested 48 h after transfection and the levels of mRNA were assessed by RT-PCR.After the proteins were extracted from frozen hearts or cultures, they were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred onto polyvinylidene difluoride membranes. A secondary antibody incubation at room temperature for 60 min followed the overnight at 4°C incubation with the primary antibodies against Tcea3. Total RNA was extracted from frozen heart tissue and primary cultured cardiomyocytes, using Trizol Reagent (Invitrogen). Reverse transcription (RT) was carried out with the First Stand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer's protocol. After the cDNA was obtained and diluted to 80ul, SYBR Green chemistry with GAPDH or Tcea3 primers were used to amplify. The primer sequences are shown in 2-free incubator. The sensor probe plate was preloaded with oligomycin, FCCP, and rotenone/antimycin. Finally, the changes of long-chain fatty acid respiration in NRVMs were detected using the Seahorse XFe24 Analyzer (Agilent).Oxygen consumption rate (OCR) were measured using a Seahorse Bioscience XF24 instrument . A density of 80,000 isolated NRVMs per well was seeded into Seahorse XF cell culture microplates. Using Seahorse XF calibration solution, a sensor probe plate was hydrated overnight at 37 °C in CO2-free conditions. Preparation of the mitochondrial stress test kit  and the assay solutions was done in advance. After washing the cells with the FAO assay solution supplemented with 2.5 mM glucose, 0.5 mM carnitine, 500 μl of the assay solution was added to the cells. Following this, we incubated the sample at 37°C for 1 h in a COThe treated cells were incubated with 5 μM MitoSox red mitochondrial superoxide indicator  at 37 °C in the absence of light for 30 min to measure the level of superoxide in the mitochondria. Following washing, the cells were observed using a conventional fluorescence microscope.t test. All statistical analyses were performed using GraphPad Prism 5.0 software. At the 95% confidence level, a P value of < 0.05 is considered to indicate a statistically significant difference.Data are expressed as the mean ± standard error of the mean of at least three independent experiments. In order to determine statistical significance, the results were compared using Student's two-tailed P < 0.05, |fold change| > 0.5). The volcano plots and heatmaps of the differentially expressed genes are displayed in Transaortic constriction (TAC) is performed by ligating the aortic arch of the heart, and the long-term high afterload of the heart causes adaptive changes in the myocardium, leading to cardiac remodeling. To detect the vital genes involved in cardiac remodeling, we employ three gene expression profiles including GSE5500, GSE1621 and GSE36074, both of which were from TAC-induced cardiac hypertrophic mouse and control mouse heart. 95 DEGs in total were identified according to screening criteria  , we selected the GSE36074 dataset to further identify the key genes among these 95 genes closely related with the transition from cardiac hypertrophy to heart failure. As a result, a total of 25 genes were significantly associated with heart failure progression (< 0.001) . Next, t< 0.001) . We furt< 0.001) . As showTo validate Tcea3 expression levels during cardiac hypertrophy, the heart samples from healthy individuals and DCM patients were used. As shown in α-actinin staining  and Tcea3-high (n = 7) groups. GSEA analysis revealed a significant association between Tcea3 and mitochondrial biogenesis, MAPK cascade, fatty acid beta oxidation (https://maayanlab.cloud/archs4/) and GSEA enrichment results, we finally speculate that Tcea3 may be related to fatty acid beta oxidation.To further understand the biological function of Tcea3 in the process of cardiac remodeling, we performed GSEA analysis based on the expression level of Tcea3 to determine the molecular mechanism of Tcea3 affecting the progression of heart failure. According to the median expression level of Tcea3, the 14 TAC samples download from GSE36074 dataset were divided into Tcea3-low  of NRVMs treated with palmitate was significantly upregulated. In the presence of palmitic acid, Tcea3 knockdown in NRVMs resulted in a significant proportional decrease in basal and maximum respiration rate. Spare respiratory capacity and ATP production derived from oxidative phosphorylation was also significantly impaired in response to Tcea3 silencing -E. SinceThe relationship between heart hypertrophy, fatty acid oxidation and mitochondrial ROS has been studied for a long time. We have shown that Tcea3 governs the expression of Ces1d and Pla2g5 involved in FAO. In addition, the results above show that that knockdown of Tcea3 from cardiomyocytes exacerbates cardiomyocyte hypertrophy. Consider the close link between fatty acid oxidation and mitochondrial ROS during cardiac hypertrophy, We next asked whether reduction in oxidative stress is a mechanism underlying Tcea3-mediated protection of cardiac function.+ in PE stimulated group. These findings collectively suggest that Tcea3 is essential for controlling oxidative stress.Firstly, we detected the level of mitochondrial ROS in isolated cardiomyocytes. The immunofluorescence staining showed that PE stimulated the increase of mitochondrial ROS compared with the normal control group, while knockdown of Tcea3 further increased MitoSOX fluorescence signal . Along tIn recent years, with the emergence and rapid development of gene chip (microarray) and high-throughput sequencing technology, it provides a valuable method for the prevention, early diagnosis, treatment and prognosis of diseases including HF. The present study points out the anti-cardiomyocyte hypertrophy effect of Tcea3, which is related to fatty acid metabolism and oxidative stress for the first time.Firstly, 78 up-regulated genes and 17 down-regulated differential expressed genes were obtained through overlap analysis of three datasets. Among these genes, studies have confirmed that some genes are related to myocardial hypertrophy. In addition to Nppb, Nppa, and Myh7, these differentially expressed genes also include some myocardial remodeling-related genes. For example, TGFB2, a multifunctional cytokine that can be involved in myocardial and vascular remodeling, has been proved to be up-regulated in peripheral blood mononuclear cells with cardiovascular structural changes in hypertensive patients, suggesting that TGFB2 monocyte production may play a role in hypertension . SERPINEIn this study, we identified Tcea3 as a potential target gene for anti-myocardial remodeling through bioinformatics analysis. Subsequently, we confirmed that knockdown of Tcea3 inhibited PE-induced cardiomyocyte hypertrophy in NRVMs. We also detected downregulation of Tcea3 expression in dilated cardiomyopathy tissue from humans, indicating the potential clinical application of Tcea3 as an anti-myocardial remodeling agent, which, however, needs to be further confirmed. Mechanistically, by using GSEA single gene enrichment analysis and the ARCHS4 online website, we found that Tcea3 is related to fatty acid degradation. Abnormalities in fatty acid metabolism are widely present in myocardial remodeling, and targeting fatty acid metabolism has become a hot topic in anti-myocardial remodeling therapy –26. InteRecent studies have shown that the process of fatty acid oxidation is accompanied by the production of NADPH –31, whicThere are also some limitations in our analysis. The small sample size may make our candidate genes inconsistent with the real situation. In addition, the function of Tcea3 needs further verification of animal research. In conclusion, we identifies Tcea3 as a novel anti-cardiac remodeling target by regulating FAO and governing mitochondrial oxidative stress. We predict that the role of Tcea3 in myocardial lipid metabolism may provide new insights into the pathogenesis of cardiovascular diseases in clinical settings. The specific mechanism by which Tcea3 regulates myocardial fatty acid oxidation and lipid metabolism, as well as its crosstalk with oxidative stress, are directions for our future research.


