The content of resistant starch (RS) was considered positively correlated with the apparent amylose content (AAC). Here, we analyzed two Indica rice mutants, RS111 and Zhedagaozhi 1B, similar in high AAC and found that their RS content differed remarkably. RS111 had higher RS3 content but lower RS2 content than Zhedagaozhi 1B; correspondingly, cooked RS111 showed slower digestibility. RS111 had smaller irregular and oval starch granules when compared with Zhedagaozhi 1B and the wild type. Zhedagaozhi 1B showed a B-type starch pattern, different from RS111 and the wild type, which showed A-type starch. Meantime, RS111 had more fa and fb1 but less fb3 than Zhedagaozhi 1B. Both mutants showed decreased viscosity and swelling power when compared with the parents. RS111 had the lowest viscosity, and Zhedagaozhi 1B had the smallest swelling power. The different fine structures of amylopectin between RS111 and Zhedagaozhi 1B led to different starch types, gelatinization properties, paste viscosity, and digestibility. In addition to enhancing amylose content, modifications on amylopectin structure showed great potent in breeding rice with different RS2 and RS3 content, which could meet the increasing needs for various rice germplasms. Resistant starch (RS) is a kind of starch and starch degradation products that are resistant to be digested in the small intestine but can be fermented in the large intestine by microbial flora to produce metabolites such as short-chain fatty acids (SCFAs). RS plays a beneficial role in controlling blood sugar and regulating intestinal flora . With thWaxy (Wx), which encodes granule-bound starch synthase (GBSS) I, can regulate RS formation through impacting amylose synthesis (BEIIb plays an important role in forming short amylopectin chains and the deficiency of BEIIb (ae mutant) in rice can significantly increase in RS content by increasing the number of B2 and B3 branched chains . These results provided opportunities to increase RS content of rice through genetic improvement.Rice is the staple food for more than half of the population worldwide and the primary source of nutrition and carbohydrates for many people. RS2 and RS3 is the main RS type that exists in raw and cooked rice, respectively . Hot cooynthesis . BEIIb pd chains . In addilopectin  has beenssIIIa mutant) and Zhedagaozhi 1B (unknown mutant), respectively, which were similar in high amylose but different in RS properties. Clarifying discrepancies in RS and starch physicochemical properties of rice similar in high amylose content will provide some guidance for high-RS rice breeding and diversified healthy and functional foods.Up to date, enhancing amylose content is an important approach to develop high-RS rice and functional foodstuffs . Some mu60Co-γ rays. RS111 is an ssIIIa mutant , milled to white rice using a Satake Rice Machine , and then ground into flour  and passed through a 100-mesh sieve.Starch was extracted according to a previous method described by Apparent amylose content (AAC) was determined by the simplified assay as Starch sample was directly adhered to double-sided adhesive tape mounted on an aluminum stub and coated under vacuum with platinum for 50 s . All coated samples were observed with a Hitachi SU8010 cold field emission SEM  operated at 3.0 kV.X-ray diffraction pattern was performed with copper Kα radiation on an x-ray diffractometer . The scanning regions of the diffraction angle 2θ were 4°–40°, which covered most of the significant diffraction peaks of the starch crystallites, with a 0.02°-step size and a count time of 0.2 s. The crystallinity was calculated according to the method of 2O and then sonicated for 5 min. The sample was loaded to the sample port by drops until 8%–12% obscuration.Granule size of starches was detected using a laser light scattering particle size analyzer . Starch (50 mg) was mixed with 15 mL of ddH-1 to 400 cm-1 with 32 scans at 4 cm-1. FTIR spectra were treated in peak half-width of 19 cm-1 and the resolution enhancement factor of 1.9. Then the relative absorbances at 1,045, 1,022, and 995 cm-1 were extracted from the deconvoluted spectra and were measured from the baseline to the peak height according with Starch granule short-range ordered structure was detected with the Fourier transform infrared (FTIR) spectrometer . The scanning range was from 4,000 cmAmylopectin fractionation was conducted as To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy change (ΔH) were analyzed with a Universal Analysis Program, Version 4.4A. The range of GT was calculated as R = Tc − Tp.Thermal properties of starch were determined by a differential scanning calorimeter (DSC)  equipped with DSC standard and dual sample cells. Starch  was weighed into an aluminum pan, and 6 μL of MilliQ water was added . The panGelatinization properties was determined according to the method of Pasting properties of rice flour were analyzed using a Rapid Visco Analyzer  according to the methods described by Digestion property was studied using a standard protocol as reported by The percentage of the starch hydrolysis was calculated by the following equation (1):Where SH is the percentage of starch hydrolysis, Sh is the amount of hydrolyzed starch at different time points in intestinal phase, Si is the initial amount of starch in the samples, and Gp is the amount of glucose released due to starch hydrolysis. The conversion factor of 0.9 was used based on the molecular weight ratio of a starch monomer to glucose.The experiment data were fitted to a first-order model according to equation (2), as previous reported by t, C0, and C∞ are the percentage of digested starch at time t, 0, and infinite time, respectively, and k is a pseudo first-order rate constant. Box Lucas model as the nonlinear curve fit model in OriginPro2022  was used for estimating k and C∞ value. Initial digestion rate in the first 20 min (IRR20) was calculated by equation (3) as described by where C20 and C0 are the percentage of digested starch at 20 min and 0 min.where CAll the tests were conducted in triplicate at least. The statistics analysis was conducted using Duncan with Tukey’s multiple comparison tests following one-way analysis of variance with SPSS version 20.0 .The AAC of R7954 and II-32B were both around 25% and significantly lower than those of their mutants. Meantime, R7954 and II-32B had similar RS content Table 1.Considering that other components in the flour might also affect the RS content, the RS content in isolated starch were also determined. The RS2 content in isolated starch was higher than those in four for all samples. The RS3 content in the isolated starches was a little lower than those in flours for R7954, II-32B, and RS111 while higher for Zhedagaozhi 1B. Meantime, the contents of RS3 in mutant RS111 were 3.67%, lower than that in Zhedagaozhi 1B starch (4.06%). The higher RS2 in isolated starch indicated that the other components in the flour might reduce the attack of amylase to the starch granules. Although the difference in RS3 content between starch and flour might because of the existence of lipids in rice flour, Zhedagaozhi 1B had significantly higher lipids content Table 1.The four varieties showed obvious different morphology of grain . Wei et −1 and 1,022/995 cm−1 can reflect the degree of order structure and the proportion of amorphous to ordered carbohydrate structure in starch granule external region, respectively , fb1 (13 ≤ DP ≤ 24), fb2 (25 ≤ DP ≤ 36), and fb3 (DP ≥ 37), corresponding to fractions A, B1, B2, and B3 and longer chains, respectively . R7954 hThe differential CLDs between RS111 and Zhedagaozhi 1B might explain the differential short ordered structure Table 1,To), peak GT (Tp), conclusion temperature (Tc), and enthalpy of gelatinization (ΔHgel) varied significantly among four materials. The To, Tp, Tc, and ΔHgel of Zhedagaozhi 1B were higher than those of RS111 for both starch and flour, which was consistent with the difference between their parents. When compared with their parents, RS111 had lower To, Tp, Tc, and ΔHgel, while Zhedagaozhi 1B had higher To, Tp, Tc, and ΔHgel. Meantime, the value of R(Tc-To)of mutant Zhedagaozhi 1B was significantly higher than other varieties, and the differences of thermal characteristics between starch and flour were more significant for Zhedagaozhi 1B, which may greatly be influenced by the non-starch components such as lipids. GT has been found to be negatively correlated with DP6-11 and positively correlated with DP12-24 . RS111 showed a lower digestion extent than that of Zhedagaozhi 1B (∞) and the digestion rate (k) of RS111 was significantly lower than that of Zhedagaozhi 1B (∞ were affected by the digestion time, the experimentally determined values of C120 were also presented, which were similar as C∞ (in vitro (Cooked flours were homogenized to mimic chewing and then digested using the standard INFOGEST2.0 method . The digin vitro  and Zhedin vitro . The RS3RS111 and Zhedagaozhi 1B had similar AAC with around 34%, significantly higher than their wild-type parent and common rice. Although two wild types had similar properties, RS111 and Zhedagaozhi 1B conferred different physiochemical properties, such as viscosity, gelatinization parameters, swelling power, and digestibility. RS111 had higher RS3 content but lower RS2 content than Zhedagaozhi 1B; correspondingly, cooked RS111 showed slower digestibility. Differences in physiochemical properties might be due to the different starch structure between RS111 and Zhedagaozhi 1B. The B-type starch granule and high proportion of fb3 in Zhedagaozhi 1B may be responsible for its high-RS3 content, while the smaller irregular and oval starch granules, high proportion of fa in RS111 might be the major contributor to its rich RS3. As RS111 and Zhedagaozhi 1B are derived from restorer and maintainer, respectively; they can be used directly as new rice varieties, or as parents for high-RS hybrid rice breeding through improving other restorers and maintainers. Fine structure of amylopectin seemed very important for determining the RS type and content when amylose content is similar. Modifications on amylopectin structure showed great potential in breeding rice with different RS2 and RS3 content, which could meet the increasingly needs for various rice germplasms.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.ML: Methodology, Data curation, Investigation, Validation, Writing – original draft. WG: Data curation, Formal Analysis, Methodology, Writing – original draft, Visualization. SZ: Investigation, Data curation, Methodology, Writing – original draft. LX: Data curation, Investigation, Writing – original draft. YS: Data curation, Investigation, Writing – original draft. DW: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing. XS: Formal Analysis, Methodology, Visualization, Writing – review & editing, Funding acquisition.


