Macaca fascicularis vaccinated with a SARS-CoV-2 RBD subunit vaccine. Further immunoinformatics analyses of these three epitopes gave rise to a method of linear B cell epitope prediction and selection. B cell epitopes in the spike (S), membrane (M), and envelope (E) proteins were subsequently predicted and confirmed using convalescent sera from COVID-19 infected patients. Immunodominant epitopes were identified in three regions of the S2 domain, one region at the S1/S2 cleavage site and one region at the C-terminus of the M protein. Epitope mapping revealed that most of the amino acid changes found in variants of concern are located within B cell epitopes in the NTD, RBD, and S1/S2 cleavage site. This work provides insights into B cell epitopes of SARS-CoV-2 as well as immunoinformatics methods for B cell epitope prediction, which will improve and enhance SARS-CoV-2 vaccine development against emergent variants.SARS-CoV-2 continues to infect an ever-expanding number of people, resulting in an increase in the number of deaths globally. With the emergence of new variants, there is a corresponding decrease in the currently available vaccine efficacy, highlighting the need for greater insights into the viral epitope profile for both vaccine design and assessment. In this study, three immunodominant linear B cell epitopes in the SARS-CoV-2 spike receptor-binding domain (RBD) were identified by immunoinformatics prediction, and confirmed by ELISA with sera from  Subsequently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was found to be the causative agent of COVID-193. It is a contagious and devastating disease that can be transmitted from human to human and was later announced as a pandemic disease by WHO2. The virus continues to spread and cause health issues worldwide. Vaccination is an effective means to prevent the infection and limit the spread of the virus.Coronavirus disease 2019 (COVID-19) is a novel disease, discovered during its initial outbreak in December 2019 in Wuhan, ChinaBetacoronavirus genus. The genus contains two other coronaviruses, SARS-CoV and MERS-CoV, which can infect humans4. It is a positive-, single-stranded RNA virus that is characterized by a spherical shape5. To date, new emerging variants of SARS-CoV-2 have been reported and four have been classified as variants of concern, which include B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta)6. These variants are circulating in numerous countries and some of which are associated with increased transmissibility9. Importantly, these variants may affect vaccine efficacy. It has been demonstrated that these emerging variants confer increased resistance to the antibodies generated through a previous natural infection and vaccination10.SARS-CoV-2 virus is a member of the 11. Among these, the S protein has been the main target for vaccine development. It is composed of 1273 amino acid residues and divided into two subunits: S1 and S212. The S1 subunit consists of the N-terminal domain (NTD), receptor-binding domain (RDB), and C-terminal domain (CTD)12. The RBD spans from amino acid residue 319 to amino acid residue 541 on the S protein and it is responsible for interacting with the host cell receptor, Angiotensin-converting enzyme 2 (ACE2), to facilitate viral entry to the cell13. The S1 subunit, particularly RBD, is thus considered the most important target for vaccine development. Indeed, neutralizing antibodies against the S protein have been characterized from individuals infected with SARS-CoV-2 16. Other main structural proteins embedded on the coronavirus membrane, M and E proteins, also play important roles in the viral assembly, budding, and replication of virus particles18. Antibodies against these two proteins have also been detected in COVID-19 patients16; however, it is not yet clear whether these antibodies confer neutralizing capacity. Therefore, they have not been the focus of vaccination strategies.The SARS-CoV-2 particle consists of multiple structural proteins; however, spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins are prevalent19. Identification of these immunogenic epitopes within a target protein can better inform and guide vaccine design. B cell epitopes can be divided into two forms: (i) a linear epitope, which is a continuous amino acid sequence fragment of an antigen, (ii) a conformational epitope, by which the residues crucial for antibody recognition are in close proximity within the folded protein 3-D structure20. Most of the B cell epitopes are identified using experimental methods, by which either overlapping peptides or short peptides are used in the immunoassays. However, this approach is labor-intensive, and is both costly and time-consuming.Generation of neutralizing antibody responses, which can neutralize