Plectranthus edulis) is an annual tuber crop indigenous to Ethiopia. The crop is underutilized and not much studied despite its high yield of starch, which has a good potential to contribute to the effort in meeting the quickly growing demand for starch. In this study, the effects of the ecotype and isolation methods on the physicochemical, functional, structural, and crystalline properties of starches were evaluated. Starches were isolated from two Ethiopian potato ecotypes (Loffo and Chanqua) using distilled water (DW), 0.01% sodium metabisulphite (SMS), and 1M sodium chloride (NaCl) in the isolation media. The results showed that the lowest starch yield was obtained from Chanqua using DW (97.4%), while the maximum was from Loffo using SMS (99.3%). The L* (lightness) and whiteness values of the starches obtained from Loffo were higher than those of Chanqua starches, with NaCl and SMS extractants yielding the highest values. The bulk density, water activity (aw), pH, proximate composition , and techno-functional properties were established. The majority of these parameters varied depending on both the isolation method and the ecotype. The crystallinity pattern of all starches showed B-type diffraction, with differences in diffraction peak intensities between all starches. FTIR tests showed structural changes as a function of the ecotype and isolation procedure used. The Loffo ecotype exhibited considerably better results, and the SMS isolation method was found to be the most effective way to acquire the highest starch quality in most of the characteristics evaluated.The Ethiopian potato ( Starch is the abundant carbohydrate resource in plants which serves as a source of carbon and energy in a variety of natural plants . The gloPlectranthus edulis) (EP) is one of the extensively grown traditional root crops in Ethiopia, with a high starch content that has a promising potential in contributing to the effort for meeting the quickly growing starch demand [Lamiaceae family and the genus plectranthus. It has various local names such as Dincha Oromo, Wolaita donuwa, Gurage dinch, Hadiya dinch, or Agew dinch. It is grown for its edible tubers [The majority of the world’s starch supply comes from corn, potatoes, wheat, cassava, and sweet potatoes . Howeverh demand ,5,6. EP e tubers ,8 in lare tubers ,10. EP he tubers , making w/v) method was significantly the highest  compared with the sodium chloride (NaCl) and distilled water (DW) methods, with different concentrations from the anchote tuber. According to Xu et al. [The application of starches from underutilized tubers like EP requires an in-depth characterization of their physicochemical properties and the selection of a suitable starch extraction method. Studies have shown that variations in the tuber variety or ecotype ,13,14 anu et al. , SMS andIn this study, the physicochemical, functional, and structural properties of starch of EP from different EP ecotypes were assessed using the NaCl, SMS, and DW isolation methods. The results of this study will help to choose the most suitable EP ecotype for starch extraction among two of the most mainly produced in Ethiopia and to identify the most appropriate solvent to carry out its isolation process.p ≤ 0.05) due to differences in the EP ecotype and starch isolation method (The starch yield ranged from 97.39% to 99.26% and varied significantly (n method . The stap ≤ 0.05) on the bulk density (BD), water activity (aw), pH, and color of the starches obtained  to 0.85 g/cm3 . Starches obtained from Loffo had significantly higher (p ≤ 0.05) BDs than those of Chanqua. The variation in BD could be due to the variation in the particle size of starch granules, the compaction profile, and the particle packing arrangement [3) > SMS (0.81 g/cm3) > DW (0.76 g/cm3). The BD of EP starch was higher than that of the anchote (0.51 g/cm3) and potato (0.71 g/cm3) starches reported by Tessema and Admassu, regardless of the ecotype and the solvent used in the isolation process [w values ranged from 0.38 (LNaCl) to 0.61 (CDW), which is comparable with the aw of the native cassava starch (0.5) reported by Ocieczek et al. [w of the starch isolates from Loffo had lower values than Chanqua. The mean aw scores of the starch isolated from the three isolation methods also varied significantly (p < 0.05), following the order: NaCl (0.40) ˂ SMS (0.51) ˂ DW (0.57). The lower aw value found in starch isolated with NaCl can be attributed to the known decreasing power of this salt on the vapor pressure and water activity of the unbound/available water of the starch, in which it remains dissolved [The EP ecotype and starch isolation method had a significant effect  between all color parameters due to the EP ecotypes and isolation methods (p < 0.05) affected L* and whiteness, and the average values of these two parameters respectively varied as: DW  < NaCl  < SMS . The lowest L* and whiteness values were obtained in DW-isolated starches, which could be due to the expected browning reaction during the isolation procedure [p ≤ 0.05) increase in L* and whiteness values for the same EP ecotypes. Different studies mentioned that SMS and NaCl could decrease the browning reactions of tissue during starch isolation and, as a result, could give whiter starches [p < 0.05) in all color parameters. Such difference in the whiteness and lightness of the starch extracts could be attributed to the presence of some co-pigmented substances, and variations in their ash and protein contents [p ≤ 0.05) a* and b* values.Whiteness is one of the physical properties of starch that is important in determining starch quality ,20. Ther methods . The ligrocedure . The SMSstarches . The stacontents ,17,20. Tp ≤ 0.05) in their moisture, protein, fiber, ash, and carbohydrate contents (p < 0.05) effect on all the compositional parameters measured. Except for the fat content, starches isolated from the two EP ecotypes had significant differences   and was p ≤ 0.05) protein content. This could be explained by the fact that NaCl eliminated the protein component by dissolving the starch–protein agglomerates and caused the removal of floating proteins during isolation [The protein content ranged between 0.65% (CSMS) and 0.95% (LDW), which is more or less in the range reported by Hellemans et al. . The measolation ,20. The solation  The values of the fiber contents of the isolated starches ranged between 0.54 (LDW) and 0.89% (CSMS), which were below the values reported by Hellemans et al.   of EP starches are given in −1, attributed to O-H stretching, and bands between 2929 and 2935 cm−1 that show the symmetric/asymmetric stretching modes of the C-H, which agreed with previous reports [−1  effect on the starch water absorption capacity (WAC) and oil absorption capacity (OAC), while the isolation methods had a significant effect on all four of the functional properties evaluated  using a laboratory-grade blender  until a smooth slurry was formed. Then, the slurries were filtered with double-layered cheesecloth and centrifuged  for 20 min at 5000× g and 20 °C. The supernatant was decanted, and the starch settled at the bottom of the centrifuge tube was washed with distilled water to remove impurities. Finally, the starch was allowed to dry overnight at room temperature, ground with a mortar and pestle into fine powder, and packed in polyethylene bags for further investigations.Material preparation was conducted following the method described by Hellemnas et al. . Fresh aThe starch yield was determined, as described in Awolu et al. , by calcThe starch bulk density (BD) was determined following the method described in Stasiak et al. . A totalThe color measurements of the starches were carried out using a Color Measuring System . L*, a*, and b* coordinates were obtained with the D65 standard illuminant and 10° standard observer. The whiteness of the starches was determined as per the method described by Kale et al. , where tThe moisture, protein, fat, crude fiber, and total ash contents of the extracted EP starches were determined according to method given by AACC, 2001 . The carg for 15 min, and the supernatant was discarded. The tube was allowed to drain for 10 min at a 45° angle. Subsequently, the sample tube was weighed, and the gain in weight was used to calculate the WAC, which was expressed as g H2O/g starch.The water absorption capacity (WAC) was measured according to the method described in Surendra Babu & Parimalavalli . A total1) was immersed in 10 mL of soybean oil at room temperature, shaken to mix well, and then left for 30 min to reach the maximal absorption. Then, the mixture was centrifuged at 1811× g for 30 min, and the clear supernatant was decanted. Additionally, the sediment was weighed (m2). Finally, the OAC was calculated using Equation (2):The oil absorption capacity (OAC) was determined according to the method used by Bikila et al. . A totalg for 15 min. The supernatant was drawn off by suction and dried in an oven at 120 °C for 4 h. The solutes that remained after evaporation were weighed, and the solubility, expressed as a percentage, was calculated using Equation (3). The SP was calculated as the weight of the sediment paste per gram of starch, as stated in Equation (4).The starch swelling power (SP) and solubility