These blends were prepared with a different content of [Emim+][Ac−] (27.5–42.5 %wt.) as a unique plasticizer. According to the results, this ionic liquid is an excellent plasticizer due to the transformation of the crystalline structure of the starch to an amorphous state, the increase in flexibility, and the drop in Tg, as the [Emim+][Ac−] amount increases. These findings show that the properties of these biomaterials could be modified in the function of [Emim+][Ac−] content in the formulations of TPS, depending on their final use, thus becoming a functional alternative to conventional polymers.The first step towards the production and marketing of bioplastics based on renewable and sustainable materials is to know their behavior at a semi-industrial scale. For this reason, in this work, the properties of thermoplastic starch (TPS)/polyvinyl alcohol (PVA) films plasticized by a green solvent, as the 1-ethyl-3-methylimidazolium acetate ([Emim Ionic liquids (ILs) are gaining interest in the field of plastic production because they have the potential to act as plasticizers in polymer matrices  due to pIn industry, ILs are commonly used as catalysts for the production and storage  of energIn the past, some researchers have investigated the effect of ILs as a solvent in the research of biodegradable and renewable materials. However, recently, ILs have been used to study their plasticizer effect in biodegradable materials ,29,30,31+][Ac−]), among other ILs, as a TPS plasticizer is interesting because it has a more biodegradable character and is less toxic than other ILs due to its carboxylic anion [2 [2 capture in a system of [Emim+][Ac−] and biopolymers . Their findings indicate that these biomaterials could be employed for food packaging and storage applications. In our research group, we have studied the prospective use of [Emim+][Ac−] with different botanical sources of starch, and have concluded that the starch characteristics are crucial in order to obtain good blend properties and the potato blend values were especially very promising [+][Ac−] showed great electrical conductivity, proving that these types of biomaterials could be used in a wide range of applications, such as lithium batteries, fuel cells, or dye-sensitized solar cells [Ac−] in thermoplastic starch/PVA blends by melt-mixing using semi-industrial scale equipment. Samples have been prepared without water to prevent interaction with the ILs. Mechanical and hydration properties, migration degree of plasticizer, dynamic mechanical analysis, morphology, and XRD and ATR-FTIR spectra of the TPS films are discussed to determine the potential capability of these materials to replace synthetic and non-biodegradable polymers. To the best of our knowledge, no studies can be found in the literature about starch/PVA mixtures plasticized by a single plasticizer consisting in IL, without an additional water supply, and produced by melt-mixing.Thus, the aim of this work is the study of the plasticization effect of [Emim+][Ac−]) (>95% purity) was supplied by loLiTec-Ionic Liquids Technologies GmbH . Zinc stearate, used as a lubricant, was also provided by Sigma-Aldrich . All chemicals were used without further purification. The potato starch was provided by Across Organics  and characterized in our previous works ,55. PVA Starch films were prepared by the method of starch melt-compounding previously described by Domene-Lopéz et al. , with soStarch, PVA, and ionic liquid were manually premixed at room temperature for 3 min and then introduced in the HAAKE rheometer. The weight ratio between the potato starch and PVA in the sample was 1:1, adding different amounts of ionic liquid as plasticizer: 27.5, 30, 32.5, 37.5, 40, and 42.5 %wt. of ionic liquid; the studied formulations were labeled as IL_27.5, IL_30, IL_32.5, IL_37.5, IL_40, and IL_42.5, respectively, and 0.5 %wt. of zinc stearate was also added to all blends.SEM images were obtained with a Hitachi Scanning Electron Microscope  using an accelerating voltage of 10 kV. To observe the microstructure and morphology, the samples were cryo-fractured by immersion in liquid nitrogen and cooled with gold.Mechanical properties of the films were determined with an Instron 3344 Universal Test Instrument  equipped with a 2000 N load cell and operated at 25 mm/min following ASTM D882-12(2012)  standardm0 is the initial mass and m1 is the mass after drying.The hydration properties were determined following the method previously described in the literature ,47,55,58fm is the final dry mass.On the other hand, the solubility in the samples of water was determined by placing the films individually in 10 mL tubes filled with 9 mL of distilled water, which were capped and stored at 25 °C for 24 h. After that, the samples were taken out and dried again at 110 °C for 5 h. Solubility in water was calculated using Equation (2):The water content and solubility values were taken as the average of at least five repetitions.m0 and fm are the initial and final weights of the sample.The degree of migration of plasticizer to the surface of films is determined as the weight loss of the sample after the migration test, which was carried out following a modified procedure based on methods described by Marcilla et al.  and RahmThe results of the migration of plasticized were obtained from an average of at least three repetitions.−1. ATR-FTIR spectra were processed using the FITYK software (1.3.1 version) and the ratio of band intensities at 995 and 1022 cm−1 was determined to study the film’s molecular rearrangement.A Bruker Spectrometer  with an ATR accessory was used to obtain the ATR-FTIR spectra of films between 400 and 4000 cmA Bruker diffractometer  was used to obtain the diffractograms of the films. This equipment has a KRISTALLOFLEX K 76080F X-Ray generator , that has an X-Ray tube with a copper anode. The dimensions of specimens were 1 × 1 cm² and the equipment was operated at 40 kV and 40 mA with 2θ varying from 4 to 50° with a step size of 0.05°.The DMA analysis was carried out in a DMA 1 Instrument . The dimensions of the samples used for this analysis were 8.5 × 25 × 1 mm and the temperature range was −100 °C to 60 °C at a rate of 3 °C/min and a constant frequency of (1 Hz).+][Ac−] percentage, the higher the transparency of the samples. In addition, they show a yellowish color attributed to the color of the [Emim+][Ac−].The visual appearance of the films is shown in SEM images of the surface of the samples are shown in SEM micrographs showed a heterogeneous matrix with some cracks and some readily visible starch granules that had not been dissolved during mixing and hot pressing at IL concentrations of up to 32.5%. At higher concentrations, starch is clearly destructured, dissolved, and incorporated into a continuous phase that exhibits a smooth surface in SEM images, especially in samples with 40 and 42.5% of IL.The mechanical properties of starch/PVA with IL as a plasticizer are shown in According to Results obtained reveal a sharp drop in Young’s modulus when IL concentration is around 30%. At higher concentrations, Young’s modulus progressively decreases coinciding with a raise in elongation at break which indicates a clear plasticizing effect of the IL. Nevertheless, concentrations up to approximately 40% involve a clear excess of plasticizer, since above this value tensile strength and elongation at break markedly decrease. In fact, at these concentrations, the material starts to become a gel, extremely soft and sticky. +][Ac−] [Ac−] .Thus, the results shown in The hydration properties are the water content and the solubility in water, which are shown in +][Ac−] as a plasticizer show a plateau with a value close to 10% of moisture content, but drastically increases at a 40% content of IL reaching a value of moisture around 15%. On the other hand, solubility values progressively increase with IL concentration, observing solubility changes not only attributable to the IL extraction and dissolution. When IL concentration is 27.5%, around 35% of starch formulation dissolves, but when IL concentration raises 15 units up to 42.5% , the solubility increases to 58%. In general, this behavior could be attributed in part to the hygroscopic nature of the ILs and, in particular, [Emim+][Ac−] has a high hygroscopicity and water solubility [Ac−] as a plasticizer in the starch/PVA blends helps to reduce this effect . It must be taken into account that the superficial moisture of the samples has probably been eliminated during this test. However, this fact does not change the results or the trend obtained from the migration test. Analyzing these, the plasticizer migration mainly occurs in the early 24 h of the test.The values of IL migration are shown in As far as we know, there are no scientific findings in the literature on the migration of starch/PVA films plasticized by any IL. Nevertheless, Rahman et al.  studied Therefore, we could conclude that the problem of plasticizer migration to the surface of the films is reduced when using a small percentage of certain ILs as plasticizers in starch/PVA blends.+][Ac−] as a plasticizer. As can be seen, the spectra are similar and, as expected, show the corresponding peaks to IL and starch.−1 where two different bands overlap: on the one hand, the stretching vibration of the –OH groups of the glucose units and water [+][Ac−] [Ac−] .+][Ac-]) are localized between 2800–3000 cm−1, and this is attributed to asymmetric and symmetric stretching of –CH2 and –CH3, and aliphatic asymmetric C–H stretching vibrations [Ac−] are shown in +][Ac−] content increases, so a loss of crystallinity is observed. This fact is due to the plasticization process since when the IL content increases the disruption of hydrogen bonds between the starch molecules [+][Ac−] as a plasticizer, obtaining similar results. Xie et al. [+][Ac−] decreases the B-type crystallinity and the V-type crystallinity, hindering the formation of the single-helical structure due to interaction between the hydroxyl groups of starch and acetate anion in [Emim+][Ac−].The XRD spectra of the starch/PVA blends plasticized by [Ac−] are shown in g of IL_27.5 was −4 °C while the Tg of IL_42.5 was −30 °C. Jaramillo et al. [Ac−] plasticized-starch films appear in only one transition. As mentioned above, water and conventional starch plasticizers do not bring about a full starch dissolution and gelation; it could be the reason for the presence of two different glass transitions readily observable in glycerol-starch compounds. In the case of the IL-starch, they seem to be fully dissolved and it could be the reason for the presence of one single glass transition. It is worth mentioning that there is not much literature on the DMA of starch films plasticized by ILs. However, the DMA analysis of the TPS plasticized by glycerol among other plasticizers has been extensively studied in the literature ,80,81,82In the present work and, for the first time, starch/PVA films have been plasticized by a single plasticizer being an ionic liquid by melt-mixing.+][Ac−]) is an efficient plasticizer to obtain these biomaterials. After the analysis of the results, it has been possible to prove that this IL provokes the disruption of hydrogen bonds between the starch molecules, changing its crystalline structure to an amorphous state, which is possible to observe in the XRD spectra. This was also confirmed by the SEM images, which show that the presence of IL involves the dissolution of the starch granules, even when the [Emim+][Ac−] content in the films was low. Furthermore, a clear trend has been found in accordance with the IL amount added to the blends that the mechanical properties were significantly changed; when there are more crystalline domains than amorphous, the samples were stiffer (increasing Young’s modulus and maximum tensile strength). By contrast, the samples showed more flexibility when the amorphous phase was predominant, due to the increase in chain movement. [Emim+][Ac−] is a good starch plasticizer as revealed by the drop in Tg when the IL content increases; the IL_27.5 sample has a glass transition temperature at −3.17 °C, while the IL_42.5 sample has −31.4 °C, finding a difference of 28.2 °C between both. The same occurs in the diffusion and hydration properties, which are higher as the IL content increases; the values of IL_27.5 and IL_42.5 for the migration degree of plasticizer and solubility in water are 7.46–11.8% and 35.2–57.6%, respectively.According to the results obtained, it is possible to conclude that the 1-ethyl-3-methylimidazolium acetate ([Emim+][Ac−] could be considered an interesting plasticizer to obtain biodegradable polymeric blends from starch, constituting an alternative to other conventional plasticizers such as water or polyols. The results achieved through a technique prior to industrial development, such as melt-mixing, show the high potential of these materials to be a real replacement to conventional polymer in a wide range of applications such as packaging and provide the possibility to be used in applications which previously had no place in this type of biomaterial, such as in dye-sensitized solar cells, fuel cells, among others.For all these reasons, the [Emim