the virus and thus prevent virus infection, are reliant on immunogenic B cell epitopes within the S protein21. Several investigations have reported putative B cell epitopes of the SARS-CoV-2 proteins predicted by immunoinformatics approach25. Although immunoinformatics tools can be used to explore potential epitopes, the peptides predicted by these methods cannot be claimed as epitopes and further experimental validation is still required to confirm whether they are genuine epitopes. Nevertheless, employing immunoinformatics for epitope prediction and screening could reduce the number of peptides used in experimental assays, thus saving both time and cost.Immunoinformatics is an alternative and powerful tool for the identification of potential B and T cell epitopes. Currently, many tools and methods are available for the prediction of linear and conformational B cell epitopes. Among the recommended tools and resources for epitope prediction, the IEDB database provides multiple tools for the prediction of linear B cell epitopes and other associated parameters of B cell epitopes(M. fascicularis) vaccinated with a plant-produced SARS-CoV-2 RBD subunit vaccine. Competitive inhibition of RBD-ACE2 binding was performed to investigate neutralizing potentials of antibodies targeting the RBD immunodominant epitopes. Next, other characteristics of the RBD immunodominant epitopes were analyzed using multiple immunoinformatics tools. These results were then used to develop a novel immunoinformatics method for B cell epitope prediction and selection. Potential B cell epitopes in the proteins S, M, and E were predicted. The predicted B cell epitopes were verified experimentally using convalescent sera from COVID-19 patients. Finally, we then applied this workflow to identify epitopes in the S proteins of SARS-CoV-2 variants of concern . Here, we demonstrate that immunoinformatics is a potent tool for a rapid and precise identification of B cell epitopes from the SARS-CoV-2 proteins, suggesting its future utility with other pathogens.In this work, we aimed to identify linear B cell epitopes in the 3 main structural proteins of SARS-CoV-2 by coupling the immunoinformatics method for epitope prediction with immunoassays. B cell epitopes in the RBD region predicted by the BepiPred-2.0 tool were tested with the sera from cynomolgus macaques Cynomolgus macaques vaccinated with plant-produced RBD subunit vaccine and 5 sera from monkeys in the control group 26.To identify linear B cell epitopes in the RBD of SARS-CoV-2 S protein, we employed an immunoinformatics tool. Using the BepiPred-2.0 server revealed 8 potential linear B cell epitopes within the RBD Table . Two paip < 0.01). To further define a positive antibody response for an individual peptide, the OD450 obtained from a single vaccinated animal’s sera was compared to that of (i) OD450 mean of control group + SD, and (ii) OD450 mean of control group + 3SD , 5 and 2 sera from vaccinated monkeys showed reactivity with peptide CoV2_S-10 based on the analyses with criteria (i) and (ii), respectively.Sera from vaccinated macaques showed reactivity with the complete RBD protein and a distinct group of peptides Fig. A. StatisTogether, these results showed that two epitopes, CoV2_S-11 and CoV2_S-11.2, were recognized as the most immunodominant epitopes in the RBD subunit, followed by CoV2_S-13 and CoV2_S-10, respectively. It is noteworthy that epitope CoV2_S-11.2 is part of epitope CoV2_S-11, suggesting that epitope CoV2_S-11.2 is the core epitope of epitope CoV2_S-11. However, most of the monkey sera exhibited a slightly higher OD450 with peptide CoV2_S-11 than with peptide CoV2_S-11.2. Additionally, we observed that the patterns of antibody response against immunodominant epitopes in different macaques were different . The majority of the CoV2_S-11 and CoV2_S-13 epitopes’ residues are exposed on the surface of the S protein. For epitope CoV2_S-10, only a few residues are exposed on the surface  RBD alone, (ii) RBD mixed with monkey serum diluted 1:500), and (iii) RBD mixed with monkey serum and peptide . To search for common characteristics among the three immunodominant epitopes (four peptides tested), the sequences predicted by each tool were aligned to the sequence of epitopes predicted by BepiPred-2.0 . For method B, linear B cell epitope is identified based on the peptides with antigenic and hydrophilic determinants predicted by the methods of Kolaskar & Tongaonkar and Parker, respectively Fig. . For metAll three immunoinformatics methods were employed to predict B cells epitopes in the SARS-CoV-2 structural proteins S, M and E. Predicted linear B cell epitopes are shown in Fig. p < 0.05) . Statistical analysis revealed that antibody responses against epitopes