OsCKI1 in rice leaves, physiological, transcriptomic and proteomic analyses of leaves of osckI1 allele mutant lses1 (leaf starch excess and senescence 1) and its wild-type varieties (WT) were performed.The normal metabolism of transitory starch in leaves plays an important role in ensuring photosynthesis, delaying senescence and maintaining high yield in crops. OsCKI1 (casein kinase I1) plays crucial regulatory roles in multiple important physiological processes, including root development, hormonal signaling and low temperature-treatment adaptive growth in rice; however, its potential role in regulating temporary starch metabolism or premature leaf senescence remains unclear. To reveal the molecular regulatory mechanism of lses1 mutant exhibited starch excess in the leaves and an obvious leaf tip withering phenotype as well as high ROS and MDA contents, low chlorophyll content and protective enzyme activities compared to WT. The correlation analyses between protein and mRNA abundance are weak or limited. However, the changes of several important genes related to carbohydrate metabolism and apoptosis at the mRNA and protein levels were consistent. The protein-protein interaction (PPI) network might play accessory roles in promoting premature senescence of lses1 leaves. Comprehensive transcriptomic and proteomic analysis indicated that multiple key genes/proteins related to starch and sugar metabolism, apoptosis and ABA signaling exhibited significant differential expression. Abnormal increase in temporary starch was highly correlated with the expression of starch biosynthesis-related genes, which might be the main factor that causes premature leaf senescence and changes in multiple metabolic levels in leaves of lses1. In addition, four proteins associated with ABA accumulation and signaling, and three CKI potential target proteins related to starch biosynthesis were up-regulated in the lses1 mutant, suggesting that LSES1 may affect temporary starch accumulation and premature leaf senescence through phosphorylation crosstalk ABA signaling and starch anabolic pathways.Phenotypic identification and physiological measurements showed that the lses1 leaves, and emphasized the positive effect of excessive starch on accelerating premature leaf senescence. The expression patterns of genes/proteins related to starch biosynthesis and ABA signaling were analyzed via transcriptomes and proteomes, which provided a novel direction and research basis for the subsequent exploration of the regulation mechanism of temporary starch and apoptosis via LSES1/OsCKI1 in rice.The current study established the high correlation between the changes in physiological characteristics and mRNA and protein expression profiles in The online version contains supplementary material available at 10.1186/s12870-022-03510-2. Arabidopsis  was calculated accordingly, where p is the probability that an observed match is a random event.lses1 were identified by using the DESeq R package [p-value < 0.05 were regarded as DEGs. The hierarchical cluster analysis of DEGs according to the gene expression pattern in each sample was performed utilizing the pheatmap package in R [http://string-db.org/), and the functional clusters of the protein-protein interaction network containing DEPs were distinguished by the MCODE [The differentially expressed proteins (DEPs) and genes (DEGs) between WT and  package . Proteinage in R . Gene Onage in R . The prohe MCODE  plugin ilses1 mutant and WT were observed before the three-leaf stage. However, compared with WT plants, the older leaf blades of lses1 started to wither at the leaf tips from the third-leaf stage and began to exhibit a premature leaf senescence phenotype  network was constructed by integrating these proteins and the known STRING interactions and distinguishing the functional clusters via the MCODE  plugin flses1 in the following analysis.Thus far, comprehensive proteome and transcriptome studies have shown that the correlation between mRNA levels and protein expression across large data sets is generally weak , 8, 27. lses1 and WT  involved in starch metabolism, glyceraldehyde-3-phosphate dehydrogenase (GAPC) involved in glycolysis and trehalase (TRE) involved in trehalose metabolism also showed altered transcription levels , no results have been reported on the premature senescence and excess starch phenotype in the leaves of OsCKI1-deficient allelic mutants except lses1. Therefore, the lses1 mutant is an ideal material for studying the molecular mechanism of premature leaf senescence and temporary starch metabolism via LSES1/OsCKI1 and the pleiotropism of LSES1/OsCKI1 in rice.Assimilate partitioning has long been recognized as a target for crop improvement because it can limit the yield potential of crop plants. A phenotype involving an excessive accumulation of starch and growth retardation is often observed when any inhibition in the export of photoassimilate or in starch metabolism occurs in the leaves . In thisg. esl10 , ossac3  [ossac3  and ossa [ossac4 . Althouglses1 leaves , GBSSII (granule-bound starch synthase II), SSS1 (chloroplastic soluble starch synthase 1) and SSI (chloroplastic starch synthase I), all exhibited increased abundance in es1 Fig. . NotablyAM3 Fig. , which elses1 were almost all upregulated  and TRE  to maintain a stable sucrose content , which is involved in chlorophyll b biosynthesis [a degradation [lses1 mutant showed reduced transcription  in lses1 showed a higher protein abundance level than that in WT , SSS (soluble starch synthase) and GBSS (granule-bound starch synthase), causing imbalance in carbohydrate metabolism [lses1 mutant  and proteins (DEPs) revealed changes in several important metabolic mechanisms, such as carbohydrate metabolism, pigment metabolism, redox regulation and cell signaling. A possible schematic for the metabolic regulation mediated by differentially abundant genes/proteins was provided to explain the changes in multiple metabolic levels in the leaves of lses1  downstream of starch metabolism. The protein-protein interaction (PPI) network might play an auxiliary role in promoting premature leaf senescence in lses1. In addition, the possible potential roles of LSES1/OsCKI1 gene in cell signaling were discussed. LSES1/OsCKI1 might participate in the regulation of starch metabolism, cell apoptosis and ABA-mediated signaling, but future studies are required to verify it. Altogether, our study provides novel insight into the metabolic mechanism of temporary starch via LSES1/OsCKI1 in rice leaves.The es1 Fig. . The resAdditional file 1: Table S1. Detection data of various physiological characteristics in leaves of the wild type (WT) and the lses1 mutant. Table S2. Summary of the transcriptome sequencing quality. Table S3. Identification of differentially expressed genes in leaves of the wild type (WT) and the lses1 mutant. Table S4. Identification of differentially expressed proteins in leaves of the wild type (WT) and the lses1 mutant. Table S5. Protein-protein interactions of the four functional clusters. Table S6. List of partial differentially expressed genes/proteins related to carbohydrate metabolism. Table S7. List of partial differentially expressed genes/proteins involved in apoptosis-related metabolism. Table S8. List of differentially expressed genes/proteins involved in multiple metabolic regulation in schematic.


O-methylation of the hydroxyl groups in flavones, which is catalyzed by O-methyltransferases (OMTs). To examine the role of wheat OMT2 in methylated flavone biosynthesis and facilitate interrogation of tricin functions in wheat-environment interactions, loss-of-function mutants of OMT2 homoeologs, omt-A2 and omt-B2, were identified from a tetraploid wheat Targeting Induced Local Lesions in Genomes (TILLING) mutant population and crossed to generate the omt-A2omt-B2 double mutant. Although tricin and most other soluble phenolics did not differ in leaves and glumes of TILLING control and the omt-A2, omt-B2, and omt-A2 omt-B2 mutants, chlorogenic acid was increased in glumes of omt-A2 omt-B2 relative to TILLING control, suggesting that it might serve as a substrate for OMT2. The omt2 mutant lines showed similar growth phenotypes as well as comparable lignin deposition in cell walls of stems compared to TILLING control. These results collectively suggest that OMT2 and its close homolog OMT1 may possess overlapping activities in tricin production, with OMT1 compensating for the missing OMT2 activities in the omt2 mutant lines.Methylated flavones, and tricin in particular, have been implicated in protecting wheat plants against a variety of biotic and abiotic stresses. Methylated flavones are produced via  Arundo donax)  /CRISPR-associated (Cas)-based genome editing has demonstrated its utility in wheat functional genomics, providing a powerful tool to increase, decrease, or eliminate expression/function of target genes [Overall, the TILLING mutant-based genetic approach allowed exploration of OMT2 function in wheat plants and suggested the overlapping activities of OMT1 and OMT2 towards the production of methylated flavones and lignin biosynthesis. Because the current results strongly suggest compensating activities of OMT1 in the wheat et genes . A bettew/v) sodium hypochlorite and 0.1% (v/v) Triton X-100  for 15 min, followed by multiple rinses with water. The sterilized seeds were incubated at 4 °C for 1 week and then placed under a fluorescent light at room temperature for 2 days. The germinated seedlings were planted in soil and grown in a temperature-controlled greenhouse.Wheat seeds were sterilized in a solution of 1% , the number of spikes on each plant was counted, and the height of the tallest stem was recorded from the soil line to the top of the spike (not including the awns). Spikes were separated from the straw, and the straw was dried in an oven at 60 °C for 1 week and subsequently weighed. The spikes were dried at 37 °C for 1 week and weighed separately. The pseudo-harvest index was determined via dividing spike weight by total biomass .To determine the growth parameters of TILLING control and the omt-A2 and omt-B2 mutants in the Kronos background, the TILLING mutant database was searched using OMT-A2 and OMT-B2 sequences from hexaploid wheat cv. CS as a query via the program Basic Local Alignment Search Tool (BLASTn). Line 3521 (omt-A2) was backcrossed with the parental line Kronos to reduce the level of background mutations. The backcrossed plants were self-pollinated, and progenies genotyped using CAPS markers to identify homozygous omt-A2 plants.To identify tetraploid wheat omt-A2 omt-B2 double mutant, the homozygous omt-A2  was crossed with the homozygous omt-B2 . The progeny of the cross was allowed to self-pollinate and subsequently genotyped. Homozygous wild-type (OMT-A2 OMT-B2), single mutants , and double mutant (omt-A2 omt-B2) plants were identified. The polymorphic CAPS markers are OMT-A2, amplification with the primers 5′-AGTTTGCCCTCATGTAGTAGC-3′ (forward) and 5′- AATTAATGCATCAGATGGGAG-3′ (reverse), followed by digestion with the restriction enzyme BamHI; and OMT-B2, amplification with the primers 5′-TGTTCAGACAGTAGTAGCATCTCC-3′ (forward) and 5′-CATCAGAGGGGTAGACTCG-3′ (reverse), followed by digestion with the restriction enzyme BsrI.To generate the OMT-A2 and OMT-B2, transcriptome data of wheat cv. Azhurnaya based on the publication by Ramírez-González et al. [www.wheat-expression.com (accessed on 18 November 2021). Means and standard deviations of normalized read counts were calculated according to three biological replicates for each tissue type at each developmental stage and used for the preparation of