Edentulism and dental disease have a major effect on the standard of lifetime of patients. Fixed partial dentures have become the treatment of choice for many people for the replacement of edentulous space in the oral cavity. Therefore, it is of interest tocorrelate and compare the esthetics between monolithic zirconia and hand layered zirconia among fixed partial dentures in Saveetha Dental College. 100 patients who monolithic zirconia and hand layered zirconia had fixed partial dentures were included withinthe study. Pink and white esthetic scores were evaluated. Data collected were entered in SPSS and analyzed through Chi square test. It was observed that hand layered zirconia have better white esthetic score (p<0.000) and pink esthetic score (p<0.003)when compared to monolithic zirconia fixed partial dentures, which were statistically significant. It was concluded that hand layered zirconia fixed partial dentures have better esthetics than monolithic zirconia fixed partial dentures. Edentulism and dental disease have a profound effect on the standard of lifetime of patients. Tooth loss may be a common finding among individuals -2. DespiThis study was conducted in Saveetha dental college, predominantly. The advantages of the study include flexibility of the study and less time consumption. The disadvantage of the study include, it is limited to a certain population.100 Fixeddental prosthesis cemented patients were randomly included for the study.It is a retrospective study. Data was collected after viewing several patient records and analyzing data of nearly 86000 patients between the months of June 2019 to March 2020.Cross verification of data for errors was done by presence of additional reviewersand by photographic evaluation. Simple sampling was done to minimize sampling bias.Data of pink and white esthetic scores of the patients who had received fixed partial dentures with both monolithic and hand layered zirconia in conditions like horizontal and vertical bone resorption in edentulous ridges where implant supported prosthesiswas not advised or possible. Data were collected after reviewing case sheets of patients as well as their photographs after FPD fixing. Data was entered in excel in a methodical manner manually and was imported to SPSS. Incomplete or censored data was excludedfrom study.SPSS 2.0 software was used for analysis of data and to obtain results. Independent variables included age, gender and dependent variables included esthetics score (white & pink). Descriptive statistics was used to describe the distribution of age andgender distribution of the population and Chi square test was used to compare between the groups.In relation to the age distribution of the fixed dental prosthesis patients, 31-40 years age groups have received the maximum number of fixed partial dentures (38%). Next, is the 21-30 years age group, which constitutes (27%) of the population .In relation to the gender distribution of the study population it was found that out of 100 patients, 56% were males and 43% were females . In relation to the association between type of prosthesis and white esthetic score it was found thathand layered zirconia has no discrepancy in white esthetics, in maximum, which shows that it has better esthetics. The results were statistically significant  . In relation tothe association between type of prosthesis and pink esthetic score it was found that hand layered zirconia has no discrepancy in maximum in pink esthetic score, which shows that it has better esthetics than monolithic FPD. The results were statisticallysignificant  . Patients' perceptions of their oral health status and appearance of their prosthesis are important outcomes in prosthodontics. The performance of any fixed dentalprosthesis is evaluated by assessing various outcomes of chewing ability, esthetics, duration as well as technical complications. Of this esthetics, plays a vital role, by which success of prosthodontic treatment is determined [28]. In this study we observedthat hand layered zirconia FPD has better white esthetic score than monolithic zirconia FPD. We also observed that hand layered zirconia FPD has better pink esthetic score than monolithic zirconia FPD. When the type of prosthetic material was associated withwhite esthetic score, it was found that it was better for hand layered zirconia FPD. This was similar to the studies by Stawarczyk et al.  and SailThe limitations of the study include differing sample size, presence of additional examiner to assess aesthetic appearance & other scores to evaluate esthetic other than pink & white esthetic score can be included. The future scope of study is that,since esthetics is a major concern, best prosthetic material to be used, to satisfy the patients needs which results in success of prosthodontic treatment. So hand layered zirconia FPD has proved to be better in esthetics and can be implemented and used widelyin future clinical practice.Within the limits of the study, hand layered zirconia fixed partial denture has better esthetics compared to monolithic zirconia fixed partial denture and hence can be widely used in future clinical practice.


A biocomposite interference screw with an open architecture was developed to provide a greater available surface area for bone ingrowth compared with conventional solid interference screws. We herein describe a prospective, single-cohort study conducted using this interference screw in anterior cruciate ligament (ACL) reconstruction. Sixty subjects  undergoing ACL repair using the biocomposite interference screw were enrolled at 3 sites in the United States. Subjects were followed preoperatively (baseline) and postoperatively at 6 months and 1 year. The primary endpoint was graft fixation survival rate at 6 months and 1 year. Secondary endpoints included graft survival (failure defined as that occurring for any reason); International Knee Documentation Committee (IKDC) score (exam and subjective forms); Tegner Activity form; Lysholm score; and EQ (EuroQuol)-5D-5L index score and visual analogue scale. There was a 100 % rate of graft fixation survival at 6 months  and 1 year . One patient experienced a complete tear of the ACL 5 months following index surgery, resulting in graft survival rates of 98.1 %  at 6 months at 98.0 %  at 1 year. Significant improvements (p < .0001) were obtained between baseline and 6 months for the majority of patient-reported outcomes, and were maintained out to 1-year follow up. There was no significant difference over baseline in the IKDC sub-scale of symptoms. Nine patients (15.0 %) experienced serious adverse events during the course of the study; three of these patients’ adverse events were considered possibly or definitely related to the procedure device . In conclusion, this biocomposite interference screw has a favorable safety and efficacy profile at 1 year, with no failures of graft fixation, noted during that time. ConsideInterference screws are a popular method among surgeons for securing the ACL graft to obtain adequate fixation during the early postoperative and rehabilitation period . BioabsoA biocomposite interference screw with an open architecture was developed to provide a greater available surface area for bone ingrowth compared with conventional solid interference screws. In addition, the open architecture imparts circumferential graft-to-bone integration. Collectively, these features may improve graft fixation and clinical outcomes in ACL reconstruction by mitigating risk of graft slippage. We herein describe a prospective, single-cohort, multicenter study conducted using this interference screw in ACL reconstruction. The objective of the study was to provide safety and performance data with the device during the initial 1-year postoperative period, with a focus on its ability to promote graft fixation.22.1Between January 2018 and August 2019, investigators at 3 sites in the United States enrolled adult subjects (aged ≥18 years) undergoing ACL reconstruction using the biocomposite interference screw. Eligible patients were willing and able to comply with the study visit schedule and to complete study procedures and questionnaires.Patients were excluded if they met any of the following criteria: a body mass index >40; total knee arthroplasty in the study knee; conditions that may interfere with graft survival or outcome ; a known allergy to the study device or any of its components; pregnant, or plans to become pregnant during the study; an emotional or neurological condition that would pre-empt their willingness to participate in the study; entered in another investigational drug, biologic, or device study, or has been treated with an investigational product in the past 30 days; and/or known to be at risk for loss to follow-up or failure to return for scheduled visits.ClinicalTrials.gov (NCT03519555).Institutional review board approval  was obtained for each investigational site, and the study was performed in compliance with the Declaration of Helsinki. All patients provided voluntary informed written consent before enrollment. This trial was registered at 2.2Relevant demographic information, medical history, and baseline clinical outcomes were obtained preoperatively. Patients were followed up at 6 months and 1 year postoperatively.The primary endpoint was graft fixation survival rate at 6 months and 1 year. Graft reconstruction failure was diagnosed using the Lachman test, with the examiner grasping the tibia at the level of the tibial tubercle while stabilizing the femur with the other hand. The patient relaxes the leg while the examiner holds the knee flexed at 25°–30° and pulls forward on the tibia while stabilizing the femur. Excessive motion relative to the opposite knee or a soft endpoint of greater than 4 mm displacement is positive . In the event of suspected graft reconstruction failure, MRI was conducted to confirm the root cause of the failure (i.e. intraarticular graft failure or fixation failure).Secondary endpoints included graft survival at 6 months and 1 year (failure defined as that occurring for any reason) as well as the following patient-reported outcomes: International Knee Documentation Committee (IKDC) score (exam and subjective forms); Tegner Activity form; Lysholm score; EQ (EuroQuol)-5D-5L index score and visual analogue scale (VAS).Finally, serious adverse events and adverse device effects occurring intra-operatively and postoperatively were recorded.2.3All patients underwent ACL reconstruction with grafts secured using the same bioabsorbable interference screw , in accordance with the manufacturer's instructions for use. The screw's biocomposite material comprises the copolymer poly  in combination with calcium sulfate and beta-tricalcium phosphate (β-TCP), which have demonstrated osteoconductive mechanisms of action . β-TCP f2.4This study did not include a formal sample size calculation, as there are no previous studies with the implant or relevant literature to use as a basis for this. Due to this lack of prior information, a sample size of 60 subjects was recruited for this study based on feasibility only.If any individual IKDC, Tegner, Lysholm or EQ-5D-5L item responses were missing at a postbaseline visit, then the data were input from the previous assessment using the Last Observation Carried Forward (LOCF) method. If baseline was the only data point available, then no imputation methods were used, and baseline data were not carried forward. If all responses were missing at a post-baseline visit (including if a subject has discontinued from the study), then all missing scores were carried forward using the LOCF method as above, with no carrying forward of baseline values.Unless otherwise stated, all significance tests and hypothesis testing are two-sided and performed at the 5 % significance level. Where data summaries are specified, categorical and ordinal variables are summarized using frequency distributions detailing the number and percentage of subjects that fall into each category. Continuous variables are summarized using the following summary statistics: mean, standard deviation, minimum and maximum values, and number of observations. In formal analyses, resulting p-values are quoted, with statistical significance set at p < .05, and 95 % two-sided confidence intervals (CIs) generated where appropriate. All statistical calculations were made using SAS software version 9.4 .3Sixty subjects were enrolled and underwent arthroscopic single-tunnel ACL reconstruction using the study device . InformaThere was a 100 % rate of graft fixation survival at 6 months  and 1 year . One patient experienced a complete tear of the ACL 5 months following index surgery, resulting in graft survival rates of 98.1 %  at 6 months and 98.0 %  at 1 year.Significant improvements (p < .0001) were observed between baseline and 6 months for the majority of patient-reported outcomes, and were maintained out to 1-year follow up . EQ-5D VNine patients (15.0 %) experienced serious adverse events during the course of the study. In 7 patients, these adverse events were considered unrelated to the device/procedure, and included right knee joint effusion and arthrofibrosis 4 months later, which successfully resolved with arthroscopic loose body removal and synovectomy; left knee arthrofibrosis, chondromalacia, and the presence of loose bodies, which resolved following treatment including manipulation under anesthesia and chondroplasty; left knee pain 7 months after surgery; a left torn meniscus in the contralateral knee 11 months following initial surgery; left elbow pain and epicondylitis and the previously reported ACL tear; anaphylaxis as a result of food allergy; and soft-tissue mass of the finger.In addition to the ACL tear, serious adverse events related to the device/procedure included one patient who experienced pulmonary embolism and deep vein thrombosis approximately 1 month following surgery (rated as definitely procedure-related) and another patient who experienced a patellar fracture .4The most important finding from this prospective, multi-center study is that this novel biocomposite interference screw  resultedThe failure to obtain sufficient graft fixation at the tibia or femur can lead to incomplete graft fixation and an increased risk of failure . This isThese results provide the first clinical evidence with this device, and support the underlying hypothesis that its design would facilitate robust graft fixation during the initial postoperative period. Interference screws facilitate joint-adjacent fixation by compressing the graft against the tunnel wall, potentially limiting both graft-tunnel movement and the entry of synovial fluid within the tunnel. In recent years, refinements to the underlying materials and designs have been made to further improve the performance of these bioabsorbable interference screws. In vitro data suggest that the bioabsorbable polymer PLGA mixed with the bioceramic filler β‐TCP makes for an ideal material combination for achieving graft stability, accelerated degradation, and the promotion of bone ingrowth when used in interference screws in ACL reconstruction .Results from the current analysis indicate a significant improvement over baseline was achieved by 6 months in the majority of patient-reported outcomes. The wide variety of surgical techniques, fixation methods, and patient populations reported in the literature for ACL reconstructions make a comparison of studies difficult. However, these results are generally in line with those reported from other recent studies (within the last 5 years) with 1 year or less follow up and using the same patient-reported outcomes .The majority of primary ACL tears occur in those between the ages of 17 and 35 years , and patAlthough occurring at relatively low rates, authors have reported complications with biodegradable interference screws including tunnel widening, prolonged knee effusion, cyst formation, and screw migration and breakage ,30. A reThe current study has several limitations that should be noted when considering its results. Firstly, the 1-year follow-up duration may be insufficient for capturing the risk of subsequent reinjury, which has been shown to occur primarily within the first 2 years following initial ACL reconstruction . Secondl5In conclusion, this prospective, multi-center study indicates that the biocomposite interference screw, described here, has a favorable safety and efficacy profile at 1 year, with no failures of graft fixation noted during that time. Further follow-up will be needed to determine whether these effects are maintained beyond the initial postoperative period.This study was reviewed and approved by the Western Institutional Review Board  with the approval number: 20,172,619. All participants/patients  provided informed consent to participate in the study. All participants/patients  provided informed consent for the publication of their anonymised case details and images.Data associated with this study has not been deposited into a publicly available repository as the authors do not have permission to share this data.Anup Shah: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. Geoffrey Van Thiel: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Smith and Nephew provided support for this manuscript .Dr Van Thiel - Vericel and Smith and Nephew (Consultant/Speaking/Teaching).


