The increased demand for miniature components has drawn the attention of researchers, engineers, and industry users to manufacture precision micro and mesoholes on foils, sheets, and plates made from a variety of engineering materials. These days, micro-drilling is extensively being adopted as a fundamental operation in all kinds of smart manufacturing industries to make different types of microholes, such as through holes, blind holes, and taper holes on micro-parts and components. Drilled holes with a diameter of less than 1 mm are referred to as microholes, while drilled holes whose diameter ranges between 1 and 10 mm are known as mesoholes. Meso and microholes are commonly referred to as fine-holes. Modern or advanced drilling processes are mostly used to drill microholes from a variety of materials. This paper presents an extensive review of the previous research conducted on the drilling of fine holes (meso and micro size) by spark- erosion-based processes along with highlighting work and tool electrode materials, specifications of drilled holes, types of microholes (through or blind holes), process parameters, performance measures, and key findings. The paper aims to facilitate researchers and scholars by highlighting the capabilities of spark erosion machining, drilling, and its variants to fabricate miniature holes. The paper ends with a conclusion and future research directions to encourage further work in this area. Nowadays, the trend of miniaturized devices and products that are lightweight, smaller in volume, portable, cheaper, and stable in performance has become one of the fundamental necessities of several manufacturing industries, such as aerospace, automotive, medicine, biomedical, electronics, telecommunication, fluidics and consumer products ,2,3. MinThe choice and range of materials for fine holes  generally depend on types of application, material features  of workpiece and drilling tools, drilling methods, and materials cost ,8. CommoThe summary of the various methods for drilling miniature deep fine holes is presented in Mechanical erosion-based drillings are abrasive water jet drilling (AWJD) and ultrasonic drilling (USD). In these processes, materials are removed by the mechanical erosion of work material by high-pressure slurry (abrasive particles mixed with water). AWJD can produce miniature-sized holes on metallic and non-metallic materials. USD is used for machining miniature-sized holes on conductive and non-conductive materials having a hardness of more than 40 HRC. In thermoelectric erosion drilling methods, thermal energy is utilized to remove materials by melting and vaporization. These methods include ram-based spark-erosion machining (SEM), spark-erosion drilling (SED), and laser drilling. SEM and SED are used to produce miniature-sized holes on electrically conductive materials irrespective of their hardness, while laser drilling is used to produce miniature-sized holes on non-reflective metallic materials. The material removal rate in laser drilling is higher than in SED and SEM processes. The micro versions of SEM and SED are micro-spark-erosion machining (µ-SEM) and micro-spark-erosion drilling (µ-SED). Electrochemical drilling (ECD) is used to produce microholes on workpieces by ion displacement. It involves anodic dissolution where an electrolytic cell is formed by a tool (cathode) and workpiece (anode) surrounded by the continuous flowing electrolyte. In chemical drilling (CHD) methods, miniature-sized holes are produced by the chemical action of the corrosive agents.Micro spark erosion machining (µ-SEM) is used to make a different type of microhole by using similar shapes and sizes of fabricated tool electrodes. In the µ-SEM process, the non-rotating tool electrode moves downward to make a microhole on the workpiece. Micro spark erosion machining (µ-SEM) for meso and microhole drilling using hollow rotating tool electrodes is popularly known as micro spark erosion drilling (µ-SED). Z-axis. The spindle is attached with a direct current (DC) motor. The ceramic or diamond guide is used to position the electrode on the desired location of the workpiece for drilling the fine hole. The guide is located above the workpiece. The downward feed of the electrode is provided with the help of a servo drive that maintains a certain gap between the electrode and workpiece, in which sparks are generated and burn the materials from the workpiece to produce through or blind fine-hole. During drilling, the dielectric fluid continuously circulates through the rotary hollow electrode to flush away the eroded particle or debris from IEG and dissipates the heat produced by electric sparks. Only localized flushing is used in the µ-SED process. The drilling is completed after the desired depth of penetration on the workpiece by the rotary electrode and the electrode is retracted above the workpiece. The working principle of µ-SED is similar to the spark-erosion machining process (SEM) and its micro version (µ-SEM). The mechanism of material removal is based on thermoelectric erosion. A very strong electric field is formed at minimum IEG after supplying DC power to the workpiece and rotary electrode. The suspended microscopic particles in a dielectric fluid are accumulated around the location of the strongest electric field and form a conductive bridge across the electrodes gap. When the supply voltage is exceeded to dielectric breakdown voltage then the conductive bridge breaks due to excessive heat and temperature. Due to the collapse of the conductive bridge, sparks are generated between the interelectrode-gap. The material is removed from the workpiece due to melting and vaporization, and at the same time dielectric is flushed away from the eroded particles from the interelectrode-gap to ensure smooth drilling. The generation of sparks is continuously repeated until the drilling is up to the desired depth [µ-SED process is similar to µ-SEM except hollow tool electrodes rotate continuously during the drilling process. In the µ-SED process, a vertical precision hollow electrode is firmly mounted into the tool chuck and attached to the spindle located along ed depth .µ-SED is mostly used for quick, accurate, and fine deep-hole drilling applications. µ-SED can be found in several industrial applications, such as dental and medical, automobile, dies and moulds, electronics and telecommunication, and aerospace industries. The products and components which consist of microholes drilled by µ-SED are: (i) spray/injection nozzles; (ii) metal filters; (iii) fuel injectors; (iv) metallic filters; (v) dental tools; (vi) coolant holes; (vii) cooling manifolds; (viii) oil drain; and (ix) turbine blades.The major advantages of µ-SED are (i) the ability to drill deep high-aspect-ratio (HAR) fine holes, (ii) the ability to produce accurate fine circular holes, (iii) no burr formation, thus eliminating the secondary finishing process, (iv) ability to drill high precision microscopic-holes, (v) the absence of mechanical stresses, (vi) the ability to produce holes on any kind of materials irrespective of their hardness, (vii) the ability to produce holes on thin and fragile materials; (viii) the ability to drill holes at a certain angle, (ix) the high material removal rate; and (x) unattended machining for a longer period.Apart from the abovementioned advantages, µ-SED has certain limitations, such as the excessive tool electrode wear, not being suitable for non-circular holes, not being suitable for large-sized holes, the debris accumulated between IEG, and the abnormal sparks during drilling.In the past two decades, substantial research has been focused on drilling deep fine holes in a variety of materials using various conventional and advanced micro-drilling methods. Nevertheless, very limited research work is found in the literature on the drilling of deep fine holes on difficult-to-machine materials  using conventional methods. The following sections present a summary of the previous work on the fabrication of microholes by spark-erosion-based processes and their comprehensive discussion.The available literature shows the interest of several researchers in the past few decades in exploring these drilling methods due to their exceptional abilities for net-shape drilling of fine holes on difficult-to-machine materials with better geometrical and dimensional accuracy, minimum tool electrode wear, and improved productivity. Lee et al.  successfZhang et al.  fabricatNine trial experiments were designed and performed using a full-factorial design (23) to identify the significant drilling parameters for SEM and laser drilling . The triMacro-holes of 1 and 2 mm diameter were fabricated on Titanium TC4 by SEM process using a copper electrode and deionized water as the dielectric . ExperimBellotti et al.  fabricatA comparative study was conducted for drilling macroholes on die steel by SEM using nickel-coated and diamond–nickel-coated composite electrodes of 2 mm diameter with each using deionized water for flushing eroded particles during drilling . They coSánchez et al.  developeJeong et al.  proposedMeso-sized holes were fabricated on tool steel (X153CrMoV12-1) by SED process using brass (CuZn37) electrodes of 2 mm diameter with spindle rotational speeds of 500 revolutions per minute and negative polarity . A totalA comparative study was performed to evaluate the effect of dielectric  fluids on the quality of an array of microholes 256 holes) fabricated on stainless steel (17–7 PH) by µ-SED using a pure tungsten electrode of 100 µm diameter . The flo holes faIn an important study, Plaza et al.  proposedIn a comprehensive investigation, fifty-four experiments were planned and conducted using Taguchi’s L54 mixed orthogonal array to fabricate through microholes of 0.5 mm diameter on 316 L stainless steel sheet using µ-SED using a tungsten rod with a diameter of 300 µm and DCO-1000i SEM oil serving as a dielectric . TaguchiHigh-aspect-ratio through microholes on 2 mm thick Ti6Al4V alloy by µ-SED using brass tubes (outside diameter 350 µm and inside diameter 130 µm) with a 750 rpm rotational speed and hydrocarbon oil as the dielectric were fabricated . AnotherKuppan et al.  studied µ-SED can produce high-quality circular microholes on difficult-to-machine materials.µ-SEM has the potential to produce different types of microholes.Tool rotational speed is a significant parameter to improve the productivity of µ-SED.Most of the work has been focused on fabricating circular microholes by µ-SED.Most of the work focused on TWR during the drilling of microholes by µ-SEM and µ-SED.Very little work has been done on the modeling and optimization of µ-SEM and µ-SED variable parameters for drilling microholes.µ-SED is more accurate and faster than the µ-SEM process.The spark-erosion-based drilling of fine holes and a review of the past work on that have been reported in this paper. The conclusion is as follows:Investigations on fabricating microholes by micro-drilling and other advanced machining processes.Investigations on the fabrication of tapered microholes by µ-SED process.Investigations on the sustainability of µ-SED process for fabrication of microholes.Identify the effect of different dielectrics and wire materials on the performance of µ-SED process for the fabrication of microholes.Development of hybrid drilling processes, such as µ-SED and ECH processes.Analysis of microholes quality, namely geometrical accuracy, surface finish, and surface integrity.Exploring other advanced drilling processes for the fabrication of microholes.Improving the productivity of µ-SED for drilling microholes by optimizing the process parameters.Exploring µ-SEM for drilling conical microholes.Comparative evaluation among µ-SED, µ-SEM, advanced machining processes, and non-traditional drilling processes for fabricating microholes.The following aspects can be explored as future work to further establish the field of meso and microhole drilling by spark erosion based machining processes:


Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disorder. With the availability of treatment and renal replacement therapy, nephropathic cystinosis has evolved from an early fatal disease to a chronic, progressive disorder with potentially high impairment. We aim to review the literature on the health‐related quality of life and identify appropriate patient‐reported outcome measurements to assess the health‐related quality of life of patients with cystinosis. For this review, we conducted a literature search in PubMed and Web of Science in September 2021. Inclusion and exclusion criteria for the selection of articles were defined a priori. We identified 668 unique articles through the search and screened them based on title and abstract. The full texts of 27 articles were assessed. Finally, we included five articles (published between 2009 and 2020) describing the health‐related quality of life in patients with cystinosis. All studies, apart from one, were conducted in the United States, and no condition‐specific measurement was used. Patients with cystinosis reported a lower health‐related quality of life (for certain dimensions) than healthy subjects. Few published studies address the health‐related quality of life of patients with cystinosis. Such data must be collected standardized and follow the FAIR  principles. To gain a comprehensive understanding of the impact of this disorder on health‐related quality of life, it is necessary to use generic and condition‐specific instruments to measure this, preferably in large samples from longitudinal studies. A cystinosis‐specific instrument for measuring health‐related quality of life has yet to be developed. Health‐related quality of life in patients with cystinosis is an under‐researched area that could be imrproved through validated patient‐reported outcomes and qualitative research techniques.1CTNS gene, which encodes the protein cystinosin.Nephropathic cystinosis is a rare lysosomal storage disorder with an incidence of 1 in 100 000–200 000 live births and is caused by mutations in the Cystinosis is one of the few rare genetic diseases for which treatment is available.Patients with cystinosis are typically diagnosed at 12–24 months of age (mean 14 months), presenting with stunted growth, rickets, polydipsia, and polyuria resulting from proximal tubular impairment.Early diagnosis and treatment positively influence the clinical outcome of patients with nephropathic cystinosis.Patient survival into adulthood as a result of cysteamine treatment has allowed for the emergence of chronic disease effects within multiple organ systems of the body.The myriad of disease‐related health effects combined with a demanding treatment regimen can negatively impact the health‐related quality of life (HrQoL) of patients with cystinosis.This article reviews the literature on HrQoL in patients with cystinosis intending to improve overall understanding of the subjective experience of living with this rare chronic disorder. On the one hand, we aim to describe HrQoL in patients with cystinosis. On the other hand, we want to identify validated patient‐reported outcome measures (PROMs) that capture HrQoL in this population appropriate for use in research and clinical daily routine.22.1We searched the Web of Science and PubMed databases for relevant literature on 22 September 2021 and updated results as of 07 April 2022. The methodological framework of Arksey and O'Malley's five‐stage scoping review guided the literature review.a priori.We considered qualitative and quantitative empirical studies focusing on HrQoL in patients with cystinosis. For a quantitative study to be considered for inclusion, HrQoL must have explicitly been measured using validated PROMs, along with details on how it was measured. All empirical study design methods were eligible for inclusion , while reviews were excluded. There were no geographic, language, or temporal restrictions. We only considered studies published in peer‐reviewed journals, although this included published conference abstracts due to the assumed scarcity of studies. These inclusion and exclusion criteria were agreed upon As additional constructs, such as psychosocial health, mental health, and well‐being, are sometimes used as a synonym for HrQoL,2.2After using the agreed search terms in the databases and removing duplicated publications, two independent reviewers screened the search results based on the title and abstract. The same two reviewers thoroughly read the full texts of any articles that met the inclusion criteria or for which it was not possible to assess eligibility based on the title and abstract alone, with subsequent evaluation for eligibility. The reviewers discussed any discrepancies or ambiguities regarding inclusion eligibility until a consensus was reached. In addition, reference lists of included publications were also reviewed for possible inclusion using the same eligibility criteria.2.3Data on the author, year of publication, country of origin, study objective, study design, number of participants, participant characteristics, HrQoL measure, and the main study results were recorded as well as PROMs' characteristics .33.1The search terms identified 668 unique publications after removing any duplicates. Based on the title and abstract screening, 641 publications were excluded, while 27 publications were selected for full‐text screening. Of these, four publications met the inclusion criteria.3.2Table 3.33.3.1positive emotions ). Based on reports from their parents, HrQoL was impaired in four dimensions , autonomy , social function , and cognitive function ), while three dimensions were in the normal range .Two of the studies identified had an observational design and aimed at describing the HrQoL of patients with cystinosis compared to healthy subjects.p ≤ 0.05). While there was no significant difference in the physical health dimension, differences were found between patients with cystinosis (higher score) and their parents in the psychosocial health dimension . Compared with previously published reports from healthy subjects, both child‐reported and parent‐reported HrQoL was significantly lower .Elenberg et al. measured HrQoL in 14 patients using the PedsQL cross‐sectional survey tool.3.3.2social functioning (p ≤ 0.05), school functioning (p ≤ 0.01), and total functioning (p ≤ 0.05). No significant change was observed in the dimensions of physical functioning or emotional functioning.Langman et al. reported HrQoL outcomes of 40 patients with cystinosis aged 6–20 years treated with delayed‐release cysteamine bitartrate for two years.In a longitudinal follow‐up study with a “pre–post” design, Sadjadi et al. evaluated the impact of a 5‐week expiratory muscle strength training (EMST) program on HrQoL and other outcomes in patients with cystinosis using the MDADI.3.3.3A qualitative study of 22 patients aged 18–47 and their parents examined patients' experiences with cystinosis transitioning to adulthood.3.4physical functioning (eight items), emotional functioning (five items), social functioning (five items), and kindergarten (three items), respectively school, university, or work (five items). This PROM is a parent‐report form for children aged 2–4 years. Both self‐report and parent‐report forms are validated for the HrQoL asessment in children , adolescents (13–18 years), young adults (18–25 years) and adults. Cronbach's alpha ranges from 0.71 to 0.88 for the self‐report form and 0.75 to 0.90 for the parent‐report form.physical functioning, physical symptoms, emotional functioning, social functioning, and cognitive functioning). Cronbach's alpha ranges between 0.72 and 0.92.Our scoping review identified three PROMs used to assess HrQoL in patients with cystinosis Table . The Pedphysical, motor functioning, autonomy, cognitive functioning and school, social contacts, positive emotions, and negative emotions). Cronbach's alpha varies between 0.65 and 0.79 for the self‐report form and between 0.67 and 0.84 for the parent‐report form.The TACQOL is a generic PROM to assess HrQoL in healthy children and children with different chronic diseases aged 6–15 years. Parent‐report forms are validated for children aged 6–11 years, and self‐report forms are available for children aged 8–15. The questionnaire consists of 56 items, including seven scales with eight items for each scale  respectively 12 , depending if the children's age is more than 18 months. Cronbach's alpha for a general population sample varies between 0.43 and 0.84.gross motor functioning, fine motor functioning, cognition, sleep, pain, social contacts, daily activities, sex, vitality, happiness, depressive mood, and anger). Reliability ranges between 0.72 and 0.90.There are also validated versions of this tool for preschool children (TAPQOL) aged 9 months up to 6 years and adults aged 16 years and above (TAAQOL). The TAPQOL consists of 32 respectively 43 items resulting in nine scales  is a disease‐specific PROM focusing on dysphagia in adult patients. The four scales of the MDADI include 4This scoping review identified five articles describing HrQoL in patients with cystinosis.Our review shows that research on HrQoL in patients with cystinosis is still rare. To fully understand the impact of cystinosis on HrQoL, additional observational studies with larger sample sizes and longitudinal studies are needed. In this context, it is also important to clarify which domains of life are affected by the disease and to what extent adaptation to the disease and treatment side effects come into play.Validated PROMs are suitable measurement tools, particularly for the characterization of HrQoL. The three quantitative studies in our review used PROMs to assess HrQoLGeneric, chronic‐generic, and condition‐specific PROMs have unique advantages and disadvantages that should be considered depending on their intended use.In clinical trials, condition‐specific PROMs could offer important advantages. Specific PROMs can be particularly helpful for rare chronic health conditions as they address the most specific concerns of unique patient populations  and are more sensitive to condition‐specific changes.Currently, there is no cystinosis‐specific instrument for measuring HrQoL in this patient population; qualitative research is needed to develop a cystinosis‐specific HrQoL PROM. In the meantime, allowing patients to share their stories, makes it possible to understand their experiences in detail, capture the full spectrum of manifestations, and identify any possible relationships with HrQoL.In the one qualitative study included in this review, valuable insights emerged from the focus group discussions and interviews, such as how treatment progress has opened up new life perspectives and milestones in the affected individuals' lives or how side effects have impacted social relationships and consequently treatment adherence and health outcomes.When developing and validating a cystinosis‐specific HrQoL instrument, the recommendations on patient‐reported outcome measurements provided by the International Society for Pharmacoeconomics and Outcomes Research and US Food and Drug Administration need to be considered and have to include all steps; literature review, focus interviews with patients and/or parents, development of a pilot‐test, field‐ and re‐test as well as linguistic and cultural validation for use in additional languages.The experience of the cross‐cultural development, validation, and cultural adaptation of condition‐specific PROMs such as the Quality of Life in Short Stature Youth (QoLISSY) questionnaire to assess the HrQoL of short stature children and adolescentsOur scoping review did not identify any study based on registries, although we included registries due to the a prior‐defined inclusion criteria. For validation of a condition‐specific PROM, registries have an enormous potential to assess HrQoL and to monitor HrQoL regarding progression and treatment. Regular use of validated PROMs in registries is desirable. It requires a good collaboration of experts in quality of life research, clinical care, and patient organization to provide the best possible care that considers the patient's perspective.The small sample sizes and the fact that four out of five studies were conducted in the United States, while one was from Switzerland, must be considered when interpreting the results and highlighting the need for further research. The present review does not claim to identify every existing study on HrQoL in patients with cystinosis. Instead, this scoping review has allowed us to summarize research findings, make conclusions about the general state of research, and highlight any research gaps. In addition, it is essential to emphasize that quality assessments are not part of a scoping review.In summary, few studies have investigated HrQoL in patients with cystinosis. A few studies suggest that HrQoL is reduced in patients with cystinosis compared with healthy individuals. Additional observational studies describing HrQoL using mixed methods with quantitative and qualitative elements and an equal focus on all age groups are needed. Especially in rare diseases, data must be collected standardized and follow the FAIR  principles.Kaja Kristensen, Stefanie Witt, Katharina Hohenfellner, and Julia Quitmann developed the study concept and the design. Kaja Kristensen and Stefanie Witt screened the publications and identified relevant articles. Kaja Kristensen wrote the first draft of the manuscript. Stefanie Witt, Julia Quitmann, and Katharina Hohenfellner critically reviewed and revised the first draft for important intellectual content. All authors have critically revised subsequent drafts of the manuscript. All authors read and approved the final manuscript to be published and agreed to be accountable for all aspects of the work.This scoping review was funded by Cystinosis Foundation Germany. The sponsor has not influenced the content of the article.Stefanie Witt, Kaja Kristensen, Katharina Hohenfellner, and Julia Quitmann have no competing interests.Not applicable.Not applicable.Not applicable.


Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) were tested by the inhibition zone method. Our composite ZnO@PPL/Cel has superior antibacterial activity compared to ZnO/Cel. The antibacterial mechanism has also been elaborated on. The low cost, simple preparation method and good performance of ZnO@PPL/Cel suggest the potential for it to be applied as active food packaging.To investigate the efficient use of bioresources and bioproducts, plant polyphenol (PPL) was extracted from larch bark and further applied to prepare ZnO@PPL/Cel with cellulose to examine its potential as an active package material. The structure and morphology were fully characterized by XRD, SEM, FTIR, XPS and Raman spectra. It was found that PPL is able to cover ZnO and form a coating layer. In addition, PPL cross-links with cellulose and makes ZnO distribute evenly on the cellulose fibers. Coating with PPL creates a pinecone-like morphology in ZnO, which is constructed by subunits of 50 nm ZnO slices. The interactions among ZnO, PPL and cellulose have been attributed to hydrogen bonding, which plays an important role in guiding the formation of composites. The antibacterial properties against Gram-negative  Plant polyphenols (PPL), the fourth most abundant bioresource in nature other than cellulose, lignin and hemicellulose, is widespread throughout all kinds of plants. Many components of PPL, such as catechin, flavones, anthocyan, and phenolic acid, have been determined and their structures depend upon the flavan-3-ol starter and extension units. Due to the diversity of functional groups in PPL, it is believed to have beneficial health effects for humans and work as anticancer agent [With industrial development and social progress, fossil resources have become very important as raw materials for industry. Massive use of fossil resources results in the emission of carbon dioxide, which has become a serious problem for environmental and sustainable development. To reduce the production of carbon dioxide and achieve carbon neutrality, substitute resources and technology need to be developed ,2. Usinger agent ,6,7,8. Ier agent ,10,11,12+ ions from their hydroxyl groups and delocalize free electrons, which can kill bacteria.Due to structural complexity of PPL, there are many kinds of PPL and all of them have the same character: an abundance of hydrogen groups in their units. As a result of this structure, PPL can release HThough PPL has many fascinating merits, studies on PPL are limited compared with those on cellulose, lignin or hemicellulose. The reason for this is the difficulty of its extraction and separation from plants. As all PPLs have similar structures and properties, it is complex and expensive to obtain specific components from the PPL mixture. Thus, finding a new way to use PPL mixture efficiently is an ongoing challenge.Salmonella typhi, Staphylococcus aureus, Escherichia coli and Staphylococcus aureus were fully investigated. However, reports about controlling synthesis of ZnO with small size and plenty of defects, which play an important role in improving its performance, are still rare. Thus, new technology to prepare ZnO with high antibacterial activity is still in demand.As an innovative and promising technology, active food packaging would prevent food from oxidation and pathogen growth ,14,15,16In this work, we prepared an active food package material of ZnO@Plant polyphenols/cellulose (ZnO@PPL/Cel) via a hydrothermal method. The PPL were extracted from the byproduct material of larch bark and used as a mixture without separation. This method would greatly reduce of preparation costs of ZnO@PPL/Cel. In ZnO@PPL/Cel composite, PPL is found coated on ZnO via hydrogen bonding but it can also build interfacial bonding with hydroxyl groups of cellulose and construct a composite package material. However, the coating of PPL restricts the growth of ZnO and creates many defects on these nanoparticles, which can enhance the antibacterial activity. The low cost, lack of secondary pollution and benefits to human health suggests the potential application of this material as active food packaging. The synthesis route and connection of components is shown in Anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd. . Zinc acetate was purchased from Tianjin Comiou Chemical Reagent Co., Ltd. . Cellulose was purchased from Hangzhou Fuyang Beimu Pulp and Paper Co., Ltd. . Agar, beef extract and peptone were purchased from Fujian Shishi Gaoxin Agar Food Co.  and Beijing Oboxing Biotechnology Co., Ltd. . All the chemical reagents were analytically pure, and agar, beef extract and peptone are biological reagents.Larch bark polyphenols were extracted from larch bark powder by an organic solvent extraction method. A 20 g larch bark powder sample and 200 mL 50% ethanol solution were added to a 250 mL conical flask and shaken at 50 °C . After a 6 h extraction, the residue of larch bark was separated by filtration and the PPL solution was obtained. Each 100 mL PPL extraction contained about 1.863 g PPL.−1 NaOH solution. Stirring was continued for 0.5 h; then the mixture was transferred into a 100 mL hydrothermal reactor and heated at 100 °C for 10 h. After cooling, ZnO@PPL/Cel was obtained after washing and drying.Zinc acetate solution was obtained by dissolving 3.0 g zinc acetate in 50 mL of distilled water and then adding 1.0 g cellulose. After 1 h of stirring, 5 mL PPL solution was added and the pH of the mixture was adjusted to 11 with 2 mol LDPPH scavenging activity denotes the free radical scavenging rate of DPPH, A0 is the absorbance of DPPH ethanol solution at 517 nm, and A stands for the absorbance of the mixture.Antioxidant activity of the prepared samples was tested by scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals ,41. A 1.Staphylococcus aureus (S. aureas) and Escherichia coli (E. coli) were used as reference strains for antimicrobial tests. The antibacterial effect of the composite was tested and evaluated by the inhibition zone method [e method ,43. A 0.W (g) is the quantity difference between the certain intervals, T (mm) and S (m2) are the average thickness and surface area of the sample, respectively, and t (h) is the time interval.The water vapor transmission rate (WVTR) of samples was tested according to the reference . In the The crystalline structures of samples were analyzed by X-ray diffractometer . The incident light was set to CuKα, λ = 1.5418 Å, the scanning voltage was set to 40 kV, the scanning current was set to 30 mA, and the scanning rate was set to 5°/min. Fourier transform infrared spectrometer  was used to analyze the functional groups of samples. The UV spectra were measured with a TU-1901 ultraviolet-visible spectrophotometer . Laser confocal Raman spectroscopy of LabRAM HR800  was used to analyze the molecular vibrations. The microstructure of the samples was examined by scanning electron microscope . The chemical environment around PerkinElmerC, O and Zn in the composites was characterized by X-ray photoelectron spectroscopy .The structure of composite as revealed by the XRD results is shown in According to the standard ZnO, the intensity of (002) is lower than the (100) plane. However, when PPL was involved during preparation, the (002) plane in ZnO@PPL/Cel becomes stronger and higher than that of (100). This result means that ZnO has a predominant growth orientation along the C-axis. ZnO is a polar crystal and has a Zn terminus on (001) planes , which iSince the stability of PPL could be affected by alkaline conditions and the hydrothermal treatment, PPL before and after treatments was evaluated by the FT-IR and UV spectra. The sample treated by alkalinity of pH = 11 was named PPL-11, and it was further hydrothermally treated at 100 °C for 10 h and the resultant sample was named PPL-11-H. The UV spectra b show th−1 for all the PPL samples. However, this adsorption peak becomes weaker in the PPL−11 and PPL−11−H samples. This suggests that alkaline and high-temperature treatments can cause a decrease in phenol groups. Moreover, the peaks around 1600 cm−1 become broader in the range of 1600–1740 cm−1, which corresponds to the benzene skeleton and C=O vibrations. The enhancement of C=O stretching vibrations is possibly caused by the structural transformation between hydroxyl group and C=O. Due to the abundance of phenol groups in PPL, there were still hydroxyl groups remaining in the treated PPL, which make FT-IR spectra of PPL, PPL−11 and PPL−11−H appear similar in general.In the FT-IR spectra in −1. Since there is an abundance and complexity of –OH groups in PPL, the peak shows a wide and relatively strong character in the IR spectrum. The peak at 1604 cm−1 is the skeleton vibrations of the benzene ring in PPL. Functional groups of C=O in PPL around 1695 and 1785 cm−1 are also observed, indicating the multifunctional groups of PPL. The adsorption band at 1000–1300 cm−1 is C−O vibration, which is similar to proanthocyanidins [−1 is C−O vibrations and the adsorption for hydroxyl groups in cellulose displays at 3336 cm−1, similar to that of PPL. Considering ZnO@PPL/Cel, absorption bands around 1570 and 1415 cm−1 are the vibrations of a benzene ring, indicating the existence of PPL in the composite. The C−O stretching vibration band is found around 1056 cm−1, which originates from cellulose or PPL. In addition, this peak has red shift compared with PPL and ZnO@Cel, which may be caused by the coupling effect with ZnO. Moreover, the vibrations of hydroxyl groups are relatively weaker in ZnO@PPL/Cel than in PPL, PPL−11−H and ZnO/Cel. This means that hydroxyl groups in ZnO@PPL/Cel may form hydrogen bonds with each other and weaken the vibrations. The analyses of FT-IR provide further evidence that PPL works as cross-linker which can form hydrogen bonds with both cellulose and ZnO.yanidins . It can yanidins . The IR Moreover, there are slices on particles, which means that pinecone-like ZnO@PPL was constructed by subunits with the size about 50 nm and form the hierarchical structure. The ZnO@PPL covered the cellulose uniformly and was well dispersed, which finally forms the composite morphology. Thus, we deduce that PPL in the composite may work as crossing linker, which connects the ZnO and cellulose. The role of PPL enhances the connection between ZnO and cellulose and results in a more uniform morphology in the composite, which can be used as active packaging material.The XPS was also applied to analyse the elemental chemical circumstance of ZnO@PPL/Cel. −1. The peaks of 456 and 590 cm−1 are the first-order phonon frequencies and peaks at 345 and 1158 cm−1 are the second-order Raman scattering frequencies of ZnO. These vibration peaks indicate the existence of ZnO. For weak dipole symmetrical vibrations of cellulose, there is no characteristic peak of cellulose found in ZnO/cellulose. However, the Raman spectrum of ZnO@PPL/Cel shows different results: the characteristic peak of benzene in PPL is found at 1590 cm−1. Moreover, the peak of ZnO around 453 cm−1 is greatly depressed and all the peaks have a red shift of about 10 cm−1. These changes may be caused by the coupling effect between the PPL and ZnO, which weakens the vibrations. Raman analysis further provides the evidence that PPL can form interactions with ZnO.To further study the structure of the composite, Raman spectra of ZnO, ZnO/Cel and ZnO@PPL/Cel were tested and are shown in Photo luminescence spectra further revealed the microstructure of samples and the results are shown in It is known that the antioxidant property of materials is related to the shelf life of food. Thus, the antioxidant activity of ZnO@PPL/Cel was evaluated by the method of DPPH radical scavenging activity . As showEscherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) were used to evaluate the antibacterial behavior of samples by the inhibition zone method [E. coli and S. aureus. However, both ZnO/Cel and ZnO@PPL/Cel show high antibacterial activity. The diameter of the inhibition zones of ZnO/Cel