were studied by the method used by Shimels et al.  and KaurThe crystalline structure of the starches was analyzed as described by Singh et al.  using anThe crystallinity index (CI) was calculated as described by Singh et al.  from theTM software, V.10.4.3. The range of the scanning wavenumber was 4000–450 cm−1.The FTIR analysis was conducted following the procedure used by Awolu et al. . The stap ≤ 0.05) was considered.Analysis of Variance (ANOVA) was performed using the statistical software OriginPro 2019b, SAS version 9.0, and MS Excel 2016. Tukey’s multiple comparison test was used to compare the individual difference in the physicochemical properties of the starches. A 95% confidence interval (p < 0.05) impact on the yield, physicochemical properties , and functional properties . Similarly, the ecotype had a significant impact on the characteristics, with the exception of fat, SP, and solubility. The XRD pattern of all starches showed B-type diffraction, with a difference in the diffraction peak intensities between all starches, which could be attributed to the differences in the ecotype and/or isolation methods. The results from the FTIR tests confirmed differences in the location (wavenumber) and intensity of the infrared absorption peaks of all the samples, especially in sensitive regions, depending on the ecotype and isolation method of EP starches. Between the two EP ecotypes, Loffo exhibited considerably better results in terms of the starch yield, color/whiteness, and some other physicochemical properties. The SMS isolation method was the most effective in acquiring better EP starch characteristics in most of the parameters evaluated. In this study, two EP (Chanqua and Loffo ) ecotypes and three different starch isolation methods were evaluated to determine their effect on the physicochemical, functional, and structural properties of EP starches. The isolation method used had a significant (


Correction to: European Journal of Applied Physiology 10.1007/s00421-023-05210-7The original version of this article unfortunately contained a mistake. Affiliation details for Author Thomas A. W. Paulson were incorrectly given1British Paralympic Association, London, UK3UK athletics, Birmingham, UKbut should have been2Sport Exercise and Health Sciences, The Peter Harrison Centre for Disability Sport, Loughborough University, Loughborough, UK3UK athletics, Birmingham, UK


Exposure to adversity and traumatic events affects well-being across important domains of functioning, including mental, physical, social, emotional, spiritual, and neurobiological. Situated as a focal point throughout neighborhoods, recreation centers are a prime opportunity to cultivate spaces of safety and healing. However, current models of trauma-informed care largely do not map neatly onto the recreation organizational structure and functioning. This paper describes the efforts over the past five years to transform the City of Cleveland, Ohio’s 22 recreation centers into trauma-informed Neighborhood Resource and Recreation Centers (NRRCs)––places where children, youth, and adults can readily acquire the support and services they need in an environment in which trauma-informed care principles are fully embedded in the fabric of the organization’s culture. Phase 1 included transitioning the recreation centers to NRRCs, hiring of trained social workers and counselors to work within the recreation centers, and training all recreation staff about trauma. Phase 2 included development of NRRC trauma-informed standards, development of the Trauma-Informed Progress Tool to track change over time, development of Trauma-Informed Leadership Competencies for Center Managers, and ongoing training for the social workers and counselors. We discuss ideas for future work and lessons learned from each phase. In 2018, the City of Cleveland Mayor Frank Jackson, his administration, and City Council worked tirelessly to pass legislation to treat crime and violence as a public health problem [Exposure to trauma has long been known to affect well-being across important domains of functioning. A trauma is an event, or series of events, or circumstances that are experienced as emotionally or physically harmful and has an enduring impact on someone mentally, emotionally, physically, socially, and/or spiritually, and neurobiologically . In the The population of Cleveland, Ohio, faces high rates of trauma exposure, rooted in socioeconomic and racial inequalities. Approximately one-third (32.7%) of Cleveland residents live below the poverty line and 46.1% of children live in poverty . ClevelaIn Cleveland, recreation centers are situated as focal points throughout neighborhoods and offer an opportunity to cultivate spaces of safety and healing. Recreation centers serve as a major resource to communities across the United States, providing fitness centers and aquatic facilities, healthy living classes, after-school opportunities for youth, programming for older adults, access to computers and the internet, health and wellness programs, and food to community members . The CleThe trauma-informed approach involves creating a safe and supportive environment that empowers individuals to engage in their own healing process, rather than re-traumatizing them. Trauma-informed care emphasizes the importance of understanding the root causes of trauma and uses a strengths-based approach to promote resilience and recovery. However, previous initiatives within youth-serving organizations have largely consisted of training and have yet to integrate a trauma-informed care model with the National Park and Recreation Association’s standards of practice . AdditioDespite goals to transform the recreation centers into trauma-informed environments, existing models of trauma-informed approaches do not translate well to recreation center environments. As such, prior trauma-informed training for staff in youth-based after-school programs was found to be ineffective due to the inability of employees to manage personal trauma along with that of the patrons’, lack of continued funding, as well as content knowledge loss over time . RecreatThe NRPA’s Commission for Accreditation of Park and Recreation Agencies (CAPRA) is the national accreditation body that outlines metrics for parks and recreation centers to provide high-quality services to communities. Becoming accredited by the CAPRA helps recreation centers uphold the highest standards of services within communities. The CAPRA guidelines, originally created in 1989, encompass a total of 154 standards that span across ten content areas, which are (1) agency, (2) planning, (3) organization and administration, (4) human resources, (5) fiscal management, (6) programs and services management, (7) facility and land use management, (8) public safety, (9) risk management, and (10) evaluation, assessment, and research. The CAPRA model is designed to enhance parks and recreation centers’ overall services to meet or exceed national established benchmarks at the local or regional levels. In order to become accredited, a recreation center needs to provide evidence that their respected agency has met an initial 142 CAPRA standards within their first accreditation period. Currently in the United States, there are nearly 170 park and recreation agencies from 38 states that have achieved this accreditation.While the CAPRA provides the standards for creating high-quality recreation centers, they do not provide guidance on how to make recreation centers a place of support, healing, and holistic care for communities who experience high levels of traumatic events. Similarly, the current trauma-informed models that focus on organizational change fail to incorporate aspects of systems change that are necessary for recreation centers. The Substance Use and Mental Health Services Administration’s  trauma-iIn 2018, the City of Cleveland invested substantial financial resources and undertook a massive effort to provide targeted coordinated programs and supports to address community-level trauma, while bolstering and leveraging already existing strengths within the recreation centers . The ultimate goal was to cultivate spaces in which Cleveland residents could thrive. While the work has spanned nearly 5 years, there are distinct phases and lessons learned from the efforts to transition the City of Cleveland’s recreation centers to the nation’s first system of high-quality trauma-informed Neighborhood Resource and Recreation Centers (NRRCs).Phase 1 spanned 2018 to 2019, which included (1) transitioning the City of Cleveland’s 22 recreation centers to NRRCs, (2) contracting with a local non-profit behavioral health organization to hire trained social workers and counselors to work within the recreation centers, and (3) training all recreation center staff about exposure to trauma and its lasting effects. This phase was led by a local non-profit behavioral health organization who collaborated with the authors in developing and co-facilitating the training as well as conducting the evaluation.Youth and adult education, ranging from K-12 enrichment programs to post-education skills training.Job and career readiness, including career planning, job training, and job-placement services for adults.Health and wellness education, raising