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


NiV infection causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. No specific and effective vaccine has yet been reported against NiV. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have designed two Multi-Epitope Vaccines (MEVs) composed of 33 Cytotoxic T lymphocyte (CTL) epitopes and 38 Helper T lymphocyte (HTL) epitopes. Out of those CTL and HTL combined 71 epitopes, 61 novel epitopes targeting nine different NiV proteins were not used before for vaccine design. Codon optimization for the cDNA of both the designed MEVs might ensure high expression potential in the human cell line as stable proteins. Both MEVs carry potential B cell linear epitope overlapping regions, B cell discontinuous epitopes as well as IFN-γ inducing epitopes. Additional criteria such as sequence consensus amongst CTL, HTL and B Cell epitopes was implemented for the design of final constructs constituting MEVs. Hence, the designed MEVs carry the potential to elicit cell-mediated as well as humoral immune response. Selected overlapping CTL and HTL epitopes were validated for their stable molecular interactions with HLA class I and II alleles and in case of CTL epitopes with human Transporter Associated with antigen Processing (TAP) cavity. The structure based epitope cross validation for interaction with TAP cavity was used as another criteria choosing final epitopes for NiV MEVs. Finally, human Beta-defensin 2 and Beta-defensin 3 were used as adjuvants to enhance the immune response of both the MEVs. Molecular dynamics simulation studies of MEVs-TLR3 ectodomain (Human Toll-Like Receptor 3) complex indicated the stable molecular interaction. We conclude that the MEVs designed and  Nipah virus (NiV) is an emerging zoonotic virus of the genus Henipavirus of the Paramyxoviridae family . NiV infNiV infection of human cells involves several protein-protein interactions and protein cluster formation on the host cell surface. Essential proteins involved in NiV pathogenesis include C protein, Fusion glycoproteins (F), Glycoproteins (G), Matrix proteins (M), Nucleocapsid protein, Phosphoprotein, Polymerase, V protein and the W protein –21. The The Nipah virus is a zoonotic RNA virus and it infects human respiratory epithelium cells as well as differentiated neurons . Thus, as concluded from previous animal model studies, the recovery from viral infection and the clearance of viral RNA requires the presence of virus-specific antibodies and interferon gamma (IFN-γ) secretion from T cells –26. AlonIn the present study we have screened for the most potential Cytotoxic T lymphocyte (CTL) epitopes, Helper T lymphocyte (HTL) and B cell epitopes from the NiV proteome. We have shortlisted and prioritized the most potential and highest scoring 33 CTL, 38 HTL and 16 B cell epitopes. Out of these a total of novel 61 CTL and HTL epitopes, targeting nine NiV proteins were used to design CTL and HTL Multi-Epitope Vaccines (MEVs). Both the MEVs carry potential B cell linear epitope overlapping regions, B cell discontinuous epitopes as well as IFN-γ inducing epitopes. Here, the additional criteria of consensus of epitope amongst CTL, HTL and B Cell epitopes to chose final epitopes was also implemented. The designed MEVs carry potential to elicit cell-mediated as well as humoral immune response. We further evaluated several critical properties including the IC(50), immunogenicity, conservancy, Non-toxic to identify the epitopes with the highest immunogeneic potential against NiV. The chosen overlapping epitopes were validated by analyzing molecular interaction with HLA allele binders and with human Transporter Associated with Antigen Processing (TAP). These criteria for choosing and validating the CTL epitopes for NiV MEVs is novel and was not reported hitherto. The shortlisted epitopes were utilized for the design of CTL and HTL Multi-Epitope Vaccines against NiV. The designed vaccines were further studied for their stable interaction with innate immunity Human Toll-Like Receptor 3 (TLR3). The analysis of cDNA of the designed Multi-Epitope Vaccine has predicted to be highly favorable for expression in mammalian cell line. Overall in the present study we have designed and proposed potential Multi-Epitope Vaccines against Nipah virus infection.in-silico study, we may put forward the design of two MEVs, which qualify all the significant criterions for being a potential vaccine candidate against NiV infections. The corresponding workflow is shown in In the present study, we have designed two Multi-Epitope Vaccines (MEVs) composed of thoroughly screened most potential Cytotoxic T lymphocyte (CTL) epitopes and Helper T lymphocyte (HTL) epitopes derived from the nine NiV proteins (glycoprotein gi-253559848); C protein gi-1859635642); fusion protein (gi-13559813); matrix protein (gi-13559811); nucleoprotein (gi-1679387250); phosphoprotein (gi-1802790259); V protein (gi-1802790260); RNA polymerase gi-15487370); W protein (gi-374256971). CTL and HTL epitopes would be the most potential vaccine candidates since they are responsible for cell-mediated immune response by their presentation on the surface of antigen presenting cells (APCs) by their respective Class I and II human leukocyte antigen (HLA) allele binders. Both the CTL and HTL epitopes chosen for MEV design also carry overlapping regions of linear B cell epitopes. Moreover, both the MEVs also carry potential discontinuous B cell epitopes as well as IFN-γ inducing epitopes in their tertiary structure model. Hence, both the designed MEVs carry potential to elicit cell-mediated as well as humoral immune response. Furthermore, both the MEVs were designed with the vaccine adjuvant human Beta-Defensin 2 and human Beta-Defensin 3 at their N and C termini to enhance the immune response ,46. Beta87370; W 9848; C p42; fusiohttps://www.ncbi.nlm.nih.gov/protein). A total of 161 protein sequences available at NCBI, belonging to different strains and origins of NiV, were retrieved. For structural based epitope screenings available tertiary structures of NiV proteins were retrieved from Protein Data Bank (PDB) (http://www.rcsb.org/pdb/home/home.do). For the NiV proteins with no structure available, homology models were generated by Swiss-model, (http://swissmodel.expasy.org/) [In the present study, nine NiV proteins were used for epitope screening. They include C protein, Fusion glycoprotein (F), Glycoprotein (G), Matrix proteins (M), Nucleocapsid protein, Phosphoprotein, Polymerase, V protein and the W protein. The full-length protein sequences of NiV proteins were retrieved from the NCBI database  .Screening of Cytotoxic T lymphocyte (CTL) Epitope. The screening of Cytotoxic T lymphocyte epitopes was performed by the IEDB (Immune Epitope Database) tool “Proteasomal cleavage/TAP transport/MHC class I combined predictor” (http://tools.iedb.org/processing/) [http://tools.iedb.org/immunogenicity/) with all the parameters set to default [essing/) –67. Protessing/) –68. The Screening of Helper T lymphocyte (HTL) Epitopes. To identify the Helper T lymphocyte epitopes from NiV proteins, the IEDB tool “MHC-II Binding Predictions” (http://tools.iedb.org/mhcii/) was used. Peptides with IC50 values <50 nM are considered high affinity, <500 nM intermediate affinity and <5000 nM low affinity [affinity –72. The Population Coverage by T-cell epitopes. The “Population Coverage” tool of IEDB (http://tools.iedb.org/population/) was used to elucidate the world human population coverage by the shortlisted 33 CTL and 38 HTL epitopes derived from nine NiV proteins [proteins .Sequence-based B Cell epitope prediction. Protein sequence-based six different methods were utilized to screen linear B cell epitopes from nine different NiV proteins. These methods are available at “B Cell Epitope Prediction Tools” of IEDB server (http://tools.iedb.org/bcell/). The tool utilizes the propensity scale method as well as the physiochemical properties of the given antigenic sequence to screen potential epitopes using “Bepipred Linear Epitope Prediction”, “Chou & Fasman Beta-Turn Prediction”, “Emini Surface Accessibility Prediction”, “Karplus & Schulz Flexibility Prediction”, “Kolaskar & Tongaonkar Antigenicity” and “Parker Hydrophilicity Prediction” methods, available as sub-tools [ub-tools –79.Structure-based B cell epitope prediction. The Ellipro  and the DiscoTope2.0  methods available at IEDB, were used to screen the linear and the discontinuous B cell epitopes [epitopes ,81. The http://tools.iedb.org/conservancy/) of IEDB, against the respective source protein sequences of NiV proteins retrieved from the NCBI protein database [The shortlisted CTL, HTL and B cell epitopes screened from nine NiV proteins were analyzed for the conservancy of their amino acid sequence by “Epitope Conservancy Analysis” tool (database .http://crdd.osdd.net/raghava/toxinpred/multi_submit.php) was used to analyze the toxicity of shortlisted CTL, HTL and B cell epitopes. The tool allows to identifying highly toxic or non-toxic short peptides. The toxicity check analysis was done by the “SVM (Swiss-Prot) based” (support vector machine) method. utilizing dataset of 1805 sequences as positive, 3593 negative sequences from Swissprot as well as an alternative dataset comprises the same 1805 positive sequences and 12541 negative sequences from TrEMBLE [The tool ToxinPred  of EBI (European Bioinformatics Institute) [The overlapping residue analysis for the shortlisted CTL, HTL and the B cell linear epitopes was performed by the Multiple Sequence Alignment (MSA) analysis by Clustal Omega tool (stitute) . The Cluhttps://www.ebi.ac.uk/ipd/imgt/hla/allele.html). Templates for homology modelling were chosen on the basis of highest amino acid sequence similarity. All the HLA allele models were further validated by their QMEAN value. The QMEAN value gives a composite quality estimate involving both global as well as local analysis of the model [The Swiss-model was used for homology modelling of the HLA class I and II allele binders of shortlisted epitopes . The amihe model . Generathttp://osddlinux.osdd.net/raghava/pepstrmod/nat_ss.php) was utilized to generate tertiary structures for the selected CTL and HTL epitopes [The “Natural Peptides Module for Beginners” module of PEPstrMOD  and the AutoDock Vina were used for  binders ,88. The  binders ,90.Molecular interaction study of the shortlisted CTL epitopes with the TAP transporter cavity was performed by molecular docking using AutoDock Vina ,88. As shttp://crdd.osdd.net/raghava/ifnepitope/scan.php) using the “Motif and SVM hybrid”,  method [From the designed amino acid sequence of both the MEVs potential interferon gamma (IFN-γ) epitopes were screened via the “IFNepitope” server  method ,93.http://crdd.osdd.net/raghava/algpred/submission.html) and the Vaxijen (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) tools respectively [The designed MEVs were further analyzed for allergenicity and antigenicity prediction by utilizing the AlgPred  tool was utilized to analyze the physiochemical properties of the designed CTL and HTL MEVs [The ProtParam (HTL MEVs . The Prohttp://raptorx.uchicago.edu/StructurePrediction/predict/) [The tertiary structure of the designed CTL and HTL MEVs were calculated by homology modelling utilizing the RaptorX