bar graphs. Transcriptome data of hexaploid wheat cv. TAM 107 exposed to drought and heat stresses, and cv. N9134 inoculated with powdery mildew and stripe rust were obtained from Liu et al., 2015 [https://embnet.vital-it.ch/software/BOX_form.html (accessed on 18 November 2021)).To examine the expression of z et al.  were obtl., 2015 , respectl., 2015 , and thev/v) methanol with sonication for 20 min at room temperature and then centrifuged twice at 17,000× g for 10 min each. The supernatant recovered from each sample was transferred to an HPLC vial, and 10 µL was used for injection.Leaves from 4-week-old greenhouse-grown wheat plants and green glumes from spikes at approximate anthesis (plants at ~8 weeks old) were collected and immediately frozen in liquid nitrogen. The leaf and green glume tissues were ground in liquid nitrogen into a fine powder using a mortar and pestle. Approximately 100 mg of ground tissue was extracted in 350 µL of 70%  following a gradient between A (0.1% formic acid in water) and B (acetonitrile) at a flow rate of 1 mL min−1 as described previously [trans-cinnamic acid , were also analyzed using the same HPLC system. Individual peaks of interest were collected from multiple HPLC runs, concentrated in a vacuum centrifuge, and subjected to MS analysis. The MS analysis was performed with a Thermo Electron LTQ-Orbitrap mass spectrometer  in the positive mode using electrospray ionization and with a flow rate of 0.2 mL min−1. Ten μL of sample was injected for each run, and the m/z was observed between 200 and 2000 Da.Wheat metabolite extracts were separated on a reverse-phase HPLC using a Zorbax Stablebond Ceviously . Commercw/v) potassium permanganate  solution for 4 min and rinsed with water. The stem sections were then incubated in 1 mL of 3.7% (v/v) hydrochloric acid (Sigma-Aldrich) for 5 min. The hydrochloric acid solution was quickly removed, and 1 mL of ammonium hydroxide  was added immediately. Following removal of 500 µL of ammonium hydroxide, stem sections were pipetted onto 1 mm-thick glass slides and imaged using a RogRes CT Scan digital camera  with 10 × objective on an Olympus BH-2 compound microscope .Stems of 3-month-old wheat plants were cut at the midpoint between the flag leaf node and the spike into 2–3 cm sections using a razor blade. The stem sections were embedded in 5% agarose and sliced off into 50 µm sections using a Vibratome 1000 Plus sectioning system . The Mäule staining procedure was modified slightly from a previously established method . BrieflyThe plant growth and metabolite data were analyzed in R using Analysis of Variance (ANOVA) and Tukey’s Honestly Significant Difference (HSD) test.


National Science Review and to share information about the different pathways being followed by the International Union of Pure and Applied Chemistry (IUPAC) toward a worldwide sustainable society.It is a pleasure to write this contribution for IUPAC was founded in 1919 with a strong impetus from chemical industries that recognized the importance of using a common language, particularly for chemical compounds, so that they would be able to communicate with each other. After IUPAC’s beginnings in Europe, it expanded and has become a truly worldwide organization, representing the global interests of the chemistry community and scientific communities associated with chemistry. These include academic institutions, industry and society at large. IUPAC’s vision of being a global indispensable source of chemistry is embodied in our mission to provide objective scientific expertise and develop the essential tools for applying and communicating chemical knowledge to benefit humankind and the world. This is achieved through our providing a common language for chemistry, advocating the free exchange of scientific information, and fostering sustainable development. Scientific excellence and objectivity, diversity and inclusiveness, are IUPAC core values that are particularly important in order to give objective advice and recommendations as a response to the many challenges that are faced by chemists, the scientific community and society. 000 people in 136 countries. Following its huge success, a second edition was launched in June 2020 and has been equally successful; there are now versions in Chinese, Arabic, Spanish and Russian. The Chinese version has already been taken by several thousand people.Our centenary year, 2019, was also the International Year of the Periodic Table of the Chemical Elements (IYPT), of which IUPAC was the leading partner. The many events held all over the world demonstrated the enthusiasm for chemistry by all, of different ages and from all parts of society. It enabled us to show the fascination for the chemical elements and emphasized how many positive things they do for us in contributing to our lives. A Periodic Table Challenge was held, taken by over 65Other centenary year initiatives that continued in 2020 and are continuing in 2021 are the Global Women's Breakfast (GWB), and the Top Ten Emerging Technologies series of articles. Highlighting these new technologies is a way for IUPAC to help show how chemists are contributing to adapting the chemical industry to new needs, with new materials and using processes that are less energy intensive. The Periodic Table of Younger Chemists, another IYPT initiative, in collaboration with the International Younger Chemists’ Network, has led directly to a new project: ChemVoices, the international voice of the younger chemists, which began a series of webinars in September 2020.Pure and Applied Chemistry. The ‘Color Books’, provide standards and standardized nomenclature in different areas of chemistry. It will be particularly important to develop Cheminformatics standards, of which the international chemical identifier (InChI) is an excellent example. Pure and Applied Chemistry also publishes themed scientific issues, many of them linked to important contributions made in IUPAC-endorsed conferences. Our publication Chemistry International highlights important news items and the results of our projects, and Chemistry Teacher International is our publication for educators.Core IUPAC activities continue, of course, and we continue to develop chemical nomenclature, with recommendations and technical reports, all open access, appearing in our journal We value our collaborations with international organizations such as UNESCO, International Science Council (ISC), International Bureau of Weights and Measures (BIPM), the Organization for the Prohibition of Chemical Weapons (OPCW) and the International Union of Pure and Applied Physics (IUPAP). For example, IUPAP collaborates with us to determine when a new element has been synthesized and identified. IUPAC was awarded the Hague award of the OPCW in 2019 for its work in collaboration with OPCW to further the peaceful uses of chemistry.IUPAC’s 2300 volunteers are from all over the world, representing scientists in different areas of chemistry and multidisciplinary areas with the best skills and knowledge. We have 54 national adhering organizations (NAOs), and over 800 Affiliate Members, Company Associates and Associated Organizations. Together we identify current and emerging scientific issues and problems and reach consensus on approaching and solving them. The Chinese Chemical Society joined IUPAC as an NAO in 1979. Since then, chemical sciences and technology in China have undergone fast development and now China is one of the most important countries in the chemistry field worldwide. China has played an important role in IUPAC. Prof. Chunli Bai has served in the IUPAC bureau and executive committee. Prof. Qifeng Zhou has served as IUPAC President in 2018–2019; under his leadership, IUPAC coordinated the IYPT activities with great success.The world is facing many challenges in health, food and the environment. The need for a multidisciplinary and global approach, in all senses, becomes paramount. Particular challenges at the moment in chemistry are sustainability, new materials and reduction in energy needs, whilst moving to renewable energy, which need to integrate new technologies. We are fomenting activities in green chemistry and sustainable development, building on our links with industry. We have projects in responsible care, education and training. We have a particular focus on inspiring younger chemists and reaching out to those parts of the world in greatest need, a principal tenet of a sustainable society. Within chemistry, sustainable chemistry must be our guide, not only in the context of a circular economy but also by first identifying what we need and then examining which ways to achieve it, tailoring the processes and cycles to demand rather than to supply, making them more efficient and sustainable.www.iupac.org for more information about our activities in IUPAC and internationally. We very much look forward to increased involvement by Chinese chemists in our projects for the future.We can only achieve these goals, which are crucial for a sustainable society, through the strong collaboration of all who are linked to the chemical sciences. Please visit our website


Pseudomonas syringae pv. tomato (Pst), is one of the most pervasive biological adversities in tomato cultivation, in both industrial and in table varieties. In this work synthesis, biochemical and antibacterial properties of a novel organic nanostructured pesticide composed of chitosan hydrochloride (CH) as active ingredient, cellulose nanocrystals (CNC) as nanocarriers and starch as excipient were evaluated. In order to study the possibility of delivering CH, the effects of two different types of starches, extracted from a high amylose bread wheat (high amylose starch—HA Starch) and from a control genotype (standard starch—St Starch), were investigated. Nanostructured microparticles (NMP) were obtained through the spray-drying technique, revealing a CH loading capacity proximal to 50%, with a CH release of 30% for CH-CNC-St Starch NMP and 50% for CH-CNC-HA Starch NMP after 24 h. Both NMP were able to inhibit bacterial growth in vitro when used at 1% w/v. Moreover, no negative effects on vegetative growth were recorded when NMP were foliar applied on tomato plants. Proposed nanostructured pesticides showed the capability of diminishing Pst epiphytical survival during time, decreasing disease incidence and severity (from 45% to 49%), with results comparable to one of the most used cupric salt (hydroxide), pointing out the potential use of CH-CNC-Starch NMP as a sustainable and innovative ally in Pst control strategies.Bacterial speck disease, caused by  Pseudomonas syringae pv. tomato (Pst), which is able to produce symptoms on fruits, leaves and stems. Bacterial cells from the epiphytic phase can penetrate living tissue through stomata or microlesions aroused by leaves pruning and harvesting operations, but also by environmental agents, such as wind, hail or rain, causing the appearance of chlorotic specks that necrotize quickly [Punica granatum peel extracts on Pst [Magnolia tamaulipana was able to inhibit almost 90% of Pst growth at concentration of 125 ppm, while a strong in vitro antibacterial activity of essential oils extracted from different plants such as mint, thyme and eucalyptus was recorded on Pst, due to the presence of phenolic compounds [®) in oral formulations [Recent advances in nanotechnology have shown the concrete possibility of minimizing agrochemicals used in agriculture, especially those ones related to plant protection strategies . The gre quickly ,5. Necro quickly . Althoug quickly ,8,9. Cop quickly ,11. On t quickly ,13,14,15s on Pst . Morin, s on Pst . Ethanolompounds ,19. Chitompounds ,21. Due ompounds ,24,25,26ompounds ,28. Seveompounds ,30,31,32ompounds . Biologiompounds ,35,36. Tompounds ,38. Antiompounds ,41,42,43ompounds ,45,46. Aompounds ,48,49,50ompounds ,52. Addiompounds . In ordeompounds . Starch ompounds ,56. Depeulations ,58. Withulations .In this work, we evaluated the possibility of fabricating a novel ternary delivery system by the spray-draying technique, using CNC as nanocarriers for chitosan hydrochloride (CH), with the addition of standard starch or high amylose starch as possible release modifiers. Obtained nanostructured microparticles (NMP) were characterized for their biochemical properties, including effects on bacterial and plant growth in comparison with copper. To the best of our knowledge, this is the first time that a fully organic nanostructured pesticide is produced for the specific management of tomato bacterial speck disease.Chitosan hydrochloride (CAS No. 70694-72-3) was purchased at Wuhan Victory Excellence Pharmaceutical Co., Ltd. . Microcrystalline cellulose (CAS No. 9004-34-6) was purchased at Sigma-Aldrich, Inc.  