CoV2_S-15, CoV2_S-17, CoV2_S-20, CoV2_S-21, CoV2_S-21.1, CoV2_S-21.2 and CoV2_M-2 in convalescent sera are significantly higher than those in healthy control sera (05) Fig. A,B. Thusp = 0.08). On the other hand, epitope CoV2_S-11.2 showed positive reactivity with 6 convalescent sera.We went on to determine the number of convalescent sera patients with antibody responses against each individual epitope, as defined by OD450 of convalescent sera being greater than OD450 of control group mean. The number of epitope-responding sera of each epitope is summarized in Table Locations of the epitopes in the S protein are shown in Fig. Amino acid substitutions and deletions on the S protein of four variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) were localized and mapped to the B cell epitopes on the 3-D structure of wild type (Wuhan) SARS-CoV-2 S protein Fig. . The occLastly, we employed the immunoinformatics methods we developed to explore the B cell epitope profile in the four variants of concern  that plays an important role in the primary interaction with the host cell receptor ACE2, is a more attractive alternative for vaccine development. Recombinant RBD subunit vaccines have been shown to stimulate immune responses in mice, rabbits and non-human primates and also protected 34. These results suggest that natural infection and vaccination could result in different patterns and levels of antibody responses. While natural infection can give rise to antibody responses against the whole viral proteome, RBD-based vaccines contain only a few epitopes and has no competitive interference from epitopes in other proteins. Thus, utilization of the RBD as an immunogen represent a better strategy to enhance antibody response against those subdominant epitopes in the RBD.Serological assays with sera from cynomolgus macaques vaccinated with plant-produced RBD subunit revealed three immunodominant linear B cell epitopes located in the RBD of the SARS-CoV-2 S protein. Interestingly, in COVID-19 patients, these three epitopes are recognized only as subdominant ones, while other peptides in the S1 CTD and S2 have been demonstrated as immunodominant36. Decreased RBD-ACE2 interaction in the presence of peptide CoV2_S-13 may be due to the replacement of antibodies targeting this epitope with antibodies targeting other epitopes such as CoV2_S-10 and CoV2_S-11, which can inhibit RBD-ACE2 binding. As a result, binding of the RBD to ACE2 was further inhibited (this assumes that only one antibody can bind to the RBD protein at a time). However, antibodies targeting the CoV2_S-13 epitope may be a hindrance at other processes of SARS-CoV-2 infection such as conformational changes and membrane fusion37, which occur after the binding of RBD to ACE2 receptor. As sera from these vaccinated monkeys were demonstrated to neutralize SARS-CoV-226, it is possible that neutralizing activity in these sera mainly results from antibodies against the three immunodominant epitopes we identified in this study. However, there may be other neutralizing epitopes in the RBD, particularly in the form of conformational epitopes.To address neutralizing potential of the antibodies targeting these three RBD immunodominant epitopes, we performed competitive inhibition of RBD-ACE2 binding. The results showed that antibodies targeting epitopes CoV2_S-10 and CoV2_S-11 were capable of inhibiting RBD-ACE2 interaction, thus suggesting their neutralizing potency. Interestingly, the opposite effect on an interaction between RBD and ACE2 was observed for CoV2_S-13 epitope. This is not surprising because residues in the CoV2_S-13 epitope are not directly responsible for interacting with the host cell receptor ACE238. Moreover, monoclonal antibody B3814, which can neutralize SARS-CoV-2, showed interaction with multiple residues in the RBD including the residues in the CoV2_S-11 epitope. Moreover, murine antibodies induced by peptides 406–420 (EVRQIAPGQTGKIAD), 439–454 (NNLDSKVGGNYNYLYR) and 455–469 (LFRKSNLKPFERDIS), which correspond to our epitopes CoV2_S-10, CoV2_S-11.1 and CoV2_S-11.2, respectively, inhibited SARS-CoV-2 pseudovirus infection31.Although competitive inhibition of RBD-ACE2 binding can hint towards neutralizing potency of antibodies recognizing RBD epitopes, it is still necessary to perform a SARS-CoV-2 neutralization-inhibition assay to confirm protective immune response. However, neutralizing activity of antibodies against some epitopes in the RBD have been reported and confirmed. It has been shown that the peptide 404-GDEVRQIAPGQTGKIADYNYKL-425, which overlaps entirely with our epitope CoV2_S-10, could elicit neutralizing antibody responses in mice34. Here, we reported six immunodominant epitopes  located in four regions of the S protein and one immunodominant epitope located at the C-terminus of