against E. coli and S. aureus were 18.9 and 20.9 mm, respectively. When the sample diameter (15 mm) is subtracted, the widths of inhibition zones were 3.9 (E. coli) and 5.9 (S. aureus) mm. When coupled with PPL, ZnO@PPL/Cel samples had much better performance than ZnO/Cel: the inhibition zones for E. coli and S. aureus increased to 24.9 mm and 31.2 mm, respectively. The calculated width of inhibition zones was 5.9 and 8.1 for E. coli and S. aureus, respectively, which have been improved 150% and 137% compared to those of ZnO/Cel. These results are evidence that PPL in the composite would enhance the antibacterial activity greatly.In active food package materials, antibacterial performance is very important. In our work, Gram-negative e method . It can 2−, H2O2 and OH−), which can kill bacterial cells. The production of ROS depends on the illumination light and the band gap as well as defects in the ZnO. In our case, the ZnO in composites has many more defects than ZnO and ZnO/cellulose, which would produce more ROS during application. In addition, PPL has the effects of membrane lysis, inhibition of energetic metabolism, reduction of host ligands adhesion and neutralization of bacterial toxins, which also can inhibit the growth of bacteria [Some particular mechanisms increase the activity of ZnO as an antibacterial agent . A commobacteria . Thus, tTo further study the role of PPL on antibacterial activity, different dosages of PPL (2.5 mL and 7 mL) were also applied to fabricate the composite. From E. coli than S. aureus. For E. coli, antibacterial activity decreases greatly with the increase of pH. When pH is 11, only a 2.4 mm inhibition zone resulted, which decreased by 52% compared with that of ZnO@PPL/Cel in the neutral conditions. However, the inhibition zone of ZnO@PPL/Cel-5 against S. aureus decreased by 11%. This may be caused by the protons which permeate into cells and kill the bacteria.The pH sensitivity of the composite was also tested in light of its antibacterial activity. From 2 mm). The high value is caused by the cellulose in the composite. The combination with PPL allows the WVTR value of ZnO@PPL/Cels to decrease and fall within the range 41.75–40.01 g/(h m2 mm). The decrease is perhaps caused by the reduction of hydroxyl groups of cellulose, which are exploited to build hydrogen bonds with ZnO and PPL. Moreover, the high WVTR of the composite can provide a drying environment for the stored food and inhibit microbial growth.The water vapor transmission rate (WVTR) of samples was tested according to the reference . As showE. coli and S. aureus. The widths of inhibition zones for E. coli and S. aureus, were determined to be 5.9 mm and 8.1 mm, respectively, which were improved by 150% and 137% relative to those of ZnO/Cel. The newly-synthesized material, which has benefits including renewability, low cost, a simple synthesis method and good antibacterial activity, is promising as an active food package.Active package material based on the composite ZnO@PPL/Cel has been accessed via a simple hydrothermal method. It was found that PPL forms a coating layer on the surface of ZnO with the assistance of hydrogen bonds and restricts the ZnO growth. Thus 200 nm pine-like ZnO@PPL is observable, which is assembled from subunits of 50 nm ZnO slices. Moreover, PPL offers hydrogen bonding, which makes ZnO@PPL evenly distributed on the cellulose and together build a hierarchical structure. The narrowed band gap and massive defects were revealed in ZnO@PPL/Cel, which enhance its antibacterial properties. Antibacterial performance was evaluated against


Brucella is a typical facultative intracellular bacterium that can cause zoonotic infections. For Brucella, it is difficult to eliminate with current medical treatment. Therefore, a multi-epitope vaccine (MEV) should be designed to prevent Brucella infection. For this purpose, we applied the reverse vaccinology approach from Omp10, Omp25, Omp31 and BtpB. Finally, we obtained 13 cytotoxic T lymphocyte (CTL) epitopes, 17 helper T lymphocyte (HTL) epitopes, 9 linear B cell epitopes, and 2 conformational B cell epitopes for further study. To keep the protein folded normally, we linked AAY, GPGPG, and KK to CTL epitopes, HTL epitopes, and B cell epitopes, respectively. The N-terminal of the vaccine peptide is supplemented with appropriate adjuvants to enhance immunogenicity. To evaluate its immunogenicity, stability, safety, and feasibility, a final MEV containing 806 amino acids was constructed by linking linkers and adjuvants. In addition, molecular docking and molecular dynamics simulations were performed to verify the affinity and stability of the MEV-TLR4. Then, codon adaptation and in silico cloning studies were carried out to identify the possible codons for expressing the MEV. In animal experiments, the results demonstrated that the MEV had high immunogenicity. Collectively, this study provided a theoretical basis for the development of a Brucella vaccine. Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, B. inopinata, B. neotomae, B. microti, B. vulpis, B. ceti, B. papionis, and B. pinnipedialis. Thereinto, B. melitensis mainly infects goats and sheep, and human brucellosis is mostly caused by it2. The human brucellosis manifested as mild fever, itching rash, arthritis, fatigue, and muscle pain. And it is a systematic infectious disease that is transmitted to humans through ingestion of unpasteurized milk or via close contact with the primary host4. Brucellosis is a major human health problem in many countries, especially in Mediterranean areas, parts of South and Central America, and northern China5. Recently, it was reported that the incidence of brucellosis in China has risen dramatically6. However, there are still many clinical challenges in the diagnosis and treatment of brucellosis. This is because it has no specific clinical characteristics and grows slowly in blood culture. In addition, the disease is easy to develop into chronic, which can affect multiple organs at the same time. Therefore, it is necessary to develop a new therapeutic approach against Brucella in early prevention.Brucella is one of the Gram-negative bacteria that does not contain flagella and does not form spores or pods. The genus Brucella mainly includes 12 species, which are 7. Currently, vaccine research consists of many approaches, such as multi-epitope vaccines (MEV), vectored vaccines, DNA vaccines, and reverse genetics-engineered live-attenuated vaccines. MEV is an emerging vaccine development approach, which uses reverse vaccinology to improve the safety and efficacy of vaccine8. In the design of MEVs, it is necessary to select the appropriate protein as antigen. In previous studies, a series of outer membrane proteins and effector proteins were used to identify immunodominant antigens against Brucella spp. Outer membrane protein 10 (Omp10), one of the outer membrane proteins of B. melitensis, has been proved to have good immunogenicity in previous experiments and is suitable as a vaccine design candidate protein9. Outer membrane protein 25 (Omp25) is one of the outer membrane proteins of B. melitensis and has good immunogenicity. The difference is that Omp25 leads to the occurrence of an inflammatory response and the chronicization of brucellosis by activating the MAPK signaling pathway10. Therefore, based on Omp25, the vaccine may slow down disease progression. Similarly, outer membrane protein 31 (Omp31), the major outer membrane protein of B. melitensis, has been shown to have good immunogenicity in animal models of infection and is also a good choice for vaccine design12. BtpB, an effector protein of B. melitensis, is present in all species of the genus Brucella. It mainly reacts with MyD88, inhibits TLR signaling, and disrupts activation of dendritic cells BtpB, restraining TLR2, TLR4, TLR5, and TLR9 signaling. And it is also a new option for designing vaccines13. In summary, the four Brucella-associated proteins are suitable for the MEV design against B. melitensis.As biological products, vaccines can stimulate and induce specific immunity to specific pathogens, which are an effective way to prevent brucellosis14. However, epitope-linked vaccine proteins alone are susceptible to enzymatic degradation, so specific adjuvants were selected to stabilize the vaccine construct15. Then, we analyzed the physicochemical properties of the MEV, constructed its tertiary structure, and performed the molecular dynamics of the MEV-TLR4. Finally, animal experiments were carried out to validate the prediction results. The current study can provide theory and data for further research, and also can increase a new method and idea for the development of an MEV against B. melitensis.In our study, we analyzed epitopes of Omp10, Omp25, Omp31, and BtpB, using diverse bioinformatics methods which were IEDB, NetCTLpan1.1, NetMHCIIpan4.0, ABCpred, and BCPREDS. Linkers are required for epitope linkage. The B-cell epitope and CTL epitopes were linked with the AAY linker, and HTL epitopes were linked together and to the CTL epitopes with the GPGPG linkerhttp://web.expasy.org/protparam/) software was applied to analyze the physicochemical properties of proteins and MEVs. The physicochemical properties include the number of amino acids, molecular formula, acidity and basicity of amino acids, and a grand average of hydropathicity (GRAVY). VaxiJen (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) was applied to analyze the antigenicity of the proteins16. All outer membrane proteins and effector proteins of B. melitensis should be taken into account. However, based on existing studies, we compared the homology, antigenicity, hydrophilicity, and stability of some important outer membrane proteins and effector proteins. Finally, we found that these four proteins had good antigenicity, hydrophilicity, and stability in designing an MEV.ProtParam (https://www.uniprot.org/). Finally, we analyzed the homology of the obtained sequences by comparing the amino acid sequence using MAFFT in Jalview17.The amino acid sequences of Omp10 , Omp25 , Omp31 , and BtpB  were obtained by applying the Uniprot database  and LiPOP1.0  were applied to predict the signal peptide of the proteins. Based on the results of the software, we choose the merged set as the final result of the prediction18.SignalP5.0  to predict the cytotoxic T lymphocyte (CTL) epitopes and helper T lymphocyte (HTL) epitopes21. IEDB (http://tools.immuneepitope.org/) and NetCTLpan1.1 server  were applied to predict the CTL epitopes of the target protein23. During CTL epitope prediction 1 was set as a threshold value for epitope identification. Since NetCTLpan1.1 counts from 0, we add one to the starting sequence number of all NetCTLpan1.1 to facilitate comparison with the results of IEDB. Accordingly, we listed the top 10 high-score epitopes for each prediction software. IEDB and NetMHC-IIpan-4.0  were applied to predict the HTL epitopes of the target protein24. When predicting HTL epitopes, the default threshold for NetMHC-IIpan4.0 is set to the original threshold.T-cell epitopes are provided by class I (MHC I) and II (MHC II) MHC molecules that are recognized by two distinct subsets of T-cells, CD8, and CD4 T-cells, respectivelyFinally, we selected two software overlapping sequences as T cell dominant epitopes for the proteins.http://ailab.ist.psu.edu/bcpred/predict.html) was used to predict the selection of dominant B-cell linear epitopes with a specificity of 75%. Then, discontinuous  epitopes were performed by Ellipro (http://tools.iedb.org/ellipro/) of the IEDB. After prediction, we also deleted the sequences with less than 5 amino acids and selected the sequences with high scores and appropriate lengths. In the end, linear epitopes and conformational epitopes are considered for the design of MEV.B-cell epitopes are the antigen portion binding to the immunoglobulin or antibody. B-cell epitopes include linear epitopes and conformational epitopes. BCPREDS  and HLA class II (HLA-DRB1*01:01) were selected to look for structural associations between the HLA alleles and the T cell epitopes. The molecular docking was performed in the HDOCK server.25. Different linkers were used to link B-cell epitopes together, the KK linker was added; for linking HTL epitopes together, the GPGPG linker was appended; and concurrently, for connecting CTL epitopes, the AAY linker was used. Human β-defensin-3 sequence  and PADRE sequences were linked with the help of EAAAK linkers at the N-terminus. Finally, a polyhistidine tag was added at the C terminus to obtain the complete vaccine protein sequence26.Non-allergic and non-toxic epitopes were selected for the MEV. Then, the selected epitopes were joined together with appropriate linkers to construct the vaccine sequencehttp://scratch.proteomics.ics.uci.edu/) was used to predict the solubility of MEV. The server has an overall accuracy of 74.15% and the threshold is 0.527. Finally, AllergenFP was used to analyze its allergenicity.Bioinformatics software was used to synthesize and analyze the properties of the MEV. First of all, ProtParam software was applied to analyze the physicochemical properties of the MEV. VaxiJen was applied to analyze the antigenicity with an accuracy of 70 to 89%. Then, SOLpro (http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) secondary structure prediction tool was used. RoseTTAFold uses a machine learning-based method and includes a three-track network to process sequence, the distance between atoms, and coordinate information to predict the tertiary structure of the MEV28.To predict secondary structures of MEV, the SOPMA (https://swissmodel.expasy.org/assess) and ProSA-web server (https://prosa.services.came.sbg.ac.at/prosa.php)29. The assessment of the model quality is based on the Z-score and the dotted structure in the Ramachandran plot.The quality of the tertiary structure models of the MEV was evaluated using the SWISS-MODEL Structure Assessment  to perform the molecular docking between the MEV and the TLR4 (PDB ID: 4G8A) immune receptor31. Key interacting residues were deduced from the protein–ligand interaction diagram generated by LigPlot+ in combination and their 3D structure was visualized by PyMOL.When the MEV enters the organism, the body recognizes it as a synthetic antigen and activates the immune systemhttps://kraken.iac.rm.cnr.it/C-IMMSIM/) was applied to simulate the extent and type of immune responses induced by the MEV in humans32. In the population of the Xinjiang region, high-frequency alleles of HLA-A*1101, HLA-A*0201, HLA-B*5101, HLA-B*3501, HLA-DRB1*0701, and HLA-DRB1*1501 were selected for analysis21. Simultaneously, the server simulates three sections that represent three different anatomical regions in mammals, including the bone marrow, the thymus, and the lymph. And three different intervals were 1, 84 and 16833. Finally, the parameters were set to the default values of the software, the simulation parameter random seed was set to 12,345, the simulation volume was set to be 50, and the simulation steps were set to 1050.The vaccine enters the body as an antigen and also causes a corresponding immune response. C-ImmSim  simulation studies of the MEV-TLR4 complex were performed in Gromacs 2021.3http://www.jcat.de/). The DNA sequence of the plasmid was obtained from the Plasmid Files, and pET-28a (+) was selected as the vector. Then SnapGene 5.3.2 was used to analyze the suitable endonuclease sites in the multiple cloning site (MCS) region, and the target gene sequence of the MEV was inserted into the plasmid to complete the silicon cloning. And the MEV was synthesized and purified by SynPeptide Co Ltd . Lastly, the MEV was detected as endotoxin-negative by Limulus amebocyte lysate (LAL) before use.The XHOI and BamHI restriction endonuclease sites were selected and the codons of the vaccine were analyzed and optimized using the JAVA codon adaptation tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome)36. Finally, the recombinant plasmid was simulated agarose gel electrophoresis in SnapGene 5.3.2.We designed the primers in SnapGene 5.3.2 based on the Tm value and the length size. The length of primers is generally 15–30 bp, Tm value is chosen as 72 °C, the annealing temperature of the upstream and downstream is 1 °C, GC content of upstream and downstream is about 40–60%, and protection nucleobase is added at the 5' end. The designed primers were further confirmed in Primer-BLAST  were obtained from the animal experiment center of Xinjiang Medical University. Mice were kept in standard conditions. Followed the animal experiment requirements and operating guidelines of the Animal Ethics Committee of First Affiliated Hospital of Xinjiang Medical University, the experiment was strictly actualized. This study was approved by the Animal Ethics Committee of First Affiliated Hospital of Xinjiang Medical University . The mice were randomly divided into two groups (N = 10 per group), the control group, and the MEV group. Mice were instilled intranasally  with PBS (Control group), and 30 μg multi-epitope vaccine (MEV group) respectively for 15 days. The mice were executed and the respiratory lymph nodes  and spleen were removed under sterile conditions. Then, the homogenate was prepared for cell isolation. The trachea was cannulated, and the lungs were gently lavaged 3 times with 1 mL of sterile PBS to obtain bronchoalveolar lavage fluid. Using a Pasteur capillary pipette without anticoagulant, blood was collected from the retro-orbital sinus and placed in coagulation microtubes. Finally, the blood was centrifuged and the serum was collected.To evaluate the immunogenicity of MEV, antigen-specific IgG1 and IgG2a titers in the immunized mice were tested by enzyme-linked immunosorbent assay (ELISA). Firstly, 96-well polystyrene plates  were coated with MEV (1 μg/mL) at 100 μL/well and incubated at 4 °C overnight. Secondly, the plate was rinsed with TBS-T  4 times, followed by blocking with PBS containing 10% fetal bovine serum (FBS) at 37 °C for 2 h. Thirdly, 100 μl of mouse serum diluted at 1:100 was added to the well and incubated at room temperature for 2 h. Then, after rinsing, horseradish peroxidase-conjugated goat-anti-mouse IgG1 or IgG2a antibodies  were added and further incubated at 37 °C for 1 h. Thereafter, a substrate solution  was added to each well. The plate was subsequently incubated at room temperature for 10 min, and 20% H2SO4 was added to terminate the reaction. Finally, using a microplate reader , the absorbance value was measured at a wavelength of 405 nm. Similarly, to measure secretory immunoglobulin A (sIgA), 96-well plates were coated with 0.25 g/well MEV overnight at 4 °C. The bronchoalveolar lavage fluid (1:5 dilution) was added in triplicate into wells of the plates and incubated for 1 h at 37 °C. Then wells were washed. Horseradish peroxidase-conjugated goat anti-mouse IgA  was added and incubated for 1 h. Finally, the reaction is completed by adding substrate, etc. in the same way.5 cells/well in a final volume of 100 μL. Fifthly, cells were stimulated with the heat-killed RB51 (10 μg/mL) (a source of Brucella protein without LPS), and synthetic Toxoplasma SAG4 peptide (10 μg/mL). ConA (10 μg/mL) at 50 μL/well and PBS at 50 μL/well were used as the positive and negative controls, respectively. After that, the plates were incubated for 24 h at 37 °C in an atmosphere containing 5% CO2. After washing, 50 μL of biotin-labeled secondary antibody  was added and the plates were incubated at room temperature for 2 h. And the plates were washed with PBST and 50 μL streptavidin alkaline phosphatase  was added and incubated at room temperature for 2 h. By adding BCIP/NBT developer  to each well for incubation for 40 min at room temperature, the spots were developed. Finally, the plates were washed in water to stop the reaction and Spot forming units (SFU) in each well were counted using an ELISPOT Bio Reader-4000 . The results were expressed as IFN-γ spot forming cells (SFC) per million cells.To further evaluate the HTL response, IFN-γ levels in the immunized mice were analyzed by enzyme-linked immunospot assay (ELISPOT). First of all, ELISPOT plates were coated overnight with an anti-mouse IFN-γ capture antibody  diluted at 1:60 in PBS at 4 °C. One day later, the plate was washed four times with phosphate-buffered solution (PBST) and once with PBS. Thirdly, plates were blocked with 100 μL of RPMI-1640 medium containing 10% fetal calf serum for 1 h at 37 °C. Afterward, the respiratory lymph nodes cells and splenocytes were added in triplicate to wells at 1 × 10The animal study was reviewed and approved by The Animal Experiment Medical Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University. We confirmed that all experiments were performed in accordance with relevant named guidelines and regulations. We confirmed that the authors complied with the ARRIVE guidelines.The study found that the FASTA structures of protein from UniProt mainly include the following types: Omp2a (Accession: Q7CNU3), Omp2b (Accession: Q8YG56), Omp10 (Accession: P0A3N8), Omp16 (Accession: P0A3S7), Omp19 (Accession: P0A3P1), Omp22 (Accession: Q93SI4), Omp25 (Accession: Q45321), Omp28 (Accession: P0A3U8), Omp31(Accession: E0A8N6), BtpB (Accession: A0A1S6ZGG9), BLS (Accession: X5DV28), BCSP31 (Accession: P0A3T2), and P39 (Accession: F6N110). Through bioinformatics analysis, the antigenicity  was used to detect whether epitopes are allergens. Then, the ToxinPred (https://webs.iiitd.edu.in/raghava/toxinpred/design.php) was applied to predict the toxicity of the epitopes37. Finally, 13 CTL dominant epitopes and 17 HTL dominant epitopes were obtained , we performed molecular docking simulations to find the interaction between the HLA alleles and the T cell epitopes. The interaction between HLA-I and CTL epitopes showed that the docking score was − 232.24 and ligand RMSD was 47.94 Å. The interaction between HLA-II and HTL epitopes showed that the docking score was − 326.30 and ligand RMSD was 143.19 Å. All the results indicated that docked complexes had a good affinity Fig. .Figure 5The MEV construct contained the dominant epitopes. There were 13 CTL epitopes, 17 Th epitopes, 9 LB epitopes, and 2 CB epitopes in the vaccine. All the dominant epitopes for the construction of MEV were shown in Fig. The MEV had 806 amino acids, the molecular weight was 86,249.70 KD and the molecular formula was C3880H5980N1082O1122S16. The instability index (II) was computed to be 20.79, so the MEV was classified as a stable protein. The GRAVY was − 0.459, so the MEV belonged to hydrophilic protein. In addition, it had an antigenicity of 0.9750 (greater than the threshold) and had no allergenicity. What’s more, the solubility of the MEV was 0.99, which means that the protein antigen was soluble. Overall, the physicochemical properties of the MEV can be designed to meet our requirements.In the result of secondary prediction, there were 20.84% alpha-helix, 8.44% β-turn, 44.17% random coil, and 26.55% extended strand. The proportion of alpha-helix, β-turn, random coil, and the extended strand was consistent with the tertiary structure. This indirectly indicated that the prediction of the tertiary structure was reasonable. Then, we showed the PDB format of the MEV in Discover Studio, where pink in the model is more likely to represent the donor and green to represent the receptor , the root mean square fluctuations (RMSF), the radius of gyration (RoG), and the potential energy analysis of the MEV-TLR4 were estimated to assess their conformational and structural stability Fig. . The strThe codon adaptation index (CAI) is shown for coding sequences designed to maximize optimal codon usage (CAI = 1 indicates that the sequence was designed to have a codon adaptation index as close as possible to 1.00). The optimized CAI value of the MEV is 0.96. The ideal percentage range of GC content of the gene was between 30 and 70%, after optimization, the average GC content was changed to 