structure prediction tool (redict/) –100.https://zhanglab.ccmb.med.umich.edu/ModRefiner/) and GalaxyRefine tool  [The refinement of both the generated MEV models was performed by ModRefiner (=REFINE) ,102. Mod=REFINE) . The Gal=REFINE) ,103.Validation of CTL and HTL MEVs refined models. Both the refined CTL and HTL MEV 3D models were further validated by RAMPAGE analysis tool (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) [age.php) ,105. TheDiscontinuous B-cell epitope prediction from MEVs. Both the generated tertiary models of designed CTL and HTL MEVs were subjected to discontinuous B cell epitopes prediction. The structure-based discontinuous B cell epitopes were screened from both the MEV models by utilizing the ElliPro method as described earlier [ earlier .http://bioinfo3d.cs.tau.ac.il/PatchDock/) [Molecular interaction analysis of both the designed MEVs with TLR3, was performed by molecular docking and molecular dynamics simulation. Molecular docking was performed by PatchDock server (chDock/) –108. PatchDock/) –108. ForchDock/) . The ProchDock/) . The MD chDock/) . The LonchDock/) . The solhttp://www.jcat.de/). The generated cDNA was further analyzed with the GenScript Rare Codon Analysis Tool  which calculates the GC content, Codon Adaptation Index (CAI) and the Tandem rare codon frequency for a given cDNA [Complementary DNA of both the MEVs, codon optimized for expression in mammalian cells were generated by Java Codon Adaptation Tool  show a match with previous studies [The Cytotoxic T lymphocyte (CTL) epitopes were screened and shortlisted according to their highest “Total Score”, low IC(50) (nM) value for epitope-HLA class I allele complexes, and epitopes with the larger number of the HLA class I allele binders. The “Total Score” is a combined score of the proteasome, MHC, TAP , and processing analysis scores. It is calculated by using the combination of six different methods viz. Consensus, NN-align, SMM-align, Combinatorial library, Sturniolo and NetMHCIIpan –68. Over studies –38, indiThe screening of helper T lymphocyte (HTL) epitopes from nine different proteins of NiV was performed on the basis of “Percentile rank”. The percentile rank is generated by the combination of three different methods viz. combinatorial library, SMM_align and Sturniolo and by comparing against other random five million 15-mer peptides of SWISSPROT database. The rank from the consensus of all three methods was generated by the median percentile rank of the three methods. A smaller value of percentile rank indicates higher affinity of the peptide with its respective HLA allele binders. In total 38 epitopes out from initial screening were chosen on the basis that they had highest percentile rank and can bind to largest number of different HLA class II allele , were chosen for docking studies with epitopes  with test scores of -0.612 and -0.935 respectively while default threshold value being -0.4. The AlgPred prediction compares the sequence similarity between known epitopes with any region of the submitted protein. For the screening of allergenicity, the Swiss-prot dataset consisting of 101725 non-allergens and 323 allergens was utilized. The prediction indicated no allergy potential for humans of the designed MEVs. The CTL and HTL MEVs were also confirmed by VaxiJen as antigens with prediction scores of 0.4447 and 0.4836 respectively, while the default threshold value for viral proteins is 0.4. The VaxiJen performs an alignment-free approach, solely based on comparing the physicochemical properties of the query amino acid sequence with the Swiss-prot dataset. For prediction of antigenicity, the bacterial, viral and the tumor protein datasets is used to derive models for the prediction of whole protein antigenicity. Every dataset consists of 100 known antigens and 100 non-antigens. The prediction indicated that all the designed MEVs are antigenic for humans. Both MEVs were also predicted to be non-allergic but antigenic in nature for human and so have promising properties for vaccine development.E.coli, yeast and mammalian reticulocytes were predicted with 10 h, 20 min, and 30 h respectively; the aliphatic index has a predicted value of 58.42, and the grand average of hydropathicity (GRAVY) of was found to be -0.010, both indicating the globular and hydrophilic nature of the CTL MEV. The instability index score of the CTL MEV was 48.03 indicating its stable folding under native conditions. Similarly, the ProtParam analysis of the HTL MEV calculated for the 857 amino acids, a molecular weight of 87.62 kDa and a theoretical pI of 5.99. The expected half-life was predicted to be 10 h, 20 min and 30 h in E.coli, yeast and mammalian reticulocytes, respectively. The aliphatic index was calculated as 82.99, and the grand average of hydropathicity (GRAVY) was found to be 0.188, indicating that HTL MEV has a globular and hydrophilic nature. The instability index of the HTL MEV was 45.66 indicating its stable nature. Overall, the physiochemical properties of the proposed MEVs indicate that both genes might be expressed in their native forms adopting a stable fold essential for future protein purification using standard protocols.Physiochemical properties of both the MEVs were analyzed. Therefore, ProtParam calculations were performed for the designed MEVs. The CTL MEV is composed of 576 amino acids, has a molecular weight of 58.57 kDa and a theoretical pI of 8.19. The expected half-life of the CTL MEV in Further, tertiary structural models were generated for both the CTL and HTL MEVs by utilizing the RaptorX modelling tool  predictist domain, template-1fd3:A), 47 to 520  and 521 to 576  , 47 to 801 , 802 to 857   indicates a relative score of the generated strucutres in terms of modeling error, combining the global distance test (GDT) and the un-normalized global distance test (uGDT) indicating the error involved at each residue. The smaller the P-value indicates greater quality of a predicted model. Here the P-values for both MEVs indicate protein models with good quality. Since for the CTL and HTL MEV design, the CTL and HTL epitopes used also show overlapping common regions with the linear B cell epitopes  . Similar-1kj6:A) . The P-Vepitopes , both thThe generated CTL and HTL 3D models were further refined using ModRefiner to fix structural gaps followed by GalaxyRefine refinement. Modrefiner improves the physical realism and structural accuracy of the model using a two-step atomic-level energy minimization employing atomic-level, high-resolution protein tertiary structure refinement. Both the side-chain and the backbone atoms are kept flexible during the structure refinement simulations complying with a composite knowledge based force field. The further refinement was performed with the GalaxyRefine program optimizing the initial tertiary model by repeated structure perturbation as well as by utilizing the subsequent structural relaxation by the molecular dynamics simulation. The MolProbity score generated for a given refined model indicates the log-weighted combination of the clash score (the number of atomic clashes per 1000 atoms), the Ramachandran favored backbone torsion angles and the percentage of bad side-chain rotamers (the percentages of rotamer outliers). The ‘GDT-HA’  generated by the tool indicates the backbone structure accuracy; ‘RMSD’ (Root mean Square Deviation) indicates the overall structural deviation in refined model from the initial model and the ‘Rama favored’ indicates percentage of Ramachandran favored residues.The first refinement with ModRefiner showed a TM-score of 0.9703 and 0.8934 for the CTL and HTL MEV models, respectively, indicating high similarity of the refined and the input model. Scores for the second CTL and HTL MEV model refinement calculated with Galaxy are given in S10 Table in The obtained models were analyzed with the RAMPAGE analysis tool after refinement. The refined CTL MEV model has 91.5% residues in favored regions and the refined HTL MEV model has 89.5% of residues in favored region. This indicates both the CTL and HTL MEVs have adopted an acceptable conformation after refinement  supporteThe designed MEVs were further tested for their potential to generate Interferon-gamma (IFN-γ) inducing epitopes required for the adaptive and the innate immune response. Therefore 15-mer peptide epitopes derived from the amino acid sequences of the designed construct of CTL and HTL MEVs were tested ability to induce IFN-γ production in CD4+T cells. A total of 33 CTL MEV and 43 HTL MEV INF-γ inducing POSITIVE epitopes with a score of 1 or more, suggesting strong potential to elicit immune response, were shortlisted  score of 1.00 with 0% tandem rare codons. Likewise, the GC content of the optimized HTL-MEV cDNA was 70.69%, CAI score was 1.00 with 0% tandem rare codons. The CAI indicates the possibility of cDNA expression in a chosen expression system. The tandem rare codon frequency indicates the presence of low-frequency codons in the given cDNA. Since for higher possibility for cDNA expression in human expression system, the GC content of a cDNA should be within the range of 30% to 70%, the CAI score should be between 0.8–1.0, and the tandem rare codon frequency that indicates the presence of low-frequency codons, should be <30%, the cDNA constructs of CTL and HTL MPVs are expected to have high potential for expression in human expression system. The tandem rare codons may hinder the proper expression of the cDNA or even interrupt the translational machinery of the chosen expression system. According to the GenScript Rare Codon Analysis the cDNA of both the MEVs satisfy all the mentioned parameters and are predicted to have high expression in the mammalian host cell line.The refined models of CTL and HTL MEVs were further studied for their molecular interaction with the ectodomain (ECD) of human TLR3. Therefore, molecular docking of CTL and HTL MEVs models with the Human TLR3 (ECD) crystal structure model (PDB ID: 2A0Z) was performed utilizing the PatchDock tool. Generated docking conformation with highest geometric shape complementarity score of 22382 and 18264 for CTL and HTL MEVs, respectively were selected for further studies. The highest docking score predicted with the PatchDock tool indicates the best geometric shape complementarity fitting conformation of MEVs and the ectodomain of TLR3 receptor. Both, the CTL and HTL MEVs were fitting well into the ectodomain region of TLR3 after docking . The CTLin vitro and in vivo validation and trial.In the present study, we have designed and validated two Multi-Epitope Vaccines derived from CTL and HTL epitopes. The selected peptides show significant sequence overlap with screened linear B cell epitopes. Both, the CTL and HTL MEVs tertiary models carry potential to elicit profound humoral and cellular immune responses. Molecular interaction analysis of both MEVs in complex with immunoreceptor TLR3(ECD) showed stable structural and conformational fit of the MEVs molecule into the ectodomain of TLR3 receptor. We conclude that the CTL and HTL MEVs provided in the present study involves potential epitopes from the entire NiV proteome and cover up to 97.88% world population. The suggested MEVs provide a potential vaccine candidate against NiV for future S1 File(PDF)Click here for additional data file.