Standard starch and high amylose starch were extracted from the cultivar Cadenza and Cadenza SBEIIa, respectively. This latter line was recently produced by Botticella et al. (2018) through the silencing of a key gene involved in starch biosynthesis, the starch brancing enzyme of class IIa (SBEIIa) . All othCellulose nanocrystals (CNC) were synthetized by acid hydrolysis, as described by Cranston and Gray (2006) . MicrocrAcid hydrolysis yield was calculated using the method reported as UNI EN ISO 638:2009. CNC morphology was investigated by field-emission microscope Zeiss LEO 1525 equipped with a GEMINI column . Samples for SEM analysis were prepared by depositing CNC suspensions onto an aluminum specimen stub covered with a double-sided adhesive disc. The sample was coated with a chromium layer (8 nm thickness) before imaging . −1. The analyses were performed directly on the powders with no sample processing. Background spectra were acquired on the empty cell.Absorption attenuated total reflection (ATR-IR) spectra were recorded with the Infrared Spectrophotometer IRSpirit  in the spectral range of 400–5000 cmg, the pellet was rinsed twice in 20 mL deionized water. The upper yellow-gray layer deposited on the surface of the starch pellet was manually removed with a spatula, and the starch left to dry for two days at room temperature.Starch granules were extracted from whole flour using the dough ball method as described in Botticella et al. (2018) . Ten graThe morphology of starch granules was analyzed using a scanning electron microscopy (SEM) Hitachi S-4000 , as reported by Sparla et al. (2014) . Amylose content was determined from 15 mg aliquot of purified starch through a colorimetric assay based on an iodine-amylose reaction . A standFor each analysis, three biological samples, each with three technical replicates, were measured either for Cadenza or Cadenza SBEIIa. In order to define the most effective CH antibacterial concentration useful to assess the starting requirements for NMP preparation and in vivo application, as well as to investigate possible St Starch, HA Starch, and CNC antimicrobial effect, an in vitro assay was conducted.8 CFU mL−1. Concentration of bacterial suspension was adjusted until an OD600 = 1 was reached using a spectrophotometer [−1 was made. KB medium plates were amended with the different substances to reach 0.05, 0.1, 0.5 and 1% w/v concentrations. KB medium was used as negative control, while copper hydroxide at 0.09% w/v (field dose) was used alone as a positive control [Antimicrobial effects of substances were recorded using an agar incorporation method . Pst  in the following conditions: inlet temperature 140 °C; aspirator rate 50%; feed rate 2.4 mL/min; and air pressure 357 L/h. The powders were recovered and stored at room temperature and 30% relative humidity until use. An established amount of CNC was suspended in ultrapure water to obtain a concentration of 15 mg/mL. CH and St Starch or HA Starch were dissolved in the CNC suspension at 50:30:20 CH:CNC:Starch w/w ratio. The suspensions were then nebulized by a Büchi mini spray-dryer B-290 , and the solution was then shaken. Subsequently, 1 mL of dinitrosalicylic acid reagent , prepared as reported elsewhere [2 = 0.999). CH standard solutions were prepared as follows. A CH solution was pipetted into test tube, and 0.1 mL of 0.5 M NaNOlsewhere , was addThe CH content (%CC w/w) in the CH-CNC-Starch NMP formulations was performed by dispersing an exactly weighed amount of NMP in 0.1 M acetic acid solution. The solution was incubated for 1 h at r.t. and periodically sonicated and vortexed, then treated according to the method for chitosan quantitation. All analyses were performed in triplicate, and the results expressed as mean ± standard deviation (SD).6 CFU mL−1 was made, after Pst was grown on KB medium at 27 °C for 48 h. Dilutions at 0.05, 0.1, 0.5 and 1% w/v were made for each NMP with LB broth. 198 μL of each dilution were pipetted in well of a 96 multiwell microplate. Then, 2 μL was of the bacterial suspension was added. LB broth was used as negative control, while copper hydroxide at field dose was used as positive control. For each thesis, a blank was made using only substances. Plates were incubated at 27 °C for 72 h. In order to study the possible bacteriostatic and bactericidal effects of NMP on Pst and their trend over time, at 48 and 72 h absorbance of plates at 600 nm was measured using a spectrophotometer. MIC has been identified as the one where inhibition percentage was equal to 100%. Inhibition of bacterial growth was expressed as a percentage, and calculated as below after subtracting the blank:Antimicrobial effects of obtained NMP with different starches were recorded using a microdilution method ,70. A baObtained NMP were tested in vivo to assess their biocompatibility with living plant tissues. For each thesis, ten tomato seedlings of a local cultivar (San Marzano Scatolone) were picked up from an organic plant nursery, and have been acclimatized in a greenhouse with 75% air humidity, a 25/15 °C day/night thermoperiod and a 16/8 day/night photoperiod. Applied concentration of NMP was determined on results of antibacterial in vitro and quantification of loaded CH on NMP assays, as shown in the Result and Discussion paragraph. Two suspensions at 1% w/v were made with the two different NMP and sprayed manually all over the leaves and the stems, until a uniform coating was reached. Deionized sterilized water and copper hydroxide were used as controls. One, seven and fourteen days post-treatment , the following parameters were recorded as stated for each thesis: three random leaves per plant were picked up and their area was measured with the software ImageJ (version 1.51j8)  (accessed on Windows 10)  ; three m6 CFU mL−1 Pst water-suspension was obtained after the bacteria was grown on KB medium for 48 h at 27 °C, and homogeneously spray-inoculated on leaves and stems. Plastic bags were placed on plants one day before and after the inoculation to increase the air humidity and promote the stomata’s opening [In order to investigate the effect of the synthetized NMP on the disease development in a plant, an in vivo assay was performed. Ten tomato seedlings for each thesis were collected and treated as described in paragraph 2.6. 24 h after treatments, a 10 opening . The exp−2 [The effect of the NMP on Pst epiphytic survival was monitored one, seven and fourteen days post-inoculation . Three random leaves per plants were collected and washed in a sterile plastic bag with a pH 7 phosphate buffer 0.05 M using a homogenizer  set at 110 rpm for 30 s. For each sample, serial decimal dilutions were obtained, and a 100 μL aliquot was gently plated on sucrose nutritive agar dishes. Each dilution was plated three times and incubated a 27 °C for 48 h. After that, developed bacterial colonies were counted and divided for the tomato leaf area measured with ImageJ; results were expressed as CFU cm−2 .Symptomatic expression of bacterial speck disease was monitored on leaves for the entire duration of the trial. Disease severity (DS) was calculated as the number of point-like necrosis per plant at 7 dpi. Proportional disease reduction per plant (DR) was also calculated as below:Disease incidence (DI) was calculated for each thesis at 7 and 14 dpi as the percentage ratio between total symptomatic (necrotized) area and total area of the collected leaves for the assessment of Pst epiphytic survival . post hoc test. p-values less than 0.05 were considered significant, while p-values of less than 0.01 were considered highly significant. Release data were analyzed by paired Student’s t-test at 95% significance level. Collected data were analyzed using one-way analysis of variance (ANOVA). Statistical significance of means was studied with Tukey’s HSD CNC were successfully obtained through acid hydrolysis of microcrystalline cellulose with a total yield of about 20%. The method performance was consistent with relevant literature. SEM images show the typical needle shape of CNC with a certain homogeneity in size . The CNCMorphological analysis of starch granules highlighted marked differences between Cadenza and Cadenza SBEIIa . In thisp < 0.05). In particular, the amylose contents were 32% and 63.1% of the total starch respectively . CH showed a total inhibition, comparable to copper hydroxide’s one, at both 0.5% and 1% w/v concentrations. MIC was established for chitosan hydrochloride at 0.5% w/v since lower tested concentrations have shown no significant activity on Pst growth. CNC showed no inhibition effects on Pst, and so both types of ST when used at 0.5% and 1% w/v concentrations. Lower concentrations of ST instead seem to promote Pst growth. This behavior is coherent with the ones showed in previous works, where chitosan, although in different forms, was able to explicate an antibacterial effect on several plant pathogenic bacteria [3 groups present in chitosan molecules, which easily interact with negatively charged bacterial cell membrane. This interaction could lead to an alteration of membrane permeability, resulting in an inefficient capability of exchange molecules from outer to inner cell environment; Gram-negative bacteria, due to their higher presence of negative charges onto their membranes, appear to be more sensitive to chitosan [Pst growth inhibition was reported as a percentage . Experimbacteria ,32,33. Cchitosan ,74. Othechitosan ,76,77,78The obtained NMP showed good CH content very closed to the theoretical value of 50% w/w, as shown in The NMP showed overlapping size distributions with average size ranging around 2.9–3.8 µm . The ana−1 are predominant in the NMP profiles regardless of the starch type employed  on Pst growth was recorded for CH-CNC-HA Starch NMP and CH-CNC-St Starch NMP when used at 1% w/v (p < 0.01). Eventually, no bactericidal effect was recorded for tested substances, except for copper hydroxide, since Pst colonies formation was observed in plates after incubating the bacterial suspensions. The decrease in Pst inhibition during time and the absence of recorded values of MBC for tested NMP could find an explanation if CH antimicrobial effects are considered as bacteriostatic rather than bactericidal [In vitro antibacterial activity of both proposed NMP was tested using a microdilution method. Results were expressed as percentage as reported in t 1% w/v a. Similat 1% w/v b. Howeveericidal ,78. ObtaBiological parameters linked to plant basal physiological functions, such as leaf development and nitrogen