70%. The BamHI and XHOI restriction enzyme sites were inserted at the N and C ends of the optimized codons, respectively. Then, the target gene was inserted into the MCS domain. Finally, specific restriction enzyme cutting sites were considered Fig. .Figure 1According to the above principles, we designed the forward primer (5′-CTTATCTCGAGGAGGCCG-3′) with a length of 18, Tm value of 56.31, and a GC content of 60%; the reverse primer (5′-CATGGATCCGTGGTGGT-3′) with a length of 17, Tm value of 56.05 and the GC content of 58%. BLAST results showed that the MEV shares the highest homology with the Homo sapiens zona pellucida glycoprotein 2 (ZP2). Afterward, the target gene of the MEV was amplified using SnapGene. We carried out the simulated agarose electrophoresis at a concentration of 1% and selected TBE with the better buffering ability of the target gene, vector, and recombinant plasmid in buffer solution. Finally, the amount of DNA was consistent with previous predictions. The sequence of the target gene was 2418 bp, the sequence of pET-28a (+) was 5639 bp, and the sequence of recombinant plasmid was 7753 bp Fig. .Figure 1The MEV has to be tested in an animal model to see if the humoral and cellular immune response can be elicited. ELISA was used to verify whether humoral immunity could be produced. The results showed that the MEV group produced significantly higher levels of IgG1 and IgG2a Fig. A than th39. The vaccine is still preferred for brucellosis prevention, but there are no existed Brucella vaccines for humans41. Several latest types of research have been reported recently, which encouraged the reverse vaccinology top down approach to get dominant epitopes from the whole proteome of viral and bacterial44. In our study, MEV was innovatively designed and its immunogenicity was evaluated in animal models.Brucellosis is a reemerged zoonotic infectious disease with complex clinical symptoms and high mortality rates45. Therefore, based on the current research, we analyzed 13 major Brucella-associated proteins. After that, we chose Omp10, Omp25, Omp31, and BtpB with high antigenicity, stability, and hydrophilicity to build the MEV. At present, based on these four candidate proteins, there are no relevant reports for the construction of MEVs. In the current study, Omp25 of B. melitensis was shown to have significant homology with Omp31 of B. melitensis. At length, we again confirmed the high homology of these proteins when comparing the sequences, which provides a theoretical basis for future mechanisms of brucellosis pathogenesis46.Proteins with high antigenicity are possible targets for the construction of MEVs47. Therefore, we removed the signal peptide sequences of Omp10 (1–31), Omp25 (1–30), and Omp31 (1–31), respectively. However, BtpB does not possess the signal peptide, so we considered the whole amino acid sequence can be further analyzed.The signal peptide influenced the start of protein translation, and the different primary structures of the signal peptides even affected the folding and transport of the proteins48. Furthermore, the prediction of B cell epitopes of pathogenic organisms broadens the types of antigenic peptides and helps to improve the immunogenicity of antigens49. Therefore, four proteins were analyzed using IEDB and NetCTLpan1.1 server, which obtained 13 dominant CTL epitopes. Using IEDB and NetMHCIIpan4.0, 17 dominant HTL epitopes were acquired from four proteins. Using BCPREDS, 9 dominant linear B-cell epitopes were obtained from four proteins. Afterward, 2 dominant conformational B-cell epitopes from four proteins were obtained using IEDB. Ultimately, docked complexes between HLA alleles and T-cell epitopes had a good affinity.The objective of predicting HTL epitopes and CTL epitopes is to find the short peptide sequence within an antigen that stimulates CD4+ or CD8+ T cells in vivo50. Therefore, an effective MEV should be designed to include effective epitopes that can induce HTL, CTL, and B cells51. Three types of linkers that can keep proteins in are intrinsically ordered and normally folded states, including flexible linkers, rigid linkers, and cleavable (or self-cleavable) linkers53. The vaccine sequence has been constructed by combining CTL epitopes, HTL epitopes, and B-cell epitopes with the AAY, GPGPG, and KK linkers, respectively. After that, we constructed a vaccine with multiple epitopes. Adjuvants maintain the stability of the peptides and increase their immunogenicity54. There are several adjuvants used in reverse vaccinology studies, such as human β-defensin-3 (hBD3) and PADRE sequence56. HBD3 is a cationic peptide with immunomodulatory effects on both innate and acquired immune responses55. The PADRE sequence can reduce the effect of human HLA-DR polymorphism, and enhance the long-term immune response by inducing CD4+ T cells57. Finally, a complete MEV was obtained by adding adjuvants to the peptide sequence and adding the histidine sequence at the end.It has shown that single-epitope-based vaccines had weak immunogenicity in the present study, but an MEV that consists of multiple epitopes can induce the activation of both cellular and humoral immune responses and enhance its immunogenicity58. The results of this project revealed that the MEV with 806 amino acids in length and molecular weight of 86 KD was a soluble protein. In addition, it had an antigenicity of 0.9750 (greater than the threshold) and had no allergenicity. These results indicated that the MEV had good stability, hydrophilicity, antigenicity, solubility, and non-allergenicity. After that, the prediction of the secondary structure showed that the β-turn and random coil accounted for 8.44% and 44.17%, respectively. The high proportion of β-turn and random coil in the MEV suggests that the protein is likely to form antigenic epitopes59. The RoseTTAFold had ultra-high tertiary structure prediction accuracy by successively integrating and converting the three-track network information of one-dimensional sequence, two-dimensional distance, and three-dimensional coordinates. Then, we used RoseTTAFold to predict the tertiary structures of four proteins and the MEV. Finally, the ProSA-web and the structure assessment service of SWISS-MODEL were used to verify the tertiary structure quality of the MEV. The results showed that the tertiary structure of MEV has high accuracy and a high approximation coefficient with the correct structure. In general, the tertiary structure of the MEV also exhibited a high number of β-turn and random coil, which is consistent with the results predicted by the secondary structure, indicating that the MEV has efficient antigen potential.In the early stages of vaccine design, some features of the MEV should be considered. In reverse vaccinology, theoretically, the molecular weight of the protein used to design the vaccine should be less than 110 KD60. TLR4 recognizes lipopolysaccharides (LPS) of gram-negative bacteria and acts as a receptor for molecular docking with the MEV. Through docking, it can be seen that there is a strong interaction force between molecules through the atomic interaction interface between TLR4-MEV. The results suggest that the vaccine can develop stable connections with immune receptors so that it can be transported throughout the host body61.TLR4 is a transmembrane receptor protein with extracellular leucine-rich repeated domains and a cytoplasmic signaling domain, specifically recognizing endogenous molecules released from damaged or ischemic tissues termed pathogen-associated molecular patterns (PAMPs), and damage-associated molecular patterns (DAMPs)62. By carrying out the immune simulation experiment, we found that B and T cells in the body gradually increased after three doses of the vaccine and peaked at the time of the last dose of the vaccine. In addition, the MEV could also increase the levels of IFN-γ, TGF-β, IL-10 and IL-18. In particular, IFN-γ plays a major role in cell-mediated immunity by enhancing the bactericidal responses of macrophages, promoting the differentiation of Th1 cells, inducing B cell antibody class switching, enhancing cytotoxic responses of NK cells, and stimulating antigen presentation to T cells63. High levels of immune cells and cytokines indicate that the vaccine has great potential to induce a good immune response in humans. To further analyze the structural stability of the docked TLR4-MEV, a 10 ns MD simulation of the complex was performed. Taking into account the starting structure as reference for the TLR4-MEV, RMSD, RMSF, and ROG were computed to evaluate TLR4-MEV conformation changes and their stability. All the results suggested that the TLR4-MEV has strong stability.To understand and explore the immune response profile and immunogenicity, an immune simulation study was conductedE. coli expression vector is pivotal for the production of recombinant proteins64. JCat was used for the optimization of codons, to increase the expression of MEV in the E. coli. CAI of optimized vaccine protein was 0.96 and the average GC content of our sequence was 70% showing good expression possibility of vaccine protein in E. coli host. After amplification, we obtained a 7753 bp recombinant plasmid that provided a favorable theoretical basis for further research.The expression of the MEV in a suitable To evaluate the effects of the MEV, we chose the specific antibodies as reliable indicators for humoral immunity and used IFN-γ as representative indices of cellular immunity. Higher IgG1, IgG2a, and sIgA demonstrated that MEV can elicit good humoral immunity in animal models. Simultaneously, higher levels of IFN-γ was demonstrated that MEV can elicit good cellular immunity in animal model.65. Because of this, we chose B. melitensis for further study, which is often infected by humans. In the present study, an MEV including CTL epitopes, HTL epitopes, and B cell epitopes, coupled with appropriate adjuvants, was designed to enhance the immune response in B. melitensis.Structure-based reverse vaccinology is an effective approach using rational vaccine design, but the resulting immune response does not represent effectiveness against all strainsB. melitensis. Dominant epitopes were fused using linkers and adjuvant to enhance the MEV’s immunogenicity. Physiochemical properties, antigenicity, allergenicity, as well as solubility and tertiary structure analysis, of MEV were found to be very satisfactory. Moreover, the docked complexes during the simulation revealed that a strong and stable binding interaction of MEV with TLR4. In addition, this study used the C-ImmSim server to define the MEV, which is highly immunogenic. Finally, the study demonstrated that MEV can elicit both humoral and cellular immune responses in animal models.Brucella is a gram-negative coccobacillus that can cause zoonotic diseases. Reverse vaccinology presented in the present work may produce new knowledge about vaccines against