Acinetobacter baumannii as a critical MDR bacteria [Globally, multidrug-resistant (MDR) bacteria represent a menace to public health. Recently, the World Health Organization recognized the ubiquitous, non-fermenting, rod-shaped Gram-negative coccobacillus bacteria .Acinetobacter baumannii can be considered an opportunistic pathogen, predominantly affecting critically ill patients or patients with immune system deficiency [Acinetobacter baumannii frequently causes healthcare-associated infections, including pneumonia, bloodstream infection, meningitis, urinary tract and wound infection.ficiency ; as a coAcinetobacter baumannii threat, including the current nosocomial burden, prevention of transmission in hospitals, and clinical management.In this Editorial, we present a bundle of recent articles on different aspects of the Recently, Jean et al. provided a picture of the current burden of hospital-acquired pneumonia due to MDR bacteria worldwide . The autStaphylococcus aureus, followed by Pseudomonas spp. and Acinetobacter spp. [They found that the most frequently isolated bacteria causing hospital-acquired pneumonia was ter spp. .Acinetobacter baumannii [In several Asian countries, including China, Thailand and Taiwan, Gram-negative bacteria are currently the most common cause of hospital-acquired pneumonia, with a trend towards an increase in MDR aumannii .Acinetobacter baumannii pneumonia, the authors identified a higher co-morbidity index, prolonged hospital stay, previous administration of broad-spectrum antibiotics and intensive care unit stay [Among the risk factors associated with the onset of MDR nit stay .Acinetobacter baumannii in the intensive care units was also confirmed by a prospective respiratory infections’ surveillance study at a 30-bed intensive care unit in Poland [Acinetobacter baumannii was the most frequently isolated bacteria. During the study, the reported mortality rate among patients with non-ventilator hospital-acquired pneumonia and ventilator-associated pneumonia was 44% and 38%, respectively [The burden of respiratory tract infections due to Acinetobacter baumannii spread in the intensive care setting represents a main issue that the COVID-19 pandemic has worsened. In fact, a recent observational, multicenter, retrospective study performed in Italy reported an increased burden of MDR Acinetobacter baumannii in intensive care units during the COVID-19 pandemic [pandemic .Acinetobacter baumannii during the intensive care unit stay, either colonization  or infection . As a further issue, the observed mortality rate in carbapenem-resistant Acinetobacter baumannii patients was extremely high, i.e., 64.7% [Overall, the study included 913 COVID-19 patients admitted to the intensive care units; of them, 176 (19%) became positive for carbapenem-resistant Acinetobacter baumannii.In addition to ventilator-associated pneumonia or bloodstream infections, hospital associated urinary tract infections can be caused by A prospective study performed over six years in a large private hospital in Greece investigated the correlation between the incidence rate of catheter-associated urinary tract infections, specific infection-control measures and the incidence rate of MDR bacteremia . InfectiAcinetobacter baumannii [This study provided comforting results. The intervention bundle was effective, determining a decrease in the incidence of catheter-associated urinary tract infections . The decaumannii .Acinetobacter baumannii the most concerning issue is represented by the resistance to most antibiotics, including carbapenems and, in some cases, polymixins and, as a consequence, the limited therapeutical options [In almost all the articles dealing with  options .Acinetobacter baumannii are restricted to few antimicrobial classes, i.e., aminoglycosides, polymyxins, tetracyclines and phosphonic antibiotics. Unfortunately, these antimicrobials have important limitations, including toxicities, site-specific pharmacokinetics and emergence of resistance. A meta-analysis by Huang et al. compared the efficacy and the safety of colistin monotherapy versus colistin and meropenem combination for the treatment of patients with MDR Acinetobacter baumannii infection [At present, possible antibiotic options with in vitro activity against MDR nfection . The metnfection .Acinetobacter baumannii [Recently, two novel antimicrobials, cefiderocol and sulbactam-durlobactam, have been considered for the treatment of infections due to MDR aumannii .Acinetobacter baumannii, but unfortunately the CREDIBLE trial reported variable in vivo efficacy [Cefiderocol showed valuable activity against efficacy .Acinetobacter baumannii isolates, and possesses valuable pharmacokinetic and pharmacodynamic profiles for the treatment of bloodstream infections and pneumonia [Acinetobacter baumannii.Sulbactam-durlobactam showed in vitro potency against neumonia . TherefoAcinetobacter baumannii [Recently, a multicentre, randomised, phase 3 clinical trial was performed to evaluate the efficacy and safety of sulbactam-durlobactam in patients with bloodstream infections or hospital-associated bacterial pneumonia due to aumannii .Acinetobacter baumannii [The trial aimed to assess the non-inferiority of sulbactam–durlobactam versus colistin, both given with imipenem–cilastatin for the treatment of infections caused by aumannii . The triaumannii .However, although these findings confirmed sulbactam-durlobactam as a promising treatment, important limitations should be highlighted.Acinetobacter baumannii isolated in sputum or in the respiratory tract. As polymicrobial infections were reported in almost 30% of the patients included, the isolation of Acinetobacter baumannii in respiratory samples could represent a colonization. Therefore, it cannot be excluded that a relevant percentage of the included cases may represent Acinetobacter-baumannii-colonized patients, with an ongoing infection due to other bacteria. Further randomized clinical trials are needed to confirm the validity of sulbactam-durlobactam to treat Acinetobacter baumannii infections [Most of the patients enrolled had fections .Acinetobacter baumannii bacteremia was recently studied by Wang et al. in a retrospective observational study [Acinetobacter baumannii bacteremia. Among them, 290 patients (76.5%) had monomicrobial bacteremia and 89 patients (23.5%) had polymicrobial bacteremia, including Acinetobacter baumannii. No significant difference in mortality at two weeks was reported between patients with monomicrobial and polymicrobial bacteremia ; however, higher mortality rates were found among polymicrobial bacteremic patients with concomitant isolation of Escherichia coli, Pseudomonas aeruginosa, and Enterobacter spp. The logistic regression analysis demonstrated that appropriate antibiotic treatment was independently associated with a decrease in mortality [The differences between monomicrobial and polymicrobial al study . Overallortality .Acinetobacter baumannii strains causes severe infections, with restricted antibiotic options and high mortality rates.To conclude, the global spread of MDR Acinetobacter baumannii in the hospitals. Surveillance, infection prevention and control, as well as knowledge of the mechanisms of resistance and novel antimicrobial compounds against Acinetobacter baumannii, are the main means of tackling this worldwide menace.More than ever, infection-control measures are needed to minimize the spread of


The X-ray diffraction results revealed that the V-type characteristic diffraction peaks were more obvious for samples including PC-EHSC and βA-EHSC, which was in line with their lowest polydispersity index (DPn). PC-EHSC and βA-EHSC also showed an increased peak intensity of the scattering maximum on the small-angle X-ray scattering spectra, whereas CCLSC exhibited an overall lower peak intensity within the studied q range of scattering. The highest XRD crystallinity and the lowest DPn value obtained for PC-EHSC indicated that the starch polymers modified by pancreatin could produce glucan chains with a comparatively homogenous Mw distribution that are readily recrystallized by hydrogen bonding through chain aggregation. Comparatively, the lowest relative crystallinity for HS-EHSC obtained from XRD suggested that thermostable α-amylolysis was unfavorable for the formation of starch structure with a higher degree of molecular order. This study could provide useful information for the needed research to obtain a deeper understanding of the impact of different amylolysis actions on the structural organization of starch hydrolysates and to provide a theoretical foundation for the development of fermentable enzymatically hydrolyzed starch with well-tailored physiological properties.This study was undertaken to investigate the enzymatic hydrolysis of lentil starch concentrates from conventional cooked seeds (CCLSC) by the action of different types of enzymes, including pancreatin (PC-EHSC), heat-stable α-amylase (HS-EHSC), β-amylase (βA-EHSC), amyloglucosidase (AMG-EHSC), and multi-enzymes (βA-HS-AMG-EHSC); their multi-scale structural characteristics of the enzymatic hydrolysis products of lentil starch concentrates were compared. The morphological features distinguished among different samples. The Fourier-transform infrared spectroscopy and solid-state  Lens culinaris. Lentil seeds contain approximately 67–69% carbohydrates (44% of which are contributed by the presence of starch) and 24–30% protein [Lentil, one of the important crops belonging to leguminaseae family, is botanically classified as  protein . The higE.C. 3.2.1.1) randomly hydrolyzes the α(1→4) glycosidic linkages of starch concentrates substrate by an endo-action, with dextrin products having α-configuration at the anomeric carbon of the newly produced reducing end (E.C. 3.2.1.2) is an exo-hydrolase that specifically hydrolyzes the α(1→4) linkage of the two D-anhydroglucose monomers inward from the non-reducing end of starch polymers. The catalysis is able to produce a maltose each time hydrolysis is carried out, and is then stopped when an α(1→6) linkage or substitution group is encountered [A number of processing methods including conventional cooking, extrusion, autoclaving, microwave cooking and acid hydrolysis have been applied to enhance the RS content of starch from different botanical origins . Followicing end . α-Amylaountered .Factors including the molecular composition and structural characteristics of the starch substrate, the diffusion of the enzyme onto the substrate surface, the mechanism of enzyme action and its catalytic efficiency could all influence the kinetics of enzymatically hydrolyzed starch formation and consequently the fine molecular structure of starch hydrolysis products. Compared with native starch, the accessibility of the gelatinized starch substrate to enzymes is greater and not restricted by α-glucan associations such as double helices  . From thTM . Thermostable