the M protein. Importantly, these immunodominant epitopes are in the functionally important regions of the S1 CTD and S2 domain. Epitope CoV2_S-15 (673-SYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSN-709) is located at the S1/S2 cleavage site, where the sequence RRAR can be recognized and cleaved by furin protease, resulting in separation of S1 and S2 domains during virus assembly40. In addition, this epitope region also contains cleavage site 2 IAYTMSL, which is recognized and cleaved by Cathepsin L in the endosome and this process is also essential in S protein priming during cell entry41. Thus, antibodies targeting epitope CoV2_S-15 could possibly block the cleavage processing of the S protein during cell entry of the virus. Epitope CoV2_S-17 is found in the S2’ cleavage site (residues 809–815), the target for cleavage by proteases including TMPRSS2, a serine protease, for S protein prime43. Consequently, antibodies binding to this region may result in inhibition of the S2’ cleavage, a crucial process during viral-cell entry. The epitope 809-PSKPSKRSFIEDLLFNKV-826, which overlaps with epitope CoV2_S-17, has been demonstrated as a neutralizing epitope33. Epitope CoV2_S-20 is located at the upstream region of the HR2, a domain involving in membrane fusion of SARS-CoV-2 during virus entry45. Antibodies against this epitope may lead to inhibition of the membrane fusion and virus infection. Epitopes that overlap with CoV2_S-20 have been previously characterized as immunodominant as well as neutralizing epitopes34. Another immunodominant epitope CoV2_S-21, which is located at the cytoplasmic domain of the S protein, has also been identified as one of the immunodominant epitopes in other studies34. Although this epitope has not been clarified to elicit neutralizing antibodies, research into an alphacoronavirus suggests its potential. B cell epitopes located at the cytoplasmic domain of the S protein of the porcine epidemic diarrhea virus (PEDV), a member of alphacoronaviruses, is shown to be a target for recognition by the neutralizing antibody 2C1046. It remains possible that the epitope CoV2_S-21, which is in the same location as epitope 2C10, can elicit antibodies with neutralizing activity. For the immunodominant epitope in the M protein, further studies are needed to address whether the antibodies elicited by this epitope confer neutralizing activity. Based on these findings, the immunodominant epitopes CoV2_S-15, -17, -20 and -21 may represent valuable candidates for both vaccine development and assessment of the antibody response in COVID-19 patients and vaccine recipients.Testing the synthetic peptides corresponding to the predicted epitopes in the S and M proteins with convalescent sera allowed us to determine their immunodominant properties. In agreement with other studies, our results showed that linear B cell epitopes in the RBD are less dominant compared to those within the S2 domain19. Amino acid substitutions P681H in B.1.1.7 and P681R in B.1.617.2 are located in the CoV2_S-15.1 epitope that contains the sequence RRAR for S1/S2 furin cleavage, while A701V in the B.1.351 variant is located in the CoV2_S-15.2 epitope, which contains Cathepsin L cleavage site41. These findings may help explain why these new variants are more resistant to antibodies induced by either natural infection or by vaccines based on wild type SARS-CoV-2 as reported in several studies10.Here, we demonstrated that our immunoinformatics method is a valuable tool for predicting linear B cell epitopes both in wild type virus and new emerging strains. Mapping amino acid changes in the S protein of variants of concern to our B cell epitopes revealed that sequences within the S1 NTD, RBD and S1/S2 cleavage site are the main targets for amino acid substitutions and deletions. Some of these B cell epitopes with amino acid changes have been well characterized as neutralizing epitopes. For instance, epitopes CoV2_S-10 and -12 are located in the RBD. Additionally, the CoV2_S-3 epitope is a part of the epitope recognized by monoclonal neutralizing antibody 4A8Taken together, we identified three linear B cell epitopes in the RBD of the SARS-CoV-2 S protein by coupling the immunoinformatics approach with the immunoassay testing sera from macaques vaccinated with plant-produced RBD. This combined method enabled the identification of six immunodominant epitopes in the S1/S2 cleavage site and S2 domain recognized by antibodies in the COVID-19 convalescent sera. The immunoinformatics method described here could be a useful tool for identification of antibody epitopes in new virus variants and also other target proteins. Linear B cell epitopes discovered in this study may find future applications both in designing a new candidate SARS-CoV-2 vaccine focusing