use efficiency, were monitored in tomato seedlings in order to study the compatibility of tested NMP with plant growth, as reported in p < 0.05) reduction in terms of bacterial epiphytic survival, with 1.37 × 101 CFU cm−2. At 7 dpi, both tested NMP in a comparable way to the copper hydroxide, showed an highly significant (p < 0.01) effect on Pst epiphytic survival. N° of CFU cm−2 in plants treated with copper was equal to 1.20 × 103, while for CH-CNC-HA Starch NMP and CH -CNC-St Starch NMP was 2.58 × 103 and 2.88 × 103, respectively. At 14 dpi, NMP with high amylose starch and copper hydroxide showed both a highly significant (p < 0.01) reduction of bacterial epiphytic survival recording values of 9.59 × 103 and 5.84 × 103 CFU cm−2  in developed point-like necrosis on tomato seedlings treated with both NMP with respect to the untreated sample; in particular, CH-CNC-HA Starch NMP has registered a mean value of 20.6 necrosis per plant, while CH-CNC- St Starch NMP has registered a mean value of 19.1 necrosis per plant. Necrosis count on plants treated with copper showed a reduction of 34.1%, while for NMP with high amylose starch and for NMP with standard starch values of 49.9% and 45.9% were reached  started to appear since the fifth day after inoculation. At 7 dpi, all plants belonging to different theses showed symptoms. DS calculated at 7 dpi showed a highly significant difference . At 7 dpi, theses treated with both NMP showed a disease incidence of 3%, while copper hydroxide at field dose 2.3%. Similar trend was recorded at 14 dpi, with value of disease incidence equal to 3, 2.7 and 3.5% for theses treated with CNC-SHA-CH NMP-HA Starch, CH-CNC-St Starch NMP and copper hydroxide, respectively . In order to facilitate chitosan hydrochloride dispersion on plant canopy and to control its release during time, a nanostructured pesticide was synthetized by the spray-drying technique. Cellulose nanocrystals, due to their chemical properties—among which was the already documented biocompatibility—were proposed as nanocarriers. At the same time, two different types of starch, extracted from a standard and a high amylose content bread wheat cultivars, were investigated to assess their effects on chitosan hydrochloride release. Results confirm the possibility of using the Cadenza SBEIIa genotype as source of high amylose content starch. Obtained nanostructured microparticles were characterized for their biochemical properties, revealing a mean size of 2.9–3.8 µm, and an achieved loading of 50% w/w in terms of chitosan hydrochloride content. In vitro chitosan release assay has, as expected, shown a higher release for microparticles embedded with high amylose starch, capable of releasing half of loaded chitosan after 24 h, while microparticles with standard starch reached the 30%. Both of microparticles when used at 1% w/v were able to fully inhibit bacterial growth after 48 h, showing a bacteriostatic effect that persists at 72 h, with a slightly decrease for microparticles with standard starch. In vivo experiments assessed the complete biocompatibility of proposed nanostructured pesticides when foliar is applied at 1% w/v, demonstrating no harmful effects on the vegetative development of tomato plants. At the same time, both microparticles were able to diminish the epiphytical bacterial survival and the disease incidence at seven and fourteen days after artificial Pst inoculation. At seven days after inoculation, a significant drop in disease severity with a proportional symptoms reduction was observed, ranging from 45% to 49%, comparable to the one showed by copper hydroxide. This study highlights the concrete possibility of using an organic nanostructured pesticide in replacement of copper, opening new scenarios in sustainable tomato crop protection strategies against bacterial diseases. Further studies have been programmed in order to better clarify chitosan hydrochloride effects on tomato resistance induction, and to investigate nanostructured microparticles behavior in field, as well as to study, through the application of fluorescent markers, uptake and translocation of proposed nanopesticides in living plant tissues.The present study describes the effects of an innovative green organic nanostructured compound to counteract tomato bacterial speck disease. Obtained results showed how chitosan hydrochloride, a more water-soluble form of chitosan, possesses a full inhibitory activity on


Journal completes an overwhelmingly successful launch year and sets our trajectory straight toward the calmer waters enjoyed by more established publications, with a steady equilibrium of the right type of papers in different stages of processing. As predicted, the Journal is indeed filling an important gap in the scientific literature by providing a forum for practitioners and translational researchers in the wide area of preventive cardiology to feature work ranging from state-of-the-art reviews to practice recommendations, from teaching and training to pragmatic research. We are approaching the 100 mark on submissions, representing not only the diversity of specialists in preventive cardiology – physicians, pharmacists, dietitians, and researchers - but also the global reach of our discipline, with contributions from several countries, including Turkey, India, Australia, and Argentina. The Journal could not have gotten to this sustainable point so fast without the tremendous support from the American Society for Preventive Cardiology, our expert colleagues at Elsevier Publishing, the diverse international editorial board, and in particular our tight team of associate editors.This fourth issue of the American Journal of Preventive Cardiology, on schedule and on target with more than a dozen contributions, including several reviews on, among other issues, the European and US cholesterol guidelines, the burden of cardiometabolic risk in the Asia-Pacific region, nutritional strategies for cardiovascular disease prevention, and the hot topic of inflammation in cardiovascular disease. Some excellent original contributions are included, touching on issues ranging from racial disparities in cardiovascular disease to hospitalization rates in persons with diabetes to the cardio-metabolic risk in those with non-alcoholic fatty liver disease.We are pleased to present to you this year’s final issue of the Journal, and the large and growing number of quality submissions currently in process promises to make the AJPC the “to go” place for implementable work in preventive cardiology. We are now in the process of applying for PubMed indexing, as testimony to the incredible progress achieved in just one year.We are working hard at making 2021 a boom year for the Journal to continue its outreach strategy to those engaged in the practice of preventive cardiology around the world, encouraging submissions featuring practice models, training systems, pragmatic research, and practice recommendations representing our field at an international level. Further, the Journal will continue to be a key educational resource for the members of the American Society for Preventive Cardiology, and a forum to feature their research and perspectives on the practice of this medical art we call Preventive Cardiology.It is the hope of the Journal and to all in this beautiful world of ours.We count on the continued support of our editorial board and of experts in the field from around the world in reviewing and submitting work and in helping us establish AJPC as the top target publication for those who live and breathe preventive cardiology. With all challenges that 2020 has thrown into our professional and daily lives, we must celebrate the small victories and everything that brings us together, and look into the future with love, confidence, and passion. Our best wishes of a healthy 2021 to our


Thermoplastic starch composites have attracted significant attention due to the rise of environmental pollutions induced by the use of synthetic petroleum-based polymer materials. The degradation of traditional plastics requires an unusually long time, which may lead to high cost and secondary pollution. To solve these difficulties, more petroleum-based plastics should be substituted with sustainable bio-based plastics. Renewable and natural materials that are abundant in nature are potential candidates for a wide range of polymers, which can be used to replace their synthetic counterparts. This paper focuses on some aspects of biopolymers and their classes, providing a description of starch as a main component of biopolymers, composites, and potential applications of thermoplastics starch-based in packaging application. Currently, biopolymer composites blended with other components have exhibited several enhanced qualities. The same behavior is also observed when natural fibre is incorporated with biopolymers. However, it should be noted that the degree of compatibility between starch and other biopolymers extensively varies depending on the specific biopolymer. Although their efficacy is yet to reach the level of their fossil fuel counterparts, biopolymers have made a distinguishing mark, which will continue to inspire the creation of novel substances for many years to come. Plastic manufacturing and distribution across the world have been steadily increasing throughout time. Petroleum-based plastics are commonly employed as single-use plastics in our daily lives as they have become incredibly useful due to certain qualities, such as versatility, durability, flexibility, and toughness . They arTherefore, to overcome this issue, a change needs to be made by replacing the commonly used petroleum-based plastic with biodegradable plastic to sustain a better environmental condition for future generation and also to provide less plastic disposal techniques. Among biopolymers, starch is the one of promising possibility and alternatives to replace petroleum-based plastics, which is due to properties of starch that is completely biodegradable and are found to be abundant in nature. It is widely accessible in plants such as corn, cassava, potato, tuber, and many more. In plants, starch is stored in the granule packed form present in an amorphous and crystalline condition . Due to As part of the awareness of global environmental issues concerning plastic waste, bio-based polymers are now promoted as alternatives for replacing petrochemical-based (fossil) polymers to supply increasing demand . This inMany research works have been previously conducted on biopolymer products derived from natural sources. In general, the animal-based biopolymer refers to the gelatin, which can be extracted from the skin and bone of porcine, bovine, poultry, and fish ,15,16. MStarch is the main component of the biopolymer structure that belongs to the polysaccharides group and is considered the second most crucial renewable sources after cellulose. It is inexpensive and completely biodegradable, generating great interest to make it into a component of bioplastic. Starch is known to be a versatile material since it can be converted into chemicals such as ethanol, acetone, and organic acids used in synthetic polymer production, such as polylactic acid (PLA)  and can The hydroxyl group in the starch structure exhibit the reaction of oxidation and reduction processes as well as the formation of hydrogen bonds, ethers, and esters. Native starches contain about 70–85% amylopectin and 15–30% amylose. The presence of amylopectin is mainly responsible for the crystallinity of the starch granules . HoweverNeat starch displays some disadvantages due to its high solubility in water, brittleness, poor melting point, and lower mechanical properties as compared to materials made of synthetic polymers. To enhance the properties of starch, various physical or chemical modifications, such as plasticisation, blending, derivation, and graft copolymerisation were investigated ,32,33,34+][Ac−]) [+][Cl−]) [Ac−]) ,37 and 1+][Cl−]) . Besides+][Cl−]) .