α-amylase from Bacillus licheniformis , β-amylase , amyloglucosidase  and pancreatin from porcine pancreas  were purchased from Sigma-Aldrich Chemical Co. . All other reagents used in this study were of analytical grade. The lentil seeds were from Daohuaxiang Co.w/v) for 20 min, followed by freeze-drying and enrichment of lentil starch concentrates according to the method described by Miao et al. (2015) [The whole lentil seeds were conventionally cooked in boiling water  . The obtThe crude protein content of CCLSC was measured by the nitrogen combustion method using a nitrogen analyzer . A nitrogen conversion factor of 5.40 was used to calculate the crude protein content. The total lipid content of CCLSC was Soxhlet extracted in a Buchi B-811 extractor  with petroleum ether, at 80 °C. The amount of total lipids was calculated as a sum of the extracted lipids after the evaporation of organic solvent from the recipients and drying to a constant weight. The protein and lipid contents of CCLSC were 17.96% ± 0.7281 and 1.08% ± 0.0954, respectively.g for 10 min, and the sediment was washed with distilled water for three times before oven drying at 42 °C, grounding and sieving (149 μm). The obtained starch sample was referred to as βA-EHSC . Ten grams of CCLSC was dispersed in 100 mL sodium acetate buffer , and β-amylase  was then added to the dispersion. Enzymatic hydrolysis was performed in a thermostatic oscillator  at 55 °C for 12 h (128 rpm). Immediately after the hydrolysis, the same volume of 95% ethanol was added to terminate the reaction. The collected sample was centrifuged at 3000× Ten grams of CCLSC was dispersed in 20 mL sodium acetate buffer  containing 0.5 mL thermostable α-amylase, and the enzymatic hydrolysis was performed in a thermostatic oscillator at 95 °C for 1 h (128 rpm). The obtained sample was then processed following the same procedure as described above , and theA sample of CCLSC (1.5 g) was slurried in 30 mL sodium acetate buffer  and was then hydrolyzed by pancreatin at 37 °C for 16 h. The obtained sample was processed following the same procedure as described above  and the A sample of CCLSC (1.5 g) was dispersed in sodium acetate buffer , followed by the addition of amyloglucosidase. The enzymatic hydrolysis was performed at 60 °C for 72 h. The obtained sample was processed following the same procedure as described above , and theA sample of CCLSC (1.5 g) was suspended in sodium acetate buffer  and then hydrolyzed by β-amylase for 12 h at a constant temperature of 55 °C. The pH of the suspension was adjusted to 6.0, followed by the addition of thermostable α-amylase. The entire hydrolysis process was continued for 1 h at 95 °C in a thermostatic oscillator (128 rpm). The pH of the obtained solution was then adjusted to 4.5, followed by the addition of amyloglucosidase, and the hydrolysis was then conducted for 72 h at 60 °C. The obtained sample was processed following the same procedure as described above  and the The morphological features of CCLSC and EHSC samples prepared by different enzymatic treatments were obtained using a Quanta 200 instrument  at an accelerating voltage of 20 keV. The powder samples were imaged after fixing on a conductive adhesive and sputter coating with gold using an ion sputtering instrument . The magnification was set at 1500×.The dry powder of CCLSC and different EHSC samples (2 mg) was dissolved in 1 mL dimethylsulfoxide containing 0.5 wt% LiBr and was incubated at 80 °C for 12 h. A size-exclusion chromatography instrument  equipped with a refractive index detector  and a multi-angle laser light scattering detector  was used to measure the molecular weight distribution of the studied samples, following the method of Yin et al. (2018) .The crystalline properties of CCLSC and EHSC samples were analyzed by an X-ray diffraction system , following the method of Yin et al. (2018) . The X-r−1 from 400–4000 cm−1.Samples of CCLSC and different EHSC were blended with pre-dried KBr powder at a mass ratio of 1:200 and pressed into tablets before the measurement. The spectra were achieved using an FTIR spectrometer  at a resolution of 4 cm13C CP/MAS NMR) measurement was carried out using an AVANCE III 400 MHz WB NMR spectrometer  equipped with a double resonance H/X CP-MAS 4mm probe. The spectrometer was operated at a frequency of 100.62 MHz and the samples were spun at a rate of 6 kHz. The 13C NMR parameters, including crystallinity (C2), double helix content and the proportion of amorphous region (PPA) were quantified using PeakFit software  following the method of Ren et al. (2021) [The solid-state cross-polarization and magic-angle spinning nuclear magnetic resonance (w/w), following the method of Yin et al. (2018) [q region of the SAXS spectra (Equation (1)). The self-similarity of the scattering object was characterized by the concept of the fractal; the relationship between the fractal dimension (surface/mass) and α values is illustrated by Equations (2) and (3). The SAXS data was also analyzed using the 1D correlation function (Equation (4)), and the average thickness of the crystalline layer (dc) was thereby calculated.I is the intensity of scattering; q is the magnitude of the scattering vector defined as q = (4πsinθ)/λ ; α is the power law exponent which reflects the mass/surface fractal dimensions of starch; mD and sD refer to mass fractal dimension and surface fractal dimension, respectively; r is the distance in real space; d is the long period (lamellar repeat distance) which can be determined as the z at the second maximum of r(z). The SAXS measurement was carried out on a SAXS system  at the sample concentration of 50%, . (2018) . The X-rp ≤ 0.05 using DPS software .All data were statistically analyzed by analysis of variance (ANOVA) followed by Duncan’s test at The scanning electron microscope images of CCLSC and enzymatically hydrolyzed residues obtained by different enzymatic hydrolysis methods of lentil starch concentrates from precooked seeds are presented in Mw values was in the following order: CCLSC > AMG-EHSC > βA-EHSC > βA-HS-AMG-EHSC > PC-EHSC > HS-EHSC, whereas the values of DPn (polydispersity index) showed a slight discrepancy, viz., CCLSC > HS-EHSC > AMG-EHSC > βA-HS-AMG-EHSC > βA-EHSC > PC-EHSC. In comparison to CCLSC, the markedly decreased Mw and DPn values upon amylolysis signified that the different enzymatic treatments could produce the glucan molecules with much shorter and more homogeneous molecular chains. Although many factors could influence the rate of enzymatic hydrolysis of starch concentrates, the accessibility of enzymes to the glycosidic bonds in the substrate was a major determinant [Mw value obtained for AMG-EHSC, compared to other EHSC samples.High-performance size-exclusion chromatography (HPSEC) analysis was performed to quantify the molecular weight distribution of EHSC obtained by different enzymatic hydrolysis methods, and the results are shown in erminant . Amyloglerminant . Howevererminant . It was Mw value obtained for βA-EHSC (5.690 × 104 g/mol) compared to other samples, including PC-EHSC with a Mw value of 3.309 × 104 g/mol and HS-EHSC with a Mw value of 1.711 × 103 g/mol. β-Amylase is an exo-amylase that is responsible for hydrolyzing the α(1→4) linkages of two D-anhydroglucose monomers inwards from the non-reducing ends of the glucan chain . AccordiMw value of 3.309 × 104 was noticed for PC-EHSC. A possible reason was based on the postulation that, apart from the existing amylose-lipid complex in lentil starch concentrate granules, the endogenous lipids and free proteins would also conjugate with the amylose/amylopectin chains which became more mobile and available for complexation during pre-gelatinization [n value for PC-EHSC (1.037), which was closer to 1, indicated the higher homogeneity and narrower distribution of molecular weight in the starch concentrate samples treated with pancreatin.A relatively low nization . After 1nization . The lowMw (1.711 × 103 g/mol) for HS-EHSC. Meanwhile, the enzymatic hydrolysis by heat stable α-amylase resulted in a wider molecular distribution of the polymer substrate, corresponding to its higher obtained DPn value of 1.262.For HS-EHSC, the high incubation temperature applied caused the disruption of the retrograded lentil starch concentrate, leading to an enhanced accessibility of heat stable α-amylase to the polymer substrate and a lower value of θ = 8.9° and a sharp peak at 26.6°, both of which were characteristic diffraction peaks of the A-type crystalline structure. An absorption peak at 22.0°, which resembled a typical B-type, was also observed for all studied samples. The additional peaks at 19.0° and 20.8°, derived from the V-amylose helical structure in the presence or the absence of a lipid ligand, and a typical A-type diffraction peak centered at 17.1°, derived from starch recrystallization, were also presented on the X-ray diffractograms for PC-EHSC, βA-EHSC and AMG-EHSC. The observation validated the fact that all EHSC samples consisted of a CA + V crystalline polymorph. The V-amylose structure is a single, left-handed helix with an internal cavity where the complexed ligand can reside. In the lamellar structural architecture of different EHSC residues, the crystalline regions consisting of A-, B- and V-type crystal polymorphs make up the lamellar region, whereas the amorphous un-complexed amylose components associated with amylose fractions with small molecular size under the action of enzymatic hydrolysis are arranged in another region that is unable to diffract X-rays [θ = 7.5° and 21.3° that are responsible for the un-complexed fatty acids were also noticed on the X-ray diffraction pattern. The diffractogram of CCLSC was characterized by the reflections at the spectral diffraction angles in the position of 15.1°, 17.1°, 19.0°, 22.0°, 23.7° and 26.6°, which confirmed that CCLSC contains domains of the type C and type V crystalline structure. A proportion of the B-type crystalline peaks of the pre-cooked lentil starch concentrates vanished in the EHSC samples, possibly due to the preferential hydrolysis of branch points of B-type starch concentrates, which were clustered in the amorphous domains after CCLSC being subjected to several enzymatic treatments [n could be generated, which increased the availability of the released linear α(1–4)-glucan chains incorporating into the V-type inclusion complex [n values obtained for PC-EHSC (DPn = 1.037) and βA-EHSC (DPn = 1.038), suggesting that their starch chains with a relatively homogenous molecular weight distribution might be more effective in participating in the formation of the V-type inclusion complex with free fatty acids in pre-cooked lentil starch concentrates. Liu et al. (2019) [eatments . Meanwhi complex . Studies. (2019)  also demMw value obtained for HS-EHSC during the HPSEC analyses. Based on the studies of Gelders et al. (2004) [It was also especially noticed that HS-EHSC displayed