awareness of both physical and emotional issues, to aid people of all ages.Youth leadership and development, including use of mentors who grew up in situations similar to what youth face today and can serve as role models.Arts programming, both for performing and visual arts.Sports and recreation, including both traditional recreation sports and nontraditional ones such as fencing and rowing.Given that the recreation centers are an essential resource embedded within Cleveland neighborhoods where thousands of children, youth, adult, and senior residents visit annually, the centers were identified as the prime system to extend resources to community members. Transitioning the recreation centers to NRRCs meant that programming was extended beyond traditional sports and recreation to include activities and programs aimed at addressing social determinants of health, which are the social and economic factors such as poverty, education, housing, discrimination, and access to healthcare that can have a significant impact on an individual’s health outcomes and overall well-being. Expanded programming centers around the following six areas:The expansion of programs allowed for the NRRCs to begin to offer holistic care and resources for community members.Staff training on trauma-informed care is a key element, and often the first step, in trauma-informed organizational transformation . ResearcDuring the first three months of the transformation effort, a series of 3 h professional development sessions were held on topics including an introduction to trauma-informed care, developmental impacts of trauma, signs and symptoms of trauma, conflict mediation, and strategies for de-escalation. Nearly 300 individuals––from recreation center desk staff, coaches, custodians, and security personnel to community partners such as violence interrupter teams––received training. Pre- and post-test evaluations were performed for each training session and showed an increase in knowledge. Each session also included space for City employees to reflect on what impact the previous training has had on their work. Some staff shared that they were utilizing aspects of trauma-informed care in how they treat youth and children; the training further benefited the staff by giving them a shared language and boosting positive energy and pride. Other recreation staff stated that while they knew the children they served had significant challenges in their lives, they had not known how to react or respond to the behaviors they saw resulting from those challenges. The trauma-informed care framework offered them information to better understand that the behaviors of these youth may be linked to traumatic experiences and provided techniques for de-escalation. Staff reported that they noticed that their own shifts in behavior changed the outcomes of youth each day.An essential component of the Phase 1 transformation effort was the decision to staff all of the NRRCs with trained social workers or counselors to provide short-term direct services such as case management, crisis intervention, psychoeducational support groups, and resource referral to residents in order to assist them in accessing the resources and supports they need. These bachelor’s or master’s level providers, initially given the title Trauma Coaches, were employed by a local non-profit behavioral health organization. The Trauma Coaches split their time between two to three NRRCs each, while also providing services in K-12 schools and in the community as needed. After the completion of the training series, the Trauma Coaches acted as stewards of the trauma-informed initiative, holding monthly professional development sessions for the staff at their respective NRRCs. The objective of these sessions was to reinforce the messages from the initial training and introduce a variety of topics to expand the understanding of developing a trauma-informed system for youth and adults who visit the NRRCs.Given the innovative nature of transforming recreation centers into high-quality, trauma-informed NRRCs, there were bound to be lessons learned from the early phase of this work. Over the course of Phase 1, the City leaders from the Division of Recreation and the Office of Prevention, Intervention, and Opportunity for Youth and Young Adults met nearly weekly to discuss the ongoing work and identify ways to make improvements in the next phase. Through these discussions, there were key areas that were identified. It became apparent that training about trauma was not enough to become trauma-informed and that there needed to be a stronger focus on organizational cultural changes within the system. There also needed to be a focus on elevating the quality of the NRRCs, which needed to happen simultaneously as the centers worked towards becoming trauma-informed. City leaders also acknowledged the importance of sustainability of the Trauma Coaches. The current model meant that the Trauma Coaches were employed through the local non-profit behavioral health organization and then contracted by the City to work year-to-year. It was determined that a better model was to have the Trauma Coaches employed by the City in order to support the sustainability of the program.Reflecting back over the past five years, we identified additional lessons learned from Phase 1 that could be useful recommendations for other cities that may be considering transforming their recreation centers.Adequate time for strategic planning between legislation passing and program starting should be built into the first year of the program. Strategic planning should at minimum include a 5-year plan with mapped-out goals for each year of the programs.Measuring organizational readiness for change and providing support in this area is recommended.Rather than training all recreation staff at the same time, training should be conducted in stages starting with city leadership, followed by Center Managers, and then the remaining recreation staff.Tools, resources, and strategies should be available to staff early on to address secondary trauma experienced by staff, particularly for recreation systems located within cities with high levels of violence and crime.The city should adopt a trauma-informed city-level ordinance so that all departments and divisions have a shared understanding and language around trauma.While Phase 2 was intended to start in 2020, the COVID-19 pandemic delayed the continuation of the project until 2021. During the first months of the pandemic, the NRRCs closed down and were subsequently utilized as COVID-19 testing sites and later as vaccination centers. Some NRRCs opened during the period when schools were closed and offered space for children to do their online learning. When the trauma-informed program resumed, Phase 2 included (1) development of NRRC trauma-informed standards, (2) development of a progress tool to track change over time, (3) development of trauma-informed leadership competencies for Center Managers, and (4) ongoing training for the Trauma Coaches. Phase 2 was led by the authors who developed trauma-informed standards, the progress tool, leadership competency, and provided the ongoing training.Phase 2 of the NRRC organizational change effort began with the development of NRRC Trauma-Informed Standards, which are guiding principles in which the recreation center policy and practice across all levels of leadership and staff should operate. These standards were created by converging the CAPRA Standards see  and SAMHThe development of the NRRC Trauma-Informed Standards began with organizing CAPRA standards into congruous SAMHSA trauma-informed organizational implementation domains. This approach allowed us to develop a framework that attended to both trauma-informed practice and CAPRA’s recommended best practices. Prior to integrating SAMHSA principles and CAPRA standards, we benchmarked other metropolitan parks and recreational facilities with patrons of similar demographics. Benchmarking was important to further align NRRC’s trauma-informed initiatives with those of high-performing parks and recreation facilities as well as to survey challenges faced by similar organizations in integrating trauma-informed approaches with operational and regulatory  demands. However, importantly, we found that no other parks and recreation system to our knowledge in the country was engaging in as comprehensive of an effort as the Cleveland NRRCs.We further took into account the level of community violence in the neighborhoods in which the NRRCs are located. Given that this organizational change effort is trauma-informed, we considered how different forms of trauma exposure may be incorporated into the NRRC Standards and accordingly into organizational learning and staff awareness. For instance, a recreation center situated in a neighborhood with higher crime rates may be stronger within the CAPRA standard for public safety, law enforcement, and security.In addition to benchmarking, it was also critical to gain input on the proposed trauma-informed standards from NRRC managers and staff, including Regional Managers, Center Managers, and program staff. We utilized a change management approach, where research has shown that practicing inclusion during change management processes