(Coccinia abyssinica) starch film. Glycerol, 1-ethyl-3-methylimidazolium acetate, sorbitol, and tri-ethylene glycol were utilised at concentrations of 30% and 40% w/w in the starch mixture. The thermoplastic film samples were then dried overnight in the oven at 50 °C before they were kept in a desiccator for at least two days for film characterisation. The prepared film was transparent, homogenous, and flexible. The same sample preparation was reported by Reference [3 among other samples. It was found that the water solubility of glycerol and tri-ethylene glycol plasticised film had reduced from 32.57 g/cm3 to 20.97 g/cm3 and 34.19 g/cm3 to 18.92 g/cm3, respectively. According to Reference [A comprehensive study was conducted by Demash and Miyake  regardineference . The obt2) using an activated sludge degradation method. The samples are pre-weighed before being immersed in the sludger under an aerobic condition for 5, 10, and 15 days and taken out to be weighed again after being cleaned and dried in an oven at 75 °C. The result obtained showed a degradation range between 6.2–16% for 0, 0.5, 1.0, 1.5, and 2.0 of lithium perchlorate. It presents that the percentage of weight lost increased as the amount of lithium perchlorate was added. This might be attributed to the presence of lithium perchlorate that promoted free volume in the polymeric matrix and, thus, enhance the biodegradability rate [Nevertheless, biodegradability of the film plasticized with ILs does not specify in the study which could be one of the interesting points that need to be highlighted. Across the journal available, there is also limited research that working on the biodegradability of ILs as a plasticizer. However, Rynkowska et al.  reportedity rate . On thatIn the production of biopolymers, starch is mainly used as a matrix or resin in bio-composite structures. Throughout past decades, numerous types of natural starch have been investigated, such as cassava starch, corn starch, sugar palm starch, and much more. However, cassava starch is the highest contributor in terms of productivity yield compared to other sources of starch . Some reRecent studies have been accomplished on polymer composites of treated oil palm mesocarp fibres (OPMF) and thermoplastic cassava starch properties prepared by using the screw extrusion rheometer method . Two typThe obtained results from the Scanning Electron Microscopy (SEM) for raw and alkaline-treated OPMF and Energy Dispersive Spectroscopy (EDS) are presented in Corn starch is seen as a promising source of biopolymer matrix that is environmentally friendly and suitable for replacing petroleum-based plastics. Pure corn starch is similar to other natural starches, which have some drawbacks such as high-water sensitivity and poor mechanical behaviour. Thus, modification of starch is required by introducing plasticisers in the mixture. Plasticised starch properties have enhanced abilities. Thermoplastics from corn starch have been widely explored and various modifications were accomplished to improve the properties of the resulting TPS. The recent studies conducted had utilised waxy corn products in their works. It was reported that waxy corn produces starch that is nearly 100% amylopectin . Table 2Fitch-Vargas et al.  studied On the other hand, a moisture adsorption test is carried out to identify the sample’s affinity toward the surrounding moisture condition. The samples are placed into two different relative humidity (RH) desiccators conditions at 53% RH and 100% RH. Weight gained among the samples are taken until constant weight are found, indicating that the samples have reached an equilibrium state. As per To date, numerous research works utilising sugar palm starch in the production of bio-composite materials as an alternative to petroleum-based polymers have been accomplished. Natural starch from sugar palm trees was found to be abundant within the tree trunk . Many auSanyang et al.  briefly w/w%. The results from this work revealed that, from the fracture surface analysis, as the glycerol content increased, acquiring a smooth surface also increased. At 15 w/w% glycerol loading, a brittle surface was the outcome, while an exceptionally smooth surface appeared at 40 w/w% glycerol loading. The glycerol, which acts as a plasticiser, effectively reduced the internal hydrogen bond while increasing the intermolecular spacing. This resulted in the appearance of a smooth surface.Thermo-mechanical studies have been conducted by Sahari et al.  in an atIn terms of the product’s mechanical properties, as illustrated in Modifying thermoplastic starch can be accomplished through blending, which widens its application at lower cost. Compatibility of blends must be tested to examine the enhancement of the composite’s properties such as water resistance, high tensile strength, and high tensile modulus. Blending is also important for modifying bioplastics in terms of biodegradability, enabling them to decompose easily through natural processes so as to cater to the issue of limited disposal technology. Starch blending with a polar polymer containing hydroxyl groups such as polyvinyl alcohol, ethylene copolymer, and partial hydrolysed vinyl acetate have been prepared since the 1970s . BlendinPVA is a synthetic biodegradable polymer that has the advantages of good film formation, strong conglutination, high thermal stability, and good gas barrier properties . PVA is The compatibility of PVA and starch enables them to form a continuous phase at blending  even thoPLA, a biodegradable polyester produced from renewable resources, is used for various applications such as biomedical, packaging, textile fibres and technical items. PLA is industrially obtained through the polymerisation of lactic acid through the ring opening polymerisation (ROP) method . PLA is,To improve PLA’s flexibility and impact resistance, numerous plasticisers are incorporated, such as poly (ethylene glycol), glycerol, glucose monoesters, citrate esters, and oligomers . TPS, asPBS can be found commercially as a thermoplastic-based polymer, which provide a high degree of crystallinity and takes a long time to degrade. PBS is a synthesis of polycondensation succinic acid and 1,4-butanediol where succinic acid can be obtained from either a monomer derived through a petroleum system or bacterial fermentation. Meanwhile, 1,4-butanediol is derived from fossil fuel extraction from formaldehyde or acetylene . PBS proThe implementation of natural fibre as a promising alternative for replacing synthetic fibre in the composite industry has been increasing in the past decades. Synthetic fibres such as glass, carbon, and aramid reinforced with polymer matrix materials provide advantages of high stiffness and strength-to-weight ratio in numerous applications as compared to conventional construction materials . ChangesThe production of synthetic polymers utilise a large quantum of energy, which produces environmental pollutants during the production and recycling of synthetic composites . Table 5Natural fibres reinforced composites are also applied as fillers in a matrix to provide high strength and stiffness on a weight basis to a composite product . Fibre rManihot esculenta C., is a root and tuber crop found in many tropical countries. Therefore, it is used as the main source of food. It is estimated that cassava could have a yield potential of up to 17,000 kg of starch per hectare per year if it is cultivated in a suitable environment with organised farming practices [Cassava, also known as ractices . Cassavaractices . Cassavaractices . The culractices . This alMany studies had focused on exploiting waste by utilising cassava bagasse as a filler or reinforcing agent to enhance the strength and mechanical properties of material products. This is especially conducted for the production of bio-based or bio-degradable materials, which have been steadily increasing. These by-products are low in market value and possess good properties with a wide range of applications, such as reinforcing agents or in the production of organic acid, bio-degradable packaging, nano particles, nanofibres, ethanol, bio-fuel, lactic acid, etc. . Paula eRecently, the valorisation of sugarcane waste for low-cost building products has been increasing due to its high potential. Since there is a vast global production of sugarcane, especially in some tropical and subtropical developing countries, high amounts of sugarcane fibres are produced as by-products . FortunaWith regard to the incorporation of natural fibres such as sugarcane into starch-based composites, numerous studies have reported and demonstrated significant improvements in the resulting bio-composite products. Fitch-Vargas et al.  stated tGrowing interest on modification of surface fibre have been implemented to increase the reinforcement strength for the bio-composite. Surface modification treatments can be classified into three categories: chemical, physical, and biological ,83. One The influence of surface treatments on bamboo fibres as reinforcement agents in TPS were conducted by Jhon et al. . In thisThe development of starch blending is more interesting since it can substitute an older material while exhibiting the same properties. w/w% with a maximum tensile value of 2.42 MPa. Besides, a reduction of transition temperature and water absorption as glycerol are added, indicating that the biocomposite has reduced its brittleness as glycerol loading increased. Meanwhile, Sanyang et al. [w/w%). The results collected show the storage modulus of plasticized SPS decreased as the plasticizer concentration was added from 15–45%, which reflect the stiffness of the material is reduced. Glycerol plasticized film portrays the highest degree mobility of the polymer chain among other plasticizers.Sahari et al.  discoverg et al.  studied Polymer films made from poly-, low-density polyethylene, poly-, or polybutylene are used for mulch manufacturing . These fOliveira et al.  studied PBS has been exhibited as mulch film, packaging, and flushable hygiene products and is considered as a non-migrant plasticizer for polyvinyl chloride (PVC) . MoreoveUtilization of natural fibre incorporated with advantages as compared to synthetic fibre in terms of tensile properties, less health hazard, acceptable insulating properties, low density, and less energy consumption . A possiThis paper conducts a general overview on biopolymers and the potential of starch-derived thermoplastics as substitutes for current petroleum-based plastic. Blending starch with other biopolymers was outlined as a viable alternative to overcome the shortcomings of native starch. However, the degree of compatibility between starch and other biopolymers extensively differs depending on the specific biopolymer. At present, mixing TPS and PLA offers significant advantages in terms of cost, properties, and biodegradability.Regarding the global environmental issue, biodegradable material properties are vital and should be considered. Although starch/biodegradable blends are a good option for solving environmental issues, their mechanical properties often have an inverse relationship to their degradability. Incorporating natural fibres as fillers in the starch matrix can be another solution. Thus, optimising their mechanical properties requires further analyses.