on antibody induction and in development of peptide-based immunoassays.Serum samples used in this study had been previously used in the project “measurement of neutralizing antibody in convalescent plasma donors for COVID-19 treatment”, which was approved by the Siriraj Institutional Review Board under COA number Si 483/2020. Informed consent was waived by the Institutional Review Board. All methods were carried out in accordance with international guidelines for human research protection, the declaration of Helsinki. Twenty serum samples collected from recovered Thai COVID-19 patients with positive neutralizing antibody test were chosen and used in this study while 11 control serum samples were prepared from healthy individuals with negative test for COVID-19. All sera were heat-inactivated at 56 °C for 30 min before used.26. Monkey sera used in this study were provided by Baiya Phytopharm Co., Ltd, under the project “Immunogenicity study of low dose of plant-produced recombinant SARS-CoV-2 RBD subunit vaccine in cynomolgus macaques (Macaca fascicularis)”. Serum samples were collected from 8 monkeys vaccinated with plant-produced RBD subunit vaccine adjuvanted with Alum and 5 monkeys in the control group receiving PBS adjuvanted with Alum26. The study was carried out in compliance with ‘The Animals in research: reporting in vivo experiments (ARRIVE)’ Guidelines. All procedures in non-human primate study were conducted in accordance with relevant guidelines and regulations, which were reviewed and approved by the NPRCT , Chulalongkorn University (NPRCT-CU) Animal Care and Use Committee (Protocol review No. 207512). The NPRCT facility has been AAALAC International Accredited (1752).Recombinant SARS-CoV-2 RBD subunit produced from plant is a SARS-CoV-2 vaccine candidate developed by Baiya Phytopharm Co., Ltd. Preclinical evaluation of this vaccine was conducted in mice and cynomolgus macaques6 and preliminary finding reported in the virology.org47.The amino acid sequence of the S protein (accession number YP_009724390), M protein (accession number YP_009724393) and E protein (YP_009724392) of SARS-CoV-2 were retrieved from National Center for Biotechnology Information (NCBI). The sequences and mutations of the S protein of the variants of concern, B.1.1.7, B.1.351, P.1 and B.1.617.2 were obtained from Center for Disease Control and Prevention (CDC)http://www.cbs.dtu.dk/services/BepiPred/). BepiPred-2.0 is an immunoinformatics tool used for predicting continuous/linear B cell/antibody epitope from protein sequences48. In this work, we used the cutoff of 0.5 and the epitope was defined based on the predicted sequence that contains at least 6 contiguous residues. It is noteworthy that prediction using BepiPred-2.0 tool through its original server (http://www.cbs.dtu.dk/services/BepiPred/) and the server provided by IEDB (http://tools.iedb.org/bcell/) could give a slight difference in the prediction result.B cell epitopes were predicted using BepiPred-2.0, (https://iupred.elte.hu/) predicts intrinsically unstructured proteins, which infers coil structure49. In the prediction with the IUPred tool, the residues of the S protein predicted with disorder prediction score higher than 0.2 were considered unstructured. Surface accessibility was predicted with two tools, BepiPred-2.0 providing the probability of exposed residues and the method of Emini providing probability for being found on the surface50. Antigenicity propensity was predicted by the method of Kolaskar & Tongaonkar, a semi-empirical method51. Hydrophilicity was predicted by the method of Parker52. Notably, the method of Emini, Kolaskar & Tongaonkar, and Parker are provided by IEDB (http://tools.iedb.org/bcell/) and the default cutoff thresholds provided by the programs were used to select the sequences. In all prediction methods, only the predicted peptides with at least 6 contiguous residues were selected and used for further analysis. All peptides obtained from predictions with different tools were aligned using AliView program53 and a method for selection of potential B cell epitopes were then created.Other characteristics including coil structure, surface exposure/accessibility, antigenicity and hydrophilicity were predicted with multiple immunoinformatics tools. Coil structure was predicted with two tools, BepiPred-2.0 and IUPred. BepiPred 2.0 provides coil probability score of each amino acid residue, while IUPred . Synthetic peptides were dissolved in sterile distilled water containing 0.1% acetic acid to the concentration of 3 nmol/μL (3 mM) and stored at -20 °C until used. The RBD subunit used in the ELISA assay was produced in the mammalian HEK 293 cell growing in DMEM supplemented with 10% fetal bovine serum (HyClone) and 1% penicillin/streptomycin antibiotics (