helps to solidify commitment to the change . It alsoWe additionally sought feedback from patrons of the NRRCs to explore the recreation facilities that provided a safe and supportive environment. Three sessions were held consisting of youth patrons, caregivers of youth patrons, and adult patrons. Feedback revolved around policies and practices that promote physical and psychological safety, transparency in communication on program offerings, as well as interactions with staff. Findings among patrons were then utilized to support the development of the standards.One feedback session was held with the NRRC leadership team to further understand the organization’s governance and leadership competencies, financing structure, progress monitoring and quality assurance of its systems, and engagement and involvement of staff and patrons. Feedback regarding the organizational dynamics aided us in refining the strategic framework to incorporate trauma-informed practices within the NRRCs overarching systems to enhance staff development and patron care.Building from the foundation of the NRRC Trauma-Informed Standards, next we developed the Trauma-Informed NRRC (TI-NRRC) Progress Tool, which included indicators that measured progress towards becoming a high-quality, trauma-informed NRRC. The purpose of the TI-NRRC Progress Tool is to identify strengths as well as opportunities for improvement in each of the NRRCs. The Tool was designed to be a collaborative assessment process, offering an opportunity for managers at each of the NRRCs to reflect on their center’s process toward becoming a high-quality, trauma-informed resource for the Cleveland community.Within each of SAMHSA’s 10 organizational implementation domains, we developed potential indicators informed by both SAMHSA’s trauma-informed principles and the CAPRA Standards. First, we reviewed trauma-informed organizational assessments such as the Trauma-Informed Practice Scales , CreatinAs with the previous stage of the Standards development, it was critical to gain input on the proposed indicators from NRRC managers and leadership. This ensured that the language and framing of each indicator was relevant to recreation and accurately reflected the scope of work conducted within the centers. During early rounds of feedback, it became apparent that some of the indicators were outside of the control of the managers, and their ability to make progress on those indicators resided at the leadership level. For example, most written documentation  is issued by the administration. Those indicators were flagged as “Division of Recreation/Public Works issues/provides,” but remained as an indicator within the TI-NRRC Progress Tool. It was determined that this method would provide a level of accountability for the leadership team to continue moving the needle toward becoming high-quality, trauma-informed NRRCs.Feedback by managers and leadership also informed our decision to structure the TI-NRRC Progress Tool into tiers. The original version was lengthy, covering all ten organizational implementation domains with a total of 272 indicators. Jointly with NRRC managers and leadership, we determined that the TI-NRRC Progress Tool needed to support building capacity over time while motivating the NRRC managers to continue making progress. Thus, we reformatted the TI-NRRC Progress Tool to five tiers, where the first tier focuses on foundational items, and then each tier builds on the previous one. Once the threshold for a tier is met, the subsequent tier is added at the next assessment period progressing towards all tiers being included. Because of the tiered approach, it was also determined that the Tool would be implemented quarterly.The TI-NRRC Progress Tool was piloted to test the methods and procedures. Baseline data were collected across all participating NRRCs. Specifically, the pilot assessed the feasibility of the tool implementation and allowed the NRRCs to determine and provide any necessary revisions to the TI-NRRC Progress Tool protocol. The TI-NRRC Progress Tool was completed collaboratively by the NRRC Regional Managers and Center Managers as intended for Tier 1, and after each domain section, the NRRC Regional Managers and Center Managers assessed the feasibility of the domain section through a series of written reflection questions. The baseline data for Tier 1 of the TI-NRRC Progress Tool was scored and assessed for expected responses and variance. The additional pilot questions were analyzed for feasibility themes, barriers, and facilitators. Results of the pilot indicated that the TI-NRRC Progress Tool functioned as intended. Center Managers reported that completing the TI-NRRC Progress Tool helped them to think through current NRRC operations and opportunities for improvement in a constructive, comprehensive, and systematic way. The TI-NRRC Progress Tool also served as a useful guide for facilitating conversations about the NRRC’s strengths, challenges, and areas for growth. Importantly, Center Managers expressed hope that the TI-NRRC Progress Tool will continue to be implemented in the future, highlighting the value and utility of the Tool for making systemic changes.Baseline results were compiled into a TI-NRRC Progress Tool Report for each NRRC that showed the current assessment. Future assessments will compare the current assessment to the previous period, allowing for a high-level view of progress made over time. The TI-NRRC Progress Tool Report is provided to the Center Manager and Regional Manager ahead of an improvement plan meeting where they discuss the results of the TI-NRRC Progress Tool and develop a plan for improvement over the subsequent 90 days. While the NRRCs have quarterly improvement plans, it is not expected that the NRRC will progress through a tier each quarter. Rather, the goal is to show continuous improvement overtime.To further support progress toward meaningful and sustainable trauma-informed systems change, we developed the TI-NRRC Leadership Competencies Framework to complement the Progress Tool and support the development of trauma-informed leadership skills for NRRC managers. Similar to the other components of the effort, these competencies integrated a trauma-informed approach with relevant NRPA standards and guidelines. Specifically, the Leadership Competencies Framework drew on Certified Park and Recreation Professional (CPRP) and Executive (CPRE) certification requirements, which encompass important leadership responsibilities and skills in the field of parks and recreation, such as communicating effectively, facilitating programming, managing finances, attending to human resources processes, and overseeing the operations of NRRCs. Similar to the CAPRA standards for recreation centers, CPRP and CPRE certification is a formal accreditation governed by NRPA and the National Certification Board (NCB). One aim of integrating these standards into the Leadership Framework was to help prepare managers for success if they wished to pursue certification formally. Another key goal of incorporating CPRP/CPRE certification standards into the Leadership Framework was to further develop the capacity for high-quality management of recreation centers, ultimately supporting the vision for a high-quality system of NRRCs. However, much like the CAPRA standards, the CPRP and CPRE standards do not naturally align with a trauma-informed approach.We began work on the development of the Leadership Framework by conducting a small focus group of managers and staff, with the goal of obtaining their input relative to desired leadership characteristics of managers at various levels, current job expectations, and ways in which trauma-informed principles were currently being enacted within the NRRCs. This feedback was synthesized into themes that informed the process of content development and the eventual structure of the framework. For example, safety—and lack thereof—arose as an important theme that was prioritized for the framework. Themes were also shared with NRRC leadership to support ongoing communication of strengths and areas of concern regarding the experiences of staff and patrons in the NRRCs. A second, larger focus group was conducted after the initial draft of the framework was constructed to provide managers the opportunity to review, discuss, and comment on each aspect of the tool. The insight, feedback, and recommendations provided by the managers was essential to every aspect of this process.Our vision for the framework was that it would support managers in progressively developing leadership competencies, while also providing opportunities to acknowledge and build upon the many skills they already had in place. Recognizing the complexity and demands of the work managers were engaged in on a daily basis, we were cautious to avoid unintentionally creating additional burdens through this process. Using feedback from the managers and insight we gained throughout our involvement with the project, we determined that designing the tool using a tiered approach to skill building would improve the overall utility and effectiveness of the tool, as well as the experience of the managers in using the framework.We then merged the specific competencies within each of the five CPRE domains  with