Reverse vaccinology is an outstanding strategy to identify antigens with high potential for vaccine development. Different parameters of five prediction programs were used to assess their sensitivity and specificity to identify B-cell epitopes of Chikungunya virus (CHIKV) strains reported in the IEDB database. The results, based on the use of 15 to 20 mer epitopes and the polyproteins to which they belong, were compared to establish the best parameters to optimize the prediction of antigenic peptides of the Mexican strain CHIKV AJV21562.1. LBtope showed the highest specificity when we used the reported epitopes and polyproteins but the worst sensitivity with polyproteins; ABCpred had similar specificity to LBtope only with the epitopes reported and showed moderate specificity when we used polyproteins for the predictions. Because LBtope was more reliable in predicting true epitopes, it was used as a reference program to predict and select six novel epitopes of the Mexican strain of CHIKV according to prediction frequency, viral genome localization, and non-homology with the human proteome. On the other hand, six bioinformatics programs were used with default parameters to predict T-cell epitopes in the CHIKV strains AJV21562.1 and AJV21561.1. The sequences of the polyproteins were analyzed to predict epitopes present in the more frequent HLA alleles of the Mexican population: DQA1*03011, DQA1*0401, DQA1*0501, DQB1*0201, DQB1*0301, DQB1*0302, and DQB1*0402. Fifteen predicted epitopes in the non-structural and 15 predicted epitopes in the structural polyprotein (9- to 16-mers) with the highest scores of each allele were compared to select epitopes with at least 80% identity. Next, the epitopes predicted with at least two programs were aligned to the human proteome, and 12 sequences without identity with the human proteome were identified as potential antigenic candidates. This strategy would be useful to evaluate vaccine candidates against other viral diseases affecting the countries of the Americas and to increase knowledge about these diseases. Chikungunya virus (CHIKV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the genus Alphavirus of the family Togaviridae ,2. It coPhylogenetic analyses have revealed the presence of three separate lineages of CHIKV strains: West Africa, Asia, and East/South/Central Africa (ESCA), and these lineages are ~92.5–98% identical at the amino acid sequence level . AnalyseAedes aegypti and Aedes albopictus circulate and can transmit this arbovirus. Patients usually have a fever, rash, and joint pain that can persist for months or years, and some patients develop destructive arthropathy/arthritis [Infections caused by CHIKV generate annual epidemic outbreaks in tropical and subtropical regions in the world where the mosquito species rthritis  that canIn 2014, more than 1 million suspected cases of chikungunya disease were reported in the Americas. In 2015, in the same region, 693,489 suspected cases and 37,480 confirmed cases of chikungunya fever were reported to the Pan American Health Organization (PAHO) regional office, of which Colombia bore the biggest burden with 356,079 suspected cases. In 2016, a total of 349,936 suspected and 146,914 laboratory-confirmed cases were reported to the PAHO regional office, i.e., half the burden compared to the previous year. Countries reporting the most cases were Brazil , Bolivia, and Colombia   and 755 Vaccination would be of great relevance for the control of this viral infection, and diverse attempts have been made to develop vaccines against CHIKV; however, no approved vaccines are available so far. Thus, it is necessary to develop strategies to design safe and effective vaccines with low cost to apply in countries where CHIKV is endemic.Since the alphavirus E2 glycoprotein is the major target of the host immune response , linear In this work, we aimed to establish the best parameters of five computational methods to optimize the prediction of linear B-cell antigenic peptides in the polyprotein of the Mexican CHIKV strain AJV21562.1. In addition, T-cell epitopes of CHIKV with wide allelic distribution in the Mexican population were predicted through six other computational methods. Sequences of 15- to 20-mer B-cell epitopes retrieved from the IEDB database and the polyprotein sequences to which they belong were analyzed using five bioinformatics programs , and the parameters were adjusted to predict a high percentage of the same sequences. The optimized parameters were used to predict B-cell epitopes of the structural polyprotein of the Mexican strain, and the results were compared with the predicted T-cell epitopes in order to identify novel B- and T-cell antigens to develop vaccine candidates.Epitopes of the alphavirus CHIKV or Sindbis virus structural proteins;Linear B-cell epitopes;Epitopes positive or highly positive by ELISA test using sera from humans infected with CHIKV (positive controls); andEpitopes negative by ELISA or another serological test using sera from humans or primates (negative controls).Epitopes from the IEDB database, Version 2.20, were selected as controls based on the following criteria:The amino acid sequences of positive and negative controls were analyzed with the programs LBtope , ABCpredABCpred predicts immunodominant linear B-cell epitopes based on artificial neural networks (machine learning technique) using fixed-length patterns from 10- up to 20-mers . LBtope The predicted epitopes with more than 80% sequence identity to the control epitopes were used to calculate sensitivity (Sn) and specificity (Sp) according to these formulas:TP  = positives by ELISA and predicted with any program;TN  = negatives by ELISA and no predicted with any program;FN  = positives by ELISA and no predicted with any program;FP  = negatives by ELISA and predicted with any program.The sequences of the structural polyproteins of the alphavirus PDB ID: 3N42 (DQ462750), PDB ID: 3N44 (DQ462746) , PDB ID:The programs LBTope , ABCPredLBtope with a threshold of 0.7 was chosen as a reference program to select similar epitopes predicted with the other programs and construct a binary matrix. Briefly, the predicted epitopes in each program were compared with the 20-mer-long epitopes predicted with the LBtope program. Epitopes predicted with at least 60% identity (12 amino acids) with the epitopes predicted by LBtope were labeled with 1; the epitopes not predicted or with less than 60% identity were labeled with 0. The prediction frequency of each epitope was calculated based on the number of programs in which it was predicted.To select epitopes with the highest antigenic potential, we clustered epitopes with more than 70% overlapping amino acids. Groups containing 10 to 14 overlapping epitopes were divided into two subgroups to homogenize the length of the epitopes in ~20-mers, whereas the epitopes with less than 75% prediction frequency were excluded.All the epitopes of each group or subgroup were superimposed forming a longer peptide that included all the sequences without repeating them. Finally, amino acids with 100% identity (core) were identified to determine representative antigenic sequences with no identity with the human proteins. To this end, the selected CHIKV sequences were aligned with the human proteome (TaxId: 9606) using the BLAST program, Version 2.10.1, under default conditions.Antigenic T-cell epitopes were identified through a similar approach conducted to identify B-cell epitopes. Briefly, the sequence of the structural and non-structural polyproteins of the CHIKV strains  retrieved from GenBank were analyzed with the default parameters of six computational programs , a combinatorial library (PROPED), and a combined consensus approach trained with the complete dataset ) to deteFifteen predicted epitopes in non-structural and 15 predicted epitopes in the structural polyprotein (9- to 16-mers), with the highest scores of each allele, were compared to select epitopes with at least 80% identity. The prediction frequency of each epitope was calculated based on the number of programs in which it was predicted, and the epitopes predicted by fewer than two programs were discarded. Finally, the selected sequences were compared with the human proteome (TaxId: 9606) using the BLAST program, Version 2.10.1, under default conditions.The sequences of the new epitopes predicted in this work, localized in the structural polyprotein of CHIKV were mapped in the crystal structure of the protein retrieved from the PDB ID 3N41 by using the PyMOL Molecular Graphics System, Version 2.0 .To determine whether the predicted epitopes induce T- and B-cell immune responses, we analyzed the sequence of the CHIKV strain AJV21562.1 with VaxiJen . In thisTwenty-four positive epitopes and 33 negative epitopes were selected from the IEDB database, in which they were registered with positive or highly positive immunoreactivity or negative against CHIKV antibodies in infected human sera .We obtained predictions with high Sp values (0.515–0.939) using a threshold of 0.7 to 0.85 . All proThe programs with the highest Sp were LBtope (Sp 0.939) and ABCpred (Sp 0.909), followed by BCPred and FBCPred (both with Sp 0.727) and AAP (Sp 0.515) . In contTo confirm the validation of the programs, we retrieved from PDB the alphavirus sequences PDB ID: 3N42 (DQ462750), PDB ID: 3N44 (DQ462746) , PDB ID:The program with the highest Sp was LBtope (Sp 0.909), followed by FBCPred and BCPred (each with Sp 0.636). The ABCpred program showed a slightly lower Sp (0.606) than FBCPred and BCPred . Based oLBtope was selected as the best predictor program due to its highest Sp using sequences of positive control epitopes and structural polyproteins of CHIKV. Therefore, this program (dataset variable_non_redundant) with a threshold of 0.7 was used to predict 171 (20-mer-long) epitopes from the CHIKV strain AJV21562.1, which were compared to epitopes predicted with ABCPred, FBCPred, and BCPred with a threshold of 0.8 each. A total of 145 epitopes with more than 60% identity were identified. Fifty-two epitopes (30.40%) were predicted with one program in addition to LBtope, 76 epitopes (44.44%) were predicted with two additional programs, and 17 epitopes (9.94%) were predicted with three or more programs.After elimination of 17 sequences with more than 80% of identity to the reported sequence in the IEDB database, used as positive and negative controls , 13 clusAn epitope with 45% similarity to the human immunoglobulin heavy chain junction human region (ID MOP20971.1 