Starch-coated magnetic iron oxide nanoparticles have been synthesized by a simple, fast, and cost-effective co-precipitation method with cornstarch as a stabilizing agent. The structural and magnetic characteristics of the synthesized material have been studied by transmission electron microscopy, Mössbauer spectroscopy, and vibrating sample magnetometry. The nature of bonds between ferrihydrite nanoparticles and a starch shell has been examined by Fourier transform infrared spectroscopy. The data on the magnetic response of the prepared composite particles have been obtained by magnetic measurements. The determined magnetic characteristics make the synthesized material a good candidate for use in magnetic separation. Starch-coated magnetic iron oxide nanoparticles have been tested as an affinity sorbent for one-step purification of several recombinant proteins  bearing the maltose-binding protein as an auxiliary fragment. It has been shown that, due to the highly specific binding of this fragment to the starch shell, the target fusion protein is selectively immobilized on magnetic nanoparticles and eluted with the maltose solution. The excellent efficiency of column-free purification, high binding capacity of the sorbent (100–500 µg of a recombinant protein per milligram of starch-coated magnetic iron oxide nanoparticles), and reusability of the obtained material have been demonstrated. The number of studies on various biomedical and biotechnological applications of magnetic nanoparticles (MNPs) has been steadily increasing ,2. Due tTB). This parameter of nanoparticles is important for biomedical applications, including magnetic separation. For use in magnetic separation, the blocking temperature of nanoparticles should be similar to room temperature.To be used as affinity sorbents, MNPs should meet certain requirements, specifically, to have desired magnetic properties, be resistant against microbes and enzymes, exhibit the “zero” adsorption capacity to prevent nonspecific interactions and the stability of the functional surface, and contain an immobilized ligand for forming specific complexes with target biomolecules. Among a wide range of MNPs, iron oxide ones were proven to be promising candidates for the role of sorbents, owing to the synthesis simplicity, high magnetization, and superparamagnetism . For useK is the anisotropy constant, V is the particle volume, The blocking temperature depends, in particular, on characteristic measurement time τ . The blo2 s for the quasi-static measurements, ~2.5 × 10−8 s for Mössbauer spectroscopy, and ~10−10 for X-band ferromagnetic resonance (FMR). Therefore, a method for determining the blocking temperature should be selected according to the field of application of nanoparticles. For example, the blocking temperature of nanoparticles for use in FMR hyperthermia [The characteristic measuring time is ~10rthermia ,12 shoulrthermia ,14 shoulAccording to Equation (1), the blocking temperature depends also on the constant of the magnetic anisotropy, which involves three contributions: the magnetocrystalline, surface, and shape anisotropies. The blocking temperature of nanoparticles can be changed by changing their size and shape. The plate-shaped and cubic nanoparticles have a large anisotropy constant and, consequently, a high blocking temperature .Since naked iron oxide MNPs are prone to oxidization and aggregation, different stabilizers and coating agents are used to enhance their stability. In addition, a coating can act as a spacer for attaching certain biomolecules to a magnetic carrier. The materials used for coating MNPs include inorganic compounds  and synthetic  and natural  polymers . DiffereE. coli malE gene [In this work, starch-coated iron oxide MNPs (starch-MNPs) were synthesized and tested as an affinity sorbent for purifying recombinant proteins genetically fused with maltose-binding protein (MBP), which is a common protein expression tag. This is a 42-kDa single-chain protein encoded by the alE gene , which salE gene , and impalE gene ,22,23. MThe obtained starch-MNPs were tested as a sorbent for affine purification of several small and medium-sized MBP-fused recombinant proteins: cardiac troponin I (cTnI), a highly specific early biomarker for acute myocardial infarction  and survThe starch-MNPs were prepared by co-precipitation of iron sulfate and cornstarch (21–24% amylose) as a stabilizing agent.The Mössbauer spectrum  shows thIn studying polysaccharide-coated MNPs, Fourier-transform infrared (FTIR) spectroscopy is used, since the presence of a matrix does not prevent obtaining information about a particle and, in addition, makes it possible to establish changes in all main types of bonds between coating molecules.−1. In the spectrum presented in −1 disappear, while bonds in the region of 800–1200 cm−1 and hydroxyl ones remain unchanged.The qualitative analysis of the spectra of starch and starch-MNPs allowed us to determine the main characteristic frequencies . Figure −1 in the spectrum of starch-MNPs correspond to O–Fe–O bending vibrations and Fe–O stretching modes and the starch chemisorption occurs on the nanoparticle surface through acetal bonds [According to the data of the spectral analysis, the absorption peaks at 388 and 570 cmal bonds ,28,29.To determine the magnetic characteristics  of the synthesized nanoparticles, the hysteresis loops were measured in fields from −2 to 2 kOe at temperatures of 80–295 K. The saturation magnetization at room temperature was found to be 29.8 emu/g.5 erg/cm3 [The blocking temperature can also be estimated using Néel-Brown formula (1). Assuming the anisotropy constant to be 4 × 10 erg/cm3  and the Thus, according to the data obtained by the two methods, the average blocking temperature of the starch-MNPs is similar to room temperature and therefore they can be used in magnetic separation.The obtained starch-MNPs were tested as affine sorbents for purification of several recombinant hybrid proteins: MBP–cTnI, MBP–Surv, and MBP–MIA consisting of maltose-binding protein (MBP), cardiac troponin I , survivin , and melanoma inhibitory activity protein , respectively. As was mentioned above, MBP is widely used as an expression tag for producing recombinant proteins in bacterial cells. MBP as an auxiliary polypeptide increases the expression level of a target protein, improving the solubility and folding of the latter  and provE. coli cells and, after ultrasonic disintegration, extracted from the cytoplasmic fraction. The proteins of interest were purified using a simple procedure, which does not require complex high-tech chromatographic equipment (see the detailed description in All the hybrid proteins were synthesized in the corresponding recombinant The purification process was controlled by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) . After wIt was found that the total amount of the protein taken for chromatography and summed up from all the eluted fractions was almost the same. This points out the absence of protein loss caused by the nonspecific irreversible adsorption on starch-MNPs.The binding capacities were found to be 100.5 ± 5.1 µg for MBP-cTnI, 177 ± 14.1 µg for MBP–MIA, and 587.2 ± 52.2 µg for MBP–Surv (per milligram of starch-MNPs). These values exceed the parameters of commercial amylose magnetic beads  and those of amylose-coated particles obtained by Lim et al.  . In all the purification experiments, the nanoparticles prepared at a starch concentration of 20 g/L were used.To evaluate the synthesis reproducibility, we obtained three starch-MNP samples in parallel experiments and tested them in the MBP–cTnI purification. The high-purity MBP-cTnI yields were found to be similar, with a variation coefficient of 14.2%.The recycling property of starch-MNPs was evaluated by measuring the MBP-binding capacity after three cycles of their use. MBP-cTnI was purified for three times using the same starch-MNPs sample see . The par3.The storage of starch-MNPs in distilled water without preservatives leads to the complete loss of the MBP binding capacity in three weeks. It was found, however, that the initial binding capacity can be kept for up to six months by adding 0.05% of NaN2, and 20 g/L of cornstarch. At a temperature of 80 °C, this solution was added with a 0.1 M of the sodium hydroxide solution until neutral pH. The starch-MNPs were thoroughly washed with distilled water to remove ions and stored with the 0.05% aqueous solution of NaN3.Iron oxide nanoparticles were prepared by co-precipitation from a solution containing 20 g/L of ammonium iron (II) sulfate, 0.2–0.4 M of sodium citrate, 20 g/L of EDTA-Na−1. The samples were pressed tablets with potassium bromide with a diameter of 13 mm and a thickness of ~0.55 mm. They were carefully ground with 0.2 g of KBr in a ratio of 2:100 in a mortar. The spectra were obtained using a Globar light source (a U-shaped silicon carbide arc), an RT-DLaTGS detector, and a KBr beam splitter. The experimental data were processed and analyzed using the OriginPro software . The static magnetic measurements were performed on an automated vibrating sample magnetometer in fields of up to 15 kOe at room temperature [The electron microscopy study was carried out on a Hitachi HT7700 transmission electron microscope at an accelerating voltage of 100 kV at the Krasnoyarsk Regional Center for Collective Use of the Krasnoyarsk Scientific Center, Siberian Branch of the Russian Academy of Sciences. Mössbauer spectra were recorded on an MS-1104Em spectrometer with a 57Co(Cr) source at room temperature. Isomer chemical shifts were referenced to α-Fe. IR absorption spectra were recorded on a Bruker VERTEX-80V vacuum FTIR spectrometer in the range of 380–7500 cmperature .The sequences of genes encoding human surviving, cardiac troponin I, and MIA optimized for bacterial expression  , were clE. coli BL21-CodonPlus (DE3)-RIPL cells  transformed by the corresponding plasmid were cultivated in an LB medium containing 2 g/L of glucose and 200 μg/mL of ampicillin at 37 °C until the culture reached an OD600 of 0.5–0.7; then, 0.33 mM of IPTG was added and the cultivation was continued at 37 °C for 3 h. The cells were harvested by centrifugation, the pellet was resuspended in buffer A  in a ratio of 1:5 (w/v), disrupted by sonication (20 s × 6) at 0 °C, and the mixture was centrifuged. The pellet was discarded and the supernatant was used in the further protein purification experiments.The cell lysate supernatant  was mixed with 500 μL of the starch-MNP suspension at 4 °C for 1 h under shaking. Then, the particles were fixed at the bottom with a magnet and the solution was removed. The particles were washed with 3 portions of buffer A (1 mL) and then the hybrid proteins were eluted with 800 μL of the elution buffer .The purity of the protein samples was controlled by Laemmli electrophoresis. The protein concentration was measured using a Bio-Rad DCTM protein assay kit.Recycling of starch-MNPs was evaluated by quantifying the purified MBP-cTnI capacity for three additional loading cycles with 0.8 mL of cell lysate to 0.5 mL of the starch-MNP suspension. After the MBP-cTnI binding and elution (as described above), the starch-MNPs were regenerated by washing with the elution buffer for several times followed by re-equilibration with buffer A. Then, the cycle of regeneration, re-equilibration, binding, washing, and elution was repeated. These experiments were carried out for three times.It was shown that starch-coated magnetic iron oxide nanoparticles synthesized by a simple and cost-effective co-precipitation method with cornstarch as a stabilizing agent represent a high binding affinity material that can ensure the high-efficiency purification of a recombinant protein bearing the maltose-binding protein as an auxiliary fragment. Starch-MNPs in the form of cubic nanocrystals with an average size of ~11.5 nm were synthesized. It was demonstrated that the saturation magnetization (29.8 emu/g at room temperature) and blocking temperature similar to room temperature  make these nanoparticles good candidates for use in magnetic separation. The FTIR spectroscopy data disclosed the formation of a bond between a nanoparticle and starch.Starch-MNPs exhibit the high specificity and purification capacity for MBP-fusion proteins (100–500 µg per milligram of the particles), as well as the reusability without the binding capacity and protein purity loss. They provide the fast, convenient, specific, and cost-effective one-step purification of recombinant proteins. The use of magnetic separation in the purification process makes it possible to exclude the centrifugation and filtration stages, as well as the need for expensive chromatographic systems and columns.Thus, the obtained starch-MNPs have a high potential for use in purification of recombinant proteins. In our opinion, this material has also good prospects as a basis for the development of specific biosensors. The proposed simple and cost-effective starch-MNP synthesis method can be easily implemented at non-specialized (resource-limited) laboratories and facilities.