trauma-informed principles and language. We overlaid the competencies with the six key trauma informed principles  to ground the framework in a trauma-informed approach. We again condensed, reframed, and re-envisioned many of the original CPRP/CPRE expectations to align with NRRC values, the essential aspects of leadership for NRRC managers, and the core aspects of trauma-informed leadership.In response to the need for sustainability, Phase 2 of the transformation process entailed making the Trauma Coaches internal employees of the City. Rather than being supported by an external agency, the City of Cleveland hired the social workers and counselors and changed their title to Social Support Services Specialists (S4) in an effort to reduce stigma. In order to build community, enhance relational health, and create shared language and knowledge, the S4 team received monthly 3 h professional development sessions based on the Trauma and the Brain Training Series . This caPhase 2 of the transformation efforts began in the context of various changes resulting from the onset of the COVID-19 pandemic. The world was changing, and while the recreation centers were able to quickly pivot to providing support around both COVID-19 testing and vaccinations, it was not without significant stress to both NRRC staff and patrons who relied on the centers for support. Furthermore, during this time, there were numerous high-profile incidents of police brutality and racial violence persistently elevated through social media and news outlets. Once the NRRCs re-opened, it was apparent that there was a need to fundamentally change the way services were provided by the city. As such, there is now even more need for culturally affirming mental health supports and programming in community sites such as the NRRCs. The S4 team is currently comprised of 11 staff, 2 supervisors, and 1 director; each staff person splits their time between two recreation centers, while also providing services in local schools and neighborhoods as needed. Expansion of this team, allowing for one S4 per NRRC, would be key in meeting the need for additional mental health support. Furthermore, having an S4 at each NRRC would further build trust within the community by having a consistent presence and access to the patrons.Phase 2 also occurred during a transition of leadership in the City of Cleveland. The election of Mayor Justin Bibb brought additional changes in top-level leadership that oversaw the trauma-informed NRRC efforts. These transitions highlighted the need for onboarding and ongoing training for city administration to create shared language and understanding around trauma and trauma-informed care. Furthermore, the initial training of the NRRC employees occurred at the beginning of the project in 2018. Phase 2 shifted to creating trauma-informed organizational tools to measure change within the NRRCs. Combined with the pause from the COVID-19 pandemic and the work conducted in Phase 2, with the exception of the S4 team, the NRRC staff did not receive ongoing trauma-informed training. While the creation of the Trauma-Informed Progress Tool and the Trauma-Informed Leadership Competencies are essential for measuring change over time and assisting in long-term change, a missed opportunity was that NRRC staff did not receive ongoing professional development on implementing trauma-informed practices within their work and day-to-day interactions.Building on our progress made from previous phases and incorporating lessons learned from Phases 1 and 2, we have identified essential areas of future work below.While Phase 1 focused on training staff about trauma and trauma-informed care, Phase 2 shifted to creating tools to measure change overtime and supporting the S4 team. The critical role of leadership in fostering trauma-informed organizational change has been well documented ,35. TrauWe recommend that management and staff members receive ongoing professional development that includes module-based learning accompanied by practical skill application. In partnership with the Neurosequential Network, NRRC Regional Managers, managers, and staff can receive a six-session training series introducing them to the Neurosequential Model of Sport (NMS), which is an evidence-informed model based on the Neurosequential Model of Therapeutics . NMS preAdditionally, the ongoing monthly professional development series with the S4 team should continue, building upon the previous focus on the impact of trauma on brain development and how to integrate the knowledge of the brain and nervous system into their work with children and youth. This training series should also focus on racialized trauma and its impacts. The professional development series should utilize a train-the-trainer format so that, upon completion, the S4 team will be prepared to take leadership in facilitating additional community building and professional development of NRRC managers, staff, and community members.Engaging youth and families in training and programming focused on trauma-informed care is important because trauma can have significant and generational impacts on health and overall well-being ,4. TraumBased on feedback gathered during a series of focus group discussions and our experiences as we developed the tool, we plan to incorporate the Leadership Competencies into the TI-NRRC Progress Tool, to support a synthesized and more holistic approach to trauma-informed organizational and leadership practices. This approach will reduce potential redundancy in processes and potential burden on managers, streamline procedures and documentation, and incorporate all aspects of organizational and leadership development into one competency building tool. A key aspect will involve support in the implementation of the TI-NRRC Progress Tool, specific to the area of leadership development. Training and consultation will be essential in supporting NRRC managers in progressing beyond the foundational leadership competencies and providing ongoing skill development, managing burnout, and applying trauma-informed leadership principles to their everyday work.The TI-NRRC Progress Tool should be implemented on a regular basis. We recommend that in addition to the TI-NRRC Progress Tool Report and Improvement Plan for each NRRC, an additional Summary Progress Tool Report be prepared for the City of Cleveland NRRC Administration that shows progress made for each NRRC as well as the overall NRRC department. The Summary Report should include the Progress Tool indicators that are specific to the City of Cleveland NRRC Administration. Following the analysis and dissemination of the Summary Report to the City of Cleveland NRRC Administration, a meeting should occur with the City of Cleveland NRRC Administration to discuss the results of the Summary Report and develop a plan for improvement over the next 90 days. Creating sustainability will be a key aspect of the next phase of work. It will be essential to train City administrators on the TI-NRRC Progress Tool data collection, online survey use, data analysis, report building, and improvement planning sessions.One of the key elements of trauma-informed organizations is resisting re-traumatization of the people served. Quite often this is conducted by daily practices and interactions with patrons. However, policies and procedures developed from a trauma-informed lens must be in place to ensure that the practices of NRRCs are not traumatizing or re-traumatizing to managers, staff, or patrons. In order for the NRRCs to operate from a trauma-informed lens, policies and procedures must align with trauma-informed care principles. Establishing an NRRC mission and vision that embraces a trauma-informed lens is one key starting point for this process. During this next phase, we also recommended that the City of Cleveland create several new policies and procedures in order to meet the CAPRA standards. These new policies and procedures will need to align with trauma-informed care principles of the NRRCs.Recreation centers serve as focal points of activity for many youths and their families. While the value of sport and physical activity is clear, more broadly, recreation centers have the potential to serve as community hubs where neighborhood residents can receive comprehensive support and care. However, if systems or organizations want to achieve trauma-informed organizational change, consideration should be given to the fact that sustainable change takes time. Ongoing commitment and effort are required in order to move the needle toward creating and sustaining trauma-informed environments. Expanded intergenerational programming, on-site mental health services, and the integration of trauma-informed practices can help recreation centers to improve outcomes for youth exposed to trauma and build stronger families and stronger neighborhoods. Recognizing these opportunities, cities can implement trauma-informed care within their recreation centers and provide services to children, youth, and adults in order to combat many of the challenges associated with experiencing trauma. Through this work, we have demonstrated that recreation centers can be potential places of support, healing, and holistic care for local families and the broader community.