and MOK15443.1) was deleted. Thus, six sequences of 20–26 amino acids were identified as novel antigens potentially immunogenic and without identity to the human proteome, which would avoid cross-reactivity with human antigens .A total of 210 T-cell epitopes predicted with bioinformatics programs were selected for the seven DQ alleles, with more frequency in the Mexican population. Ninety-five (45.2%) epitopes were predicted with the highest scores in most of the programs, of which 21% had the same length, 60% shared at least 80% identity, and 19% had >80% identity and the same scores in all programs. Finally, 12 sequences predicted by two or more programs with the highest scores were identified as potential antigenic candidates . None ofThe structural polyprotein from the PDB ID: 3N41 sequence was modeled by using the PyMOL Molecular Graphics System, Version 2.0 , to map The T-cell epitopes EC17-RPG (orange) and EC38-MTN (red) were partially exposed, whereas EC20-MVL (cyan) and EC15-EFA (blue) were embedded in the E1 protein .The epitopes pep2, EC6-CST, EC18-QPL, and EC4-GVG could not be mapped in the 3N41 protein. In addition, we identified two new epitopes of the nsP1 and one epitope in the protein nsP2, nsP3, and nsP4 proteins (data not shown) restricted to the MHC class II complex.The CHIKV strain AJV21562.1 sequence was analyzed with VaxiJen . The preIn order to improve the specificity and sensitivity of our prediction of B-cell epitopes, several thresholds in computational programs were used to analyze the prediction of control epitopes retrieved from the IEDB. As we expected, increasing the prediction thresholds in some programs allowed us to discriminate between positive and negative epitopes and to choose true antigenic epitopes. The LBtope and ABCpred programs predicted the least amount of FP and had the highest Sp using control epitopes. Therefore, they were the most reliable programs, even though they had the lowest Sn . HoweverIn general, when alphavirus polyproteins sequences were used to predict CHIKV epitopes, Sn increased and Sp decreased in all programs. This suggests that more TP and more FP could be predicted, as more neighboring amino acids flank the epitopes, since changing an amino acid disrupts the epitope/antibody interaction . In thisSeveral of the positive control epitopes have not been predicted as TP, probably due to different algorithms and models of the bioinformatics programs used, since the programs are based on different properties of the proteins or do not take into account the reconfiguration of epitope residues when an antigen is in complex with a specific antibody . HoweverMost antibodies against CHIKV recognize the region between amino acids 3 and 10 (STKDNFNVYK) of the peptide E2EP3 STKDNFNVYKATRPY , which wNarula et al.  identifiIn addition, with both strategies, we predicted a TP epitope and one FP epitope containing 30% and 50%, respectively, of the B-cell epitope PLVPRNAELGDRKGKIHIPF .On the other hand, the amino acids LPCST of the CD4+ epitope LPCSTYVQSNAATAEE  were ideBased on the above findings, we suggest that the prediction of FP with bioinformatics programs does not depend on sequences used.The CHIKV sequences used in this work had 80% amino acid conservation, even with other alphaviruses. Therefore, the epitopes predicted and selected with our strategy could be effective to bind to antibodies from patients in different endemic regions.We consider that our new epitopes of CHIKV identified in silico have high antigenic potential, since they were predicted using a strategy based on a similar prediction of epitopes already validated experimentally, which is completely different from the other in silico strategies used so far. Furthermore, as some of our new epitopes have some percentage of similarity with the epitopes reported by Narula et al. , we expeIn addition, to determine the antigenicity of all of the 18 newly identified B and T-cell epitopes, we analyzed the sequence of CHIKV strain AJV21562.1 with VaxiJen , consideWe evaluated different conditions within several computer programs to increase the probability of prediction of antigenically known positive and negative epitopes of alphavirus, and this allowed us to identify six new linear B-cell epitopes of CHIKV with high antigenic potential. Using six computational methods, we improved the identification of 12 new T-cell epitopes to induce cellular immune responses against CHIKV. This work also shows that it is necessary to adjust the threshold of the prediction programs to eliminate many FP and increase the specificity and probability of predicting true antigens by immunoinformatics for the development of safe and effective vaccines regardless of the viral strains. Our strategy would be useful for developing vaccine candidates against other viral diseases.


Increased tibial slope (TS) is believed to be a risk factor for anteriorcruciate ligament (ACL) tears. Increased TS may also promote graftinsufficiency after ACL reconstruction.To delineate the relationship between TS and single as well as multiple graftinsufficiencies after ACL reconstruction.Cohort study; Level of evidence 3.We retrospectively identified 519 patients who had sustained ACL graftinsufficiency after primary or revision ACL reconstruction . In addition, a subgroup analysis was conducted in63 patients who received all surgical interventions by 2 specializedhigh-volume, single-center ACL surgeons. TS was measured by an observer with>10 years of training using lateral knee radiographs, and intraraterreliability was performed. Multiple logistic and univariate Cox regressionwas used to assess the contribution of covariates  on repeated graft insufficiency and graftsurvival.r = 0.48; P <.0001). TS was significantly lower in group A (9.0°± 2.3°) compared withgroup B  and group C . A significant correlation was seenbetween the TS and age at index ACL tear  as well as time to graft insufficiency. A TS ≥12° had anodds ratio of 11.6 for repeated ACL graft insufficiency.The study included 347 patients, 119 female and 228 male, who were 24 ± 9years of age at their first surgery . Mean TS was 9.8°± 2.7° . TS produced thehighest adjusted odds ratio (1.73) of all covariates for repeated graftinsufficiency. A significant correlation was found between TS and the numberof graft insufficiencies (The current results indicate that patients with a markedly increased TS wereat risk of early and repeated graft insufficiency after ACL reconstruction.Because the TS is rarely accounted for in primary and revision ACLR,isolated soft tissue procedures only incompletely address recurrent graftinsufficiency in this subset of patients. The functional importance of the anterior cruciate ligament (ACL) has been widelyacknowledged. After injury, reconstruction of the ACL is deemed necessary to restorenative knee kinematics close to the physiological state and to allow athletes to returnto high-level pivoting and cutting sports. These graft insufficiencies are worrisome, because revision ACLR is associatedwith worse outcomes compared with primary ACLR.33Surgical techniques for ACL reconstruction (ACLR) continue to evolve and to improveclinical results. Even though early results appear to be satisfactory, outcomeparameters in the long term seem to deteriorate and the true incidence of graftinsufficiency after ACLR is likely underreported.31 An emerging consensus indicatesthat from a biomechanical view, an increased TS facilitates an anterior translation ofthe tibia relative to the femur.23 Clinical studies have echoedthese results, reporting an association between an increased TS and graft insufficiencyafter ACLR.29 However, there is a considerablepaucity in the current literature regarding the TS in large revision ACL cohorts, andits effect on singular and multiple graft insufficiencies has not been delineated.Even though the contributions of peripheral soft tissue restraints on sagittal kneelaxity have been validated extensively, the importance of the bony geometry of theunderlying tibial plateau is less well understood. However, a growing body of evidencehas provided a refined view on the effect of the posterior inclination of the tibialplateau—namely, the tibial slope (TS)—on anteroposterior knee laxity and loading of thecruciate ligaments. The TS is rarely accounted for in surgical decision making in revision ACLR, thuspotentially representing a nonmodifiable risk factor for repeated graftinsufficiency.Of note, the susceptibility for graft insufficiency is dependent not only on anatomicconditions but also, and to a greater degree, on age, acquired injuries, graft tissue,graft position, treatment of additional peripheral instabilities, and postoperative rehabilitation.The aim of the current study was to evaluate the TS in patients with single and multiplegraft insufficiencies after ACLR. It was hypothesized that the TS would be significantlyhigher in patients with multiple graft insufficiencies compared with those with a singlegraft insufficiency.A total of 519 patients who underwent revision ACLR between 2012 and 2018 due tograft insufficiency were retrospectively identified from a single-center, 2-surgeonseries . Inclusion criteria consisted of single or multiple ACL graftinsufficiencies after ACLR, confirmed by magnetic resonance imaging (MRI) and/orclinical as well as arthroscopic assessment.Patients with concomitant injuries of the posterior cruciate ligament (PCL), combinedlower extremity fractures, or previous osteotomy of the ipsilateral knee wereexcluded. In addition, we excluded patients whose radiographs did not include a truelateral view of the tibia as determined by proper superimposition of the femoralcondyles. Patients with poor-quality or nondigital radiographs were not enrolled inorder to decrease the number of confounding variables.Anthropometric and demographic measures including age and sex were documented.Additionally, for each patient, we reported the number of graft insufficiencies , graft choice, the patient’s age at first ACL injury orACLR, and the subsequent time interval (months) between ACLR and graftinsufficiency. This information was also collected for a subgroup of patients whounderwent both the primary ACLR and subsequent revision surgeries by either of the 2specialized high-volume ACL surgeons .The study protocol was approved by our institutional ethics committee (EA2/016/21).The study was carried out in accordance with the Declaration of Helsinki, and allparticipants provided informed consent.Lateral knee radiographs of