Aquifex aeolicus VF5)-catalyzed enzymatic polymerization. As a result of poor dispersibility of PPL in sodium acetate buffer, the enzymatically produced amylose by GP catalysis incompletely included PPL in the buffer media under the general vine-twining polymerization conditions. Alternatively, we employed an ethyl acetate–sodium acetate buffer emulsion system with dispersing PPL as the media for vine-twining polymerization. Accordingly, the GP (from thermophilic bacteria)-catalyzed enzymatic polymerization of an α-d-glucose 1-phosphate monomer from a maltoheptaose primer was performed at 50 °C for 48 h in the prepared emulsion to efficiently form the inclusion complex. The powder X-ray diffraction profile of the precipitated product suggested that the amylose-PPL inclusion complex was mostly produced in the above system. The 1H NMR spectrum of the product also supported the inclusion complex structure, where a calculation based on an integrated ratio of signals indicated an almost perfect inclusion of PPL in the amylosic cavity. The prevention of crystallization of PPL in the product was suggested by IR analysis, because it was surrounded by the amylosic chains due to the inclusion complex structure.This study investigates inclusion behavior of amylose towards, poly(β-propiolactone) (PPL), that is a hydrophobic polyester, via the vine-twining process in glucan phosphorylase (GP, isolated from thermophilic bacteria,  Amylose, which is a component of starch, is an energy storage polysaccharide in nature . Becaused-glucose 1-phosphate (G-1-P) as a monomer [n + G-1-P → [α(1→4)-G]n+1 + inorganic phosphate. We have found that amylose, thus, enzymatically produced in the GP-catalyzed polymerization, includes polymeric guests with moderate hydrophobicity in its cavity, which are dispersible in the polymerization media, via the vine-twining process  (PRHB), as the guest polyester, only its low molecular weight (approximately 500) oligomer showed ability for the formation of an inclusion complex with amylose under the following specific conditions in the GP-catalyzed enzymatic polymerization system [R)-3-hydroxybutyrate] (ORHB) was first caried out by the thermostable GP (from Aquifex aeolicus VF5) catalysis at elevated temperature (at 70 °C), higher than that for the general vine-twining polymerization (at 45–50 °C), to obtain water-soluble short α(1→4)-glucan  [l-lactic acid), poly(d-lactic acid), and poly(dl-lactic acid) , an amylosic inclusion complex is obtained only from PLLA under the general vine-twining polymerization conditions [7 primers are covalently immobilized on appropriate polymeric substrates [For example, slender polyesters with moderate hydrophobicity, e.g., poly(ε-caprolactone), poly, and poly(γ-butyrolactone) , that do not carry side substituents, can be employed as the polymeric guests to construct the corresponding amylosic helical inclusion complexes through vine-twining polymerization ,24,25. Wn system . The enzssembly) , that inssembly) ,24, cannnditions . The helnditions . PRHB alnditions . The resnditions ,32,33. Bbstrates .1H NMR, and IR analysis. The present study develops the new emulsion system with dispersing the guest polymer for vine-twining polymerization, which accelerate inclusion ability of the enzymatically produced amylose to obtain amylosic supramolecular inclusion complexes.In this study, we investigate the inclusion behavior of amylose towards poly(β-propiolactone) (PPL), which is the structural isomer of PLA in the absence of methyl substituents, as a new guest polyester in vine-twining polymerization . PPL wasAquifex aeolicus VF5), was kindly supplied from Dr. Takeshi Takaha  [Pseudomonas fluorescens was purchased from Sigma-Aldrich, Darmstadt, Germany). G7 was synthesized by selective hydrolysis of a glycosidic bond in β-cyclodextrin under acidic conditions [p-xylene. All other reagents and solvents were available commercially and used as received.GP, isolated from thermophilic bacteria  . Lipase nditions . The unr1H NMR , 4.30–4.46 , 5.83–5.85, 6.07–6.14, 6.38–6.43 ). The Mn values were calculated from integrated ratios of the main-chain signals to the terminal signals to be 1360–2230 (depending on reaction times).The guest polyester, PPL, was synthesized by enzymatic ring-opening polymerization of PL catalyzed by lipase according to the literature procedure . MixtureR CDCl3,  δ 2.63–2Mn = 2230, 0.0646 g) with ethyl acetate (2.0 mL), which was prepared by ultrasonication of the mixture, was added sodium acetate buffer  and the mixture was ultrasonicated to form an emulsion. After G-1-P , G7 , and thermostable GP (15 U) were added to the emulsion, the obtained mixture was maintained at 50 °C for 48 h with vigorously stirring. The product, precipitated, was removed by filtration, washed successively with water, acetone, and chloroform, and dried in vacuo to obtain the inclusion complex (0.0565 g). 1H NMR (DMSO-d6) δ 2.61–2.64 , 3.33–3.96 , 4.12–4.26 , 4.58, 5.41, 5.55 , 5.09 .To a suspension of PBL (λ = 0.15418 nm). Laser microscopic images were obtained by a Keyence VK-8500 laser microscope . 1H NMR spectra were recorded on a JEOL ECX 400 spectrometer . IR spectra were recorded on a PerkinElmer Spectrum Two spectrometer .Powder XRD measurements were conducted using a Rigaku Geigerflex RADIIB diffractometer  with Ni-filtered CuKα radiation (Pseudomonas fluorescens)-catalyzed enzymatic ring-opening polymerization of PL at 80 °C for 6–24 h according to the literature procedure [1H NMR spectrum to be PPL  as the guest polyester was then attempted by the procedure, the same as that, which previously used PPL, PVL, and PCL, as follows (Aquifex aeolicus VF5)-catalyzed enzymatic polymerization of G-1-P from a maltoheptaose (G7) primer (molar ratio = 280:1) was conducted at 50 °C for 48 h in the resulting aqueous mixture. The powder XRD profile of the precipitated product  at 50 °C for 48 h in the emulsion system containing the dispersed PPL (Mn = 2230) for the progress of the vine-twining inclusion of amylose towards PPL  signal to the methylene signal  of PPL (b/H1) in the 1H NMR profile of the ideal inclusion complex is calculated to be 0.60. The actual b/H1 value in the 1H NMR spectrum of the product (−1 (−1 in that of the vine-twining polymerization product (The  0.80 nm ,39, wher product a was evaduct (−1 a, that s product b. The sh1H NMR, and IR results of the precipitated product supported the structure of the amylose–PPL inclusion complex. From the integrated ratio of the signals in the 1H NMR spectrum, almost perfect inclusion of PPL by amylose was suggested. As this study has indicated that the present emulsion system strongly assists inclusion of PPL by amylose in the GP-catalyzed enzymatic polymerization, a similar system using other poorly dispersed polymers in aqueous media will be performed to obtain new amylosic supramolecular inclusion complexes in the future.In this study, we investigated the vine-twining inclusion behavior of amylose towards the hydrophobic polyester, PPL, in the thermostable GP-catalyzed enzymatic polymerization. As PPL was not sufficiently dispersed in sodium acetate buffer owing to its highly crystallinity, an ethyl acetate–sodium acetate buffer emulsion system with dispersing PPL was employed as the media for the acceleration of inclusion by the amylosic cavity in the GP-catalyzed enzymatic polymerization. The XRD,