Karenia longicanalis and showed that the ciliate Strombidinopsis sp. was dominant in a nonbloom plankton community, whereas the diatom Chaetoceros sp. dominated the after-bloom community, along with remarkable shifts in the community structure for both eukaryotes and prokaryotes. Metatranscriptomic analysis indicated that heightened energy and nutrient acquisition in K. longicanalis significantly contributed to bloom development. In contrast, active grazing by the ciliate Strombidinopsis sp. and attacks by algicidal bacteria  and viruses prevented (at nonbloom stage) or collapsed the bloom (in after-bloom stage). Additionally, nutrition competition by the Chaetoceros diatoms plausibly contributed to bloom demise. The findings suggest the importance of energy and nutrients in promoting this K. longicanalis bloom and the failure of antimicrobial defense and competition of diatoms as the major bloom suppressor and terminator. This study provides novel insights into bloom-regulating mechanisms and the first transcriptomic data set of K. longicanalis, which will be a valuable resource and essential foundation for further elucidation of bloom regulators of this and related species of Kareniaceae in the future.The development and demise of a harmful algal bloom (HAB) are generally regulated by multiple processes; identifying specific critical drivers for a specific bloom is important yet challenging. Here, we conducted a whole-assemblage molecular ecological study on a dinoflagellate bloom to address the hypothesis that energy and nutrient acquisition, defense against grazing and microbial attacks, and sexual reproduction are critical to the rise and demise of the bloom. Microscopic and molecular analyses identified the bloom-causing species as IMPORTANCE HABs have increasingly occurred and impacted human health, aquatic ecosystems, and coastal economies. Despite great efforts, the factors that drive the development and termination of a bloom are poorly understood, largely due to inadequate in situ data about the physiology and metabolism of the causal species and the community. Using an integrative molecular ecological approach, we determined that heightened energy and nutrient acquisition promoted the bloom, while resource allocation in defense and failure to defend against grazing and microbial attacks likely prevented or terminated the bloom. Our findings reveal the differential roles of multiple abiotic and biotic environmental factors in driving the formation or demise of a toxic dinoflagellate bloom, suggesting the importance of a balanced biodiverse ecosystem in preventing a dinoflagellate bloom. The study also demonstrates the power of whole-assemblage metatranscriptomics coupled to DNA barcoding in illuminating plankton ecological processes and the underlying species and functional diversities. N/dt = μ + I − Ex − G − M, where N and t are population abundance and time period, respectively. Some blooms are believed to be initiated from seed populations brought onshore by currents , which have increased in recent decades in frequency, bloom species diversity, geographic extent, and severity of ecological and economic damages , 2. HABscurrents  or dissicurrents . Currentcurrents –10. In currents –. The maiurrents ––14 and irrents ––, althougrrents –––18. It iMetabarcoding and metatranscriptome profiling with next-generation sequencing have proven to be effective ways to profile community composition and metabolic landscape , 20 and 23–Karenia are frequent bloom-forming dinoflagellates in coastal waters , and after-bloom conditions within the bay. Metabarcoding analyses based on small subunit (SSU) ribosomal DNA  were performed to profile algal and bacterial community structures and their changes across the different bloom conditions. In parallel, whole-assemblage metatranscriptomic analyses were carried out to identify critical metabolic processes associated with the different bloom conditions. This research portrays prokaryotic and eukaryotic community shifts among the bloom, nonbloom, and after-bloom stages and sheds light on what main biotic factors and metabolic processes associated with energy and nutrient acquisition, cell proliferation, and defense drive the bloom development and demise in a seriously understudied dinoflagellate.In this study, we investigated a a concentration was 16.8 μg/liter in the bloom samples, compared with 1.1 μg/liter in the nonbloom samples and 1.5 μg/liter in the after-bloom samples  in the nonbloom samples, and it declined by 137-fold (1.1 × 105 cells/liter) in the after-bloom samples. Based on 18S rDNA metabarcoding of the community  , and the after-bloom community was dominated by the diatom Chaetoceros sp. (accounting for 63.86%) (Picozoa (0.10% to 1.75%) throughout this study period  . Other my period .To explore what algae-associated microbes might be involved in the bloom development and decline, the microbial community was analyzed using 16S rDNA metabarcoding. Ten dominant families of bacteria were identified : RhodobaKarenia (based on 18S metacoding) was negatively correlated with NO3−, NO2−, and SiO43− but positively correlated with PO43− and dissolved organic carbon (DOC) concentrations , indicating active photosynthesis. The Pearson test showed that the relative abundance of Karenia was positively correlated with the abundances of bacteria from the families Flavobacteriaceae, Alteromonadaceae, Vibrionaceae, and Pseudoalteromonadaceae but negatively correlated with that of Cryomorphaceae, Planctomycetaceae, and Piscirikettsiaceae . The reference metatranscriptome showed a total read mapping rate of 77.63 to 90.88% (Table S4), indicating an overall good assembly quality. Functional annotation based on the eggNOG, Swiss-Prot, KEGG, GO, and nrMegPhylodb databases gave a total annotation rate (genes annotated by at least one of these databases) of 74.87% (Table S5).Our whole-assemblage metatranscriptome sequencing yielded a total of over 1.15 billion cDNA reads (Table S2). After quality filtering, the resulting 0.91 billion (79.11%) clean reads were assembled into a reference metatranscriptome composed of 1,389,260 unigenes with an Karenia, while the nonbloom metatranscriptomes were contributed mainly by the ciliate Strombidinopsis, the diatom Chaetoceros, the bacteria Crymorphaceae and Rhodobacteracea, and viruses, in decreasing order. The after-bloom metatranscriptomes were mainly composed of transcripts from Karenia, Chaetoceros, viruses, Crymorphaceae, and Strombidinopsis  in the nonbloom samples than in the bloom samples.Among the 1,389,260 unigenes, 74.32% were assigned taxa, of which 58.20% were from bacteria, 40.04% from eukaryotes, 1.08% from viruses, and 0.69% from archaea. The number of expressed genes and the expression levels of the genes from bacteria, viruses, and archaea decreased markedly from the nonbloom samples to the bloom samples . Gene exdinopsis . The genK. longicanalis, particularly during a natural bloom, it is of interest to catalog the expressed genes. From the metatranscriptomes, 94,785 unigenes were identified as Karenia genes. Four genes were expressed constantly at >1,500 TPM across all sample groups, including three major basic nuclear protein genes and one EF-hand domain-containing protein gene , 1,001 (47.96%), and 897 (42.98%) were highly expressed in nonbloom, bloom, and after-bloom samples, at higher percentages than the overall percentage of HEGs (25%)  involved in light harvesting, photosynthesis, proton pump rhodopsin, and carbon fixation and genes of ATP synthase; 618 nutrient uptake genes (N), such as N-, P-, and Si-related transporters; 73 defense genes (D); and 270 cell proliferation genes comprising those regulating sexual reproduction (meiosis) and cell division cycle (S) in Karenia ; Fig. S1Karenia . Among tGs (25%) .a to c binding protein gene family), photosynthesis, and carbon fixation. Nutrient uptake-related upregulated DEGs included amino acid transporter, nitrate transporter, pyrophosphatase, urease and urease accessory protein. Related to cell proliferation were DEGs of the cell cycle proteins and MSH2, which were upregulated in the bloom , followed by amidase, with both being upregulated in the after-bloom samples compared to the bloom or nonbloom samples, while nitrate-nitrite transporter genes and nitrate-nitrite reductase genes were the top two most highly expressed N-related genes in Karenia during its bloom  under their respective dominance conditions. Karenia also showed high expression of diphosphatase and 5′-nucleotidase in the bloom sample. In addition, Chaetoceros silicon transporters were more highly expressed and upregulated in the after-bloom condition than in the bloom and nonbloom conditions , were most highly expressed in the nonbloom samples. Ten of these showed the highest expression in the after-bloom stage, including Beihai picorna-like virus, major capsid protein-containing viruses, Chrysochromulina ericina virus, and Edafosvirus sp., four types of algal viruses different from those in the nonbloom stage  and viruses might have led to the release of undigested algal remnants in the nonbloom or after-bloom samples, and bacteria might have used the same enzymes (as reported above) to utilize this rich food source and support their growth.Bacteria are