the affected knee were used for radiographicassessment. An orthopaedic surgeon (C.G.) with >10 years of experience inradiographic measurements evaluated the TS using a picture archiving andcommunication system workstation . Theobserver was blinded for all other parameters. To account for readinginaccuracy, 50 patients were randomly selected and measured again at 6 monthsafter the initial assessment to establish intrarater reliability. using 2 midpoints between the anterior and posterior tibial cortex: oneat 5 cm below the tibial tuberosity and the other at 15 cm below the tibialjoint line. Subsequently, the TS was determined between the tangent line to themedial tibial plateau, which is commonly superimposed on lateral radiographs,and a line 90° to the diaphyseal axis. Although the best available radiographswere chosen, not all images included enough tibial shaft to determine the widthof the shaft at a distance of 15 cm distal to the joint line. In theseinstances, the most distal width (if ≥12 cm) was measured based on the aforementioned conditions  was used to evaluate intraraterreliability. Notably, values of 0.5 through 0.6 are regarded as moderateagreement, whereas 0.7 through 0.8 indicate a strong agreement.P < .05 was considered to be statisticallysignificant.Statistical analysis was performed using Prism Version 6 (GraphPad Software).Continuous data are expressed as mean ± SD or median according to theirdistribution. D’Agostino and Pearson omnibus normality tests were used to testGaussian distribution. Parametric data were compared with a Of the 519 patients who were identified after sustaining ACL graft insufficiency,patients were excluded due to missing (n = 137) or low-quality (n = 13) radiographs,additional PCL reconstruction (n = 17), or tibial osteotomy (n = 5). Consequently,347 patients  were enrolled in the present study . The meaAccording to the number of graft insufficiencies, 260 patients were enrolled with 1graft insufficiency (group A), 62 patients with 2 graft insufficiencies (group B),and 25 patients with ≥3 graft insufficiencies . Patientcharacteristics among the subgroups are described in Multiple logistic regression was performed to assess the relative contribution ofage, sex, bilateral ACL tears, and TS on the number of graft insufficiencies. Theresults are shown in r = 0.48; P < .0001).The mean TS was 9.8°± 2.7°  . A signiP < .0001) as wellas between groups A and C . No significant difference was seen betweengroups B and C   . we found that the rate of sustaining repeated graft insufficiencies wassignificantly higher in patients exceeding this cut-off (64%) compared withthose who had a TS <12° (13%) (P < .0001). Morespecifically, a TS ≥12° produced an odds ratio of 11.6 for repeated ACL graftinsufficiency.Using a cutoff of ≥12°, as proposed by Webb et al,r =−0.12; P = .02). The interval between ACLR and first graftinsufficiency was 57 ± 61 months. As well, we found a significant correlationbetween the TS and the time to graft insufficiency . Graft survivorship after ACLR was significantlyimpaired in patients with TS ≥12° .The interval between revision ACLR and second graft insufficiency was 37 ± 35months. This is significantly shorter than the time between ACLR and first graftinsufficiency . This is in line with theregression analysis, which showed a weak association between female patients andrepeated graft insufficiency.We compared the TS in female (n = 119) versus male (n = 228) patients. Nosignificant difference was seen regarding the TS in both cohorts . A total of 49 male (22%) and 38 female (32%) patientssustained repeated graft insufficiency, which resulted in a weak, yetsignificant, difference .Ipsilateral hamstring tendons (n = 236) were predominantly used during indexACLR, followed by ipsilateral patellar tendons (n = 74) and quadriceps tendons(n = 5). In 32 patients, the primary graft choice was unknown. In the hamstringtendon group, 56 patients (24%) had >1 graft insufficiency. In the patellartendon group, 21 patients (28%) had >1 graft insufficiency. However, thisdifference did not reach statistical difference.P = .024) and patellar tendon grafts(P < .0001). However, the TS was significantly higher inpatients with patellar tendon grafts (11.3°± 3.1°) than in patients withhamstring tendon grafts  and quadricepstendon grafts . The TS in patientsreceiving hamstring tendon grafts and quadriceps tendon grafts was notsignificantly different.For revision ACLR, autologous hamstring tendons of the ipsilateral (if notpreviously used) or contralateral side were used in 268 patients. Quadricepstendons were used in 48 patients and patellar tendons in 18 patients. The graftwas unknown in 13 patients. In this ACL revision setting, 48 (18%) of thehamstring tendon grafts, 16 (33%) of the quadriceps tendon grafts, and 18 (100%)of the patellar tendon grafts failed. In revision ACLR, hamstring tendon graftsshowed a significantly lower rate for repeated insufficiency compared withquadriceps tendon grafts (P < .001) compared with those with multiple graft insufficiencies (n = 6)  .Because the graft choice for primary and revision ACLR was predominantlyipsilateral or contralateral hamstring tendons, and bone-tendon-bone orquadriceps tendons were sparsely used, no further analysis on the graft choicewas conducted in this cohort.The results of the current study indicate an association between an increased TS andthe incidence of repeated graft insufficiency after ACLR. More specifically, kneeswith a radiographic TS ≥12° had a 64% incidence of repeated graft insufficiency,which produced an odds ratio of 11.6 for repeated ACL graft insufficiency. Notably,the TS also significantly correlated with a younger patient age at first ACL injuryand further correlated with a shorter time between primary surgery and first ACLgraft insufficiency. Consequently, patients with a markedly increased TS are at riskof sustaining an ACL tear early in their lives and are at further risk of animpaired survival rate as well as survival time of their ACL graft.20 Although proper choice of graft tissue, graft position,tensioning, treatment of additional peripheral instabilities, and postoperativerehabilitation continue to be the mainstays for good clinical outcomes, theinfluence of the underlying osseous geometry of the operated knee on clinicaloutcome has long been underestimated. Yet, the TS may be considered a common,nonmodifiable risk factor for successive failures. Numerous studies have identified an increased medial as well lateral TS to bea risk factor for both primary ACL injury and ACL graft insufficiency. Of note, these findings contrast with a recent case-control study thatdelineated the TS in 317 revisions. Cooper et al failed to observe an association between graft insufficiency and medial aswell as lateral TS as measured on MRI. Interestingly, the authors found twice asmany graft insufficiencies with a TS >12° than below this threshold. However,this result did not reach statistical significance after correction formultiplicity. The conflicting results between Cooper’s study and our findings mightbe attributable to the fact that Cooper et al recruited their cohort from a largedatabase in which the number of involved surgeons, their degree of specialization,and surgical volume were unknown. Notably, the TS of the single-center patients inthe current study, who were treated in the same surgical fashion by 2 highlyspecialized ACL surgeons, was significantly higher in patients with multiple graftinsufficiencies than in those with a single graft insufficiency.Despite recent advances in primary and revision ACL surgery, a considerable number ofpatients do not recover from revision ACLR, clinical results remain poor comparedwith primary procedures, and the risk of repeated graft insufficiency isconsiderably higher. demonstrated that a TS >12° on lateral radiographs may lead to a 5-foldincrease in odds of graft insufficiency after primary ACLR. These results have beenechoed by recent studies22 and are in line with the results of the current study using anACL revision cohort. As an implication of this, isolated soft tissue procedures mayonly incompletely address anterior stability in this subset of patients.Webb et alOur findings clearly indicate that a high TS significantly correlates with the numberof repeated graft insufficiencies and has a detrimental effect on graftsurvivorship. Even though the TS appears to be smaller if assessed on MRI, radiographs havebeen determined to provide an accurate and objective quantification of the TS aswell as a high correlation with MRI.16 In addition, the clinical useof radiography might be greater than the use of MRI assessment, which is moretime-consuming and requires specialized software. Finally, this study does not indicate whether reducing the TS can change theodds of a subsequent graft failure.A few limitations of this study should be noted. Because we did not account forclinical endpoints, the clinical applicability in regard to patient-reportedoutcomes or resumption of sporting activities may be limited. Furthermore, thecurrent cohort included only patients with symptomatic graft insufficiency who wereundergoing revision ACLR. Because we did not include an asymptomatic ACLR controlgroup, results may be only partially applicable to patients undergoing primary ACLR.Additionally, because most of the patients underwent their index ACLR in otherinstitutions, data such as tunnel positioning, associated injuries, rehabilitation,and return to activity were missing. Thus, potential differences among subgroupscannot be delineated. Of note, the results of the single-center patients whoreceived a standardized treatment and rehabilitation confirm the results of theoverall cohort. However, some potentially pertinent factors, such as activity level,could not be considered. Another drawback is that we measured the TS on a standardradiograph, thus potentially ignoring a possible asymmetry between the medial andlateral TS.In this study of 347 patients with ACL graft insufficiencies, we showed that patientswith a markedly increased TS were at risk of early and repeated graft insufficiencyafter ACLR. This was particularly distinct in patients with a TS >12°, whichproduced an odds ratio of 11.6 for repeated ACL graft insufficiency. Consequently,measurement of the TS should be an integral part of failure analysis in cases of ACLgraft failure. Future studies need to identify a threshold of the TS that predictsrecurrent graft insufficiency after isolated soft tissue revision ACLR.