a relatively flat and broad reflection in the 2θ range, between 15° and 26°. The excessive hydrolysis of pre-cooked lentil starch concentrates during enzymatic treatment with thermostable α-amylase could generate shortened polymer chains including oligosaccharides with varying lengths and branched oligosaccharides , which are not favorable for folding and organizing into an orderly, packed crystalline structure. This assumption was also supported by the relatively low . (2004) , these n4 g/mol) of PC-EHSC, indicating that the starch polymers modified by pancreatin could produce glucan chains with a comparatively homogenous Mw distribution that are readily recrystallized by hydrogen bonding through chain aggregation [From regation . On the regation , the hig−1, and the spectra are shown in −1 was observed for each sample, corresponding to the inter- and intra-molecular stretching related to the O–H and N–H vibration of the EHSC samples [−1 were susceptible to the C–O and C–C stretching vibrations, and the wavenumber of 860 cm−1 was ascribed to the C–O–C glycosidic band in the EHSC [−1 for different EHSC samples in contrast to CCLSC suggested the efficient cleavage of glycosidic bonds during different enzymatic hydrolysis treatments. The FT-IR data were collected for the EHSC samples obtained by different enzymatic treatments in the present study from 4000–400 cm samples . The banthe EHSC . A decre−1 is also observed in I band at 1658 cm−1 was associated with the C=O stretching in the proteins secondary structure, the Amide II band at 1448 and 1532 cm−1 was linked to the C–N and N–H bending, whereas the absorption at 1236 cm−1 (Amide III band) was related to the C–H vibration in the peptide bonds. All the above three amide characteristic bands were observed in the starch concentrate samples in the present study, suggesting the existence of protein in the EHSC, with enzymatically hydrolyzed starch concentrates obtained by the treatment with heat-stable α-amylase samples being subjected to cooking and enzymolysis treatments. Individual peaks shown on the spectra in the Amide I region (1700–1600 cm−1) were classified as α-helix (1650–1658 cm−1), β-sheet (1620–1635 cm−1) and random structure (1640–1650 cm−1) [−1 on the FT-IR spectrum [II band at 1532 cm−1 which is linked to N–H bending and the peak at 2790 cm−1 which is associated with the asymmetric hydrogen bonding of carboxylic acid were markedly increased after different enzymatic treatments, compared with the CCLSC. This observation suggested that the electrostatic interaction could be formed between the negatively charged carboxyl group of lipids and positively charged amino group of proteins during enzymatic hydrolysis, which might act as an important force for the additional development of the ternary complex through hydrophobic interaction between amylose and the aliphatic tail of fatty acids [The protein secondary structure in the range of 1200–1700 cm50 cm−1) . Proteinty acids .−1, 2850 cm−1 and 1745 cm−1, corresponding to the CH3 asymmetric stretch, CH2 symmetric stretching vibration, and C=O stretch vibration of fatty acids, respectively [−1 and 2850 cm−1 with the appearance of more tiny and distinguished peaks for the enzymatically hydrolyzed samples could be observed  of the EHSC samples, whereas the absorbance ratio at 986/1022 cm−1 reflected the degree of double helix (DD) [The EHSC samples had peaks at 2854 cmomplexed . The appunfolded . Furtherlix (DD) . As show13C CP/MAS NMR spectra of the EHSC samples obtained by different enzymatic treatments are illustrated in 13C CP/MAS NMR was used to measure the structural order in the amylolyzed lentil starch concentrates on a molecular level, in contrast to the XRD analysis on a crystalline level. The resonance parameters obtained from the solid-state NMR spectra could also reflect the behavior of each component, including protein, starch and lipids in the multi-component system [O-alkyl carbons starch at 60–110 ppm, aromatic carbons visible at 130 ppm, and carbonyl carbons of proteins at 172 ppm [The solid-state t system . Spectra 172 ppm ,41. The 13C NMR crystallinities, double helix content and proportion of amorphous phase (PPA) with respect to different enzymatic treatments, is presented in 13C chemical shift of the signal for C1 of the hexapyranose unit of CCLSC was especially noticed in 13C NMR spectra, due to the formation of the amylose-lipid complex, particularly due to (i) the changes in the torsion angles of the glycosidic linkages, resulting in a different pattern of electron distribution, and (ii) the structural alteration of the amylose molecule from a random coil to a helix [The quantification of the  a helix . This ob13C NMR signal resonances from 20.3 to 31.8 ppm, which were attributed to the methyl terminal groups, aliphatic ring methylene groups and methylene groups in long alkyl chains was observed, which further supported the potential formation of the V-type amylose-lipid complex during amylolysis. Compared with CCLSC, the resonances at 10–40 ppm with the contribution from lipids and at 172 ppm with the contribution from proteins showed the greatest change amongst all signal peaks after different enzymatic treatments, whereas the peak intensities were markedly higher upon the enzymatic hydrolysis treatments. Furthermore, an up-field shift of the resonance at 172 ppm was also observed for the amylolytic hydrolyzed starch concentrates compared, with CCLSC. This observation suggested that the different enzymatic treatments applied could induce several consecutive changes involving the hydrolysis of glycosidic bonds, the disruption of the molecular order of CCLSC by the breaking of the hydrogen bonds, and the self-rearrangement of starch chains and starch-protein/lipids complex formation. Additionally, in comparison to CCLSC, the broadening of the resonance band at 172 ppm observed in all the EHSC samples might be attributed to the protein conformational disorder triggered by the stabilizing effect of lipids on proteins . An incrq range centered at ca. 0.03–0.04 Å−1 corresponding to a ~14–22 nm semi-crystalline structure based on the Bragg’s law D = 2π/q was observed for CCLSC, suggesting a non-periodic structure with ordered domains randomly arranged in the amorphous matrix. The disappearance of the typical lamellar peak at 0.06–0.07 Å−1, resulting from both the thermal processing and enzymatic hydrolysis treatments, indicated the destruction of the alternating lamellar semicrystalline architecture of the native granular starch. From Imax, viz., the intensity of the scattering maximum, whereas CCLSC exhibited an overall lower peak intensity within the studied q range of scattering. The relatively higher Imax was generally ascribed to the larger electron density contrast between the amorphous and crystalline regions inside the starch granules, rather than the absolute density itself. The electron density difference between the amorphous and crystalline lamellae became prominent for the EHSC samples, possibly because that the electron density of the amorphous region decreased upon amylolysis. Furthermore, as presented in cd value, which reflect the average thickness of the crystalline lamellae within the starch concentrate samples, resembled the trend observed for the values that represent the ordered structure of EHSC, including the C1, DD and DO values. As opposed to XRD, which generally determines the crystallographic dimensions of native and processed starch granules associated with interhelix order, the small-angle X-ray scattering technique has been commonly used in measuring the orders along the chains with dimensions approaching 10 μm . A broadα values ranged from approximately −0.38 to −2.10 for the studied samples. Specifically, the α values of PC-EHSC and AMG-EHSC were less than −3, indicating that these two EHSC samples were of a surface fractal structure, in which case the surface fractal dimension Ds can be calculated as Ds = 6 + α, and is regarded as an indicator of the degree of smoothness of the starch concentrate granules. When the Ds value was close to 2, the surface of the scattering objects tended to be very smooth [α values of CCLSC, βA-EHSC, HS-EHSC and βA-HS-AMG-EHSC were in the range between −3 and −1, suggesting that these samples were characterized as a mass fractal structure, in which case the fractal dimension (Dm) can be calculated as: Dm = −α. The Dm value was 2.10 without enzymatic hydrolysis, indicating thar the packing efficiency was relatively low for CCLSC. On the other hand, the comparatively high value of Dm (2.58) for βA-EHSC suggested a relatively compact internal structure. Fractal analysis was carried out to provide an insight into the degree of surface smoothness and mass compactness of the EHSC. The power law exponent y smooth . The α vMw and DPn values upon amylolysis signified that the different enzymatic treatments could produce the starch glucan molecules with much shorter and more homogeneous molecular chains. The morphological features distinguished among different samples. The XRD analysis revealed that all EHSC samples consisted of a CA + V crystalline polymorph. In particular, the V-type characteristic diffraction peaks were more obvious for samples including PC-EHSC and βA-EHSC, which was in line with their lowest DPn values, suggesting that their glucan polymer chains with a relatively homogenous molecular weight distribution might be more effective in participating in the formation of the V-type inclusion complex with free fatty acids in pre-cooked lentil starch concentrates. Among the EHSC samples, PC-EHSC and βA-EHSC also showed an increased peak intensity at Imax on the SAXS profile, whereas CCLSC exhibited an overall lower peak intensity within the studied q range of scattering. The highest XRD crystallinity and the lowest DPn value obtained for PC-EHSC indicated that the starch concentrate polymers modified by pancreatin could produce glucan chains with a comparatively homogenous Mw distribution and are readily recrystallized by hydrogen bonding through chain aggregation. Comparatively, the lowest relative crystallinity for HS-EHSC obtained from XRD suggested that thermostable α-amylolysis was unfavorable for the formation of the molecular structure with a higher degree of order in the starch concentrate samples. Both the FTIR and solid-state 13C CP/MAS NMR spectral features indicated the possible formation of a binary and ternary complex between amylose, protein and lipids. This study was expected to provide useful information for a deep understanding of the impact of different amylolysis actions on the structural organization of starch concentrate hydrolysates and their relevant physiological properties. The present study compared the multi-scale structural characteristics of the enzymatically hydrolyzed starch concentrates obtained by the action of different types of enzymes. In comparison to CCLSC, the markedly decreased