another potential bloom killer. Highly expressed genes  from prokaryotes that encode enzymes potentially involved in algal cell wall lysis were detected, including genes with expression (listed from high to low expression): aminopeptidase, metalloprotease, sulfatase, xylanase, glucosidase, lysozyme, lipase, chitinase, trypsin, and amylase . These 1in situ measurements of these terms. However, integrative ecological and molecular analyses provide a possibility to approach the problem. Although transcriptomes are indirect indications of in situ physiological and ecological dynamics, they are still powerful evidence of these processes. By taking the molecular ecological approach, clues and insights about the most important bottom-up  and the top-down  factors can be interrogated  and growth- and development-associated EF-hand domain-containing protein  is advantaged in energy acquisition with high expression levels of genes encoding proton pump rhodopsin, nutrient transporters, and enzymes involved in the utilization of organophosphates  was depleted under the bloom condition (<5 μM) relative to the nonbloom and after-bloom conditions , diphosphatase, and 5′-nucleotidase (5NT). Pyrophosphatase, which hydrolyzes the high-energy pyrophosphate into two inorganic phosphate molecules  as potential drivers of blooms , 26, 28.e blooms , the rolK. longicanalis in the nonbloom sample group suggests that a more vigorous microbial attack might have diverted intracellular resources to defense at the cost of cell proliferation. Conversely, the considerable decreases in diversity and activity of viral genes under the bloom conditions suggest a lower infection activity during the bloom. Several viruses were transcriptionally very active under nonbloom or after-bloom conditions. Picorna-like viruses are positive-sense single-stranded RNA viruses that are major pathogens of plants, animals, insects, and marine phytoplankton  could feed on many species of dinoflagellates -resolved sampling.While the high activities of ciliates, bacteria, and viruses likely substantially contributed to the prevention and termination of the K. longicanalis and represents the first natural bloom investigation of this species. Acquisition of energy and nutrients, defense, cell proliferation, and cell mortality were explored as potential regulators of a Karenia longicanalis bloom using an integrated molecular ecological approach. Our integrative data analysis indicated that the promoted cell proliferation and bloom formation in the bloom stage were supported by high or increased expression of energy and nutrient acquisition and metabolism genes in Karenia. Cell mortality due to microbial attack and ciliate grazing appeared to be the major inhibitor of bloom formation in the nonbloom stage and the promoter of bloom demise in the after-bloom stage. The competition of the Chaetoceros sp. likely also contributed to the decline of the K. longicanalis population and the rise of the Chaetoceros sp. population to dominance in the after-bloom stage. Our metatranscriptomic data suggested that K. longicanalis was heavily “invested” in defense during the nonbloom or after-bloom stages, but defense was inadequate to overcome the overwhelming grazing and microbial attacks. Therefore, mechanisms underlying the K. longicanalis bloom appear to be different from those regulating the bloom of P. shikokuense, which requires the upregulation of genes related to energy and nutrient acquisition, defense, and sexual reproduction . The after-bloom condition was sampled in the same location as the previously sampled bloom area on June 12, when the abundance of the bloom species declined by 137-fold (1.1 × 105 cells/liter). At each sampling event, three separate samples were collected as replicates, giving a total of 9 samples. For each sample, 20 liters of water was taken from the subsurface (~0.5 m) and prescreened through a 200-μm mesh to exclude large zooplankton. Immediately afterward, the sample was filtered onto 3-μm polycarbonate filters (47 mm). Filtration was completed in 15 min to minimize changes in gene expression. Each filter was split into four equal parts using clean scissors, one for DNA analysis and the other three for RNA analysis. Cells retained on the filter membranes included planktonic eukaryotes (3 to 200 μm) and microbes that were infecting or attached onto the eukaryotic cells. Filters for RNA sequencing were immediately immersed in 1 mL TRIzol in a 2-mL tube and kept frozen in liquid N2 during the cruise and, upon return to the laboratory, were stored at −80°C until RNA extraction. Samples for DNA sequencing were transferred to a 2-mL tube containing 1 mL DNA lysis buffer  and held at −20°C until DNA extraction.Three sampling events, each in triple biological replicates, were launched to represent a nonbloom condition (nonbloom 1 to 3), a bloom condition (bloom 1 to 3), and an after-bloom condition (after-bloom 1 to 3) . Fifty-min situ using YSI Professional Plus, and surface light was measured using a digital lux meter. Two sets of 50-mL seawater samples from each of the three sampling events were filtered onto GF/F membranes and a 0.2-μm membrane and stored at −80°C for chlorophyll a and nutrient measurements, respectively. Chlorophyll a was measured using a Trilogy laboratory fluorometer . The concentrations of N nutrients , PO43−, and SiO44− were measured using the Bran-Lubbe AAIII system (Germany) on water samples that had been filtered through a 0.22-μm membrane and kept at −80°C. DOC and total dissolved nitrogen (TDN) were measured using the high-temperature catalytic oxidation method with a Multi N/C 3100 system (Germany). DON concentration was estimated based on TDN minus DIN (NH4+ + NO3− + NO2−). For particulate organic phosphorus and particulate organic nitrogen  and 806R (5′-GGACTACHVGGGTWTCTA AT-3′) (GGCAAGTCTGGTGCCAG-3′) and 18SV4-R (5′-GACTACGACGGTATCTRATCRTCTTCG-3′) (DNA was extracted as previously reported . The DNAA AT-3′) . The 18STTCG-3′) . The 28SRNA extraction was performed as previously reported , 29 with(i) Community classification and taxon interactions. The raw data from the 16S and 18S amplicon sequencing were quality filtered to eliminate the adapters and low-quality reads to obtain clean reads with TrimmomaticPE. The parameters were set as follows: leading, 5; trailing, 5; sliding window, 4:15; minlen, 50. The clean data were used for feature identification and quantification using the UPRASE with NOISE3 method  Metatranscriptomic read assembly. Sequencing reads from each RNA sample were assembled with MEGAHIT v1.0  Gene taxonomic assignment and functional annotation. To identify the species origin of the unigenes in the reference metatranscriptome, a comprehensive in-house database (nrMegPhylodb)   was creaFunctional annotation of the unigenes in the reference metatranscriptome was conducted using Diamond , based o(iv) Gene expression quantification and differential expression analysis. The raw reads of the metatranscriptomes were trimmed and quality-filtered using Cutadapt (v1.9.1) with parameters setting at “-m 75 –max-n 0.1 –discard-trimmed -q 20.” Clean reads from each sample were mapped to the above-described reference metatranscriptome using the aligner Bowtie2 and quantified using RSEM (v1.2.4) with default parameters. Gene expression at the community level (community TPM) was calculated as transcripts of the specific gene per million of total mapped reads of the community and used to compare gene expression contributions of different lineages or kingdoms to the whole community. While comparing gene expression differences in the same species between sample groups, gene expression at the species level was renormalized as transcripts of the specific gene from the specific species per million total mapped reads of the species (species TPM) was used. Only genes with an average gene expression TPM of ≥0.1 were used in differential expression analyses within species, including K. longicanalis and Chaetoceros sp., for which the DESeq package in R software was employed. DEGs were defined as those with an adjusted P value of <0.05 and fold change of ≥2. DEGs associated with energy and nutrient acquisition, defense, and cell reproduction were identified using a t test based on average gene expression (TPM) of the gene family, as previously reported (reported .PRJNA689700).Raw sequence reads of mRNA and the DNA metabarcodes were deposited in the NCBI Sequence Read Archive (BioProject ID


Philip Alexander, MD, is a native Texan, retired physician, and accomplished musician and artist. After 41 years as an internal medicine physician, Dr. Phil retired from his practice in College Station in 2016. A lifelong musician and former music professor, he often performs as an oboe soloist for the Brazos Valley Symphony Orchestra. He began exploring visual art in 1980, evolving from pencil sketches—including an official White House portrait of President Ronald Reagan—to the computer-generated drawings featured in this journal. His images, which first appeared in this journal in the spring of 2012, are his own original creations.Methodist DeBakey Cardiovascular Journal, submit your creation online at journal.houstonmethodist.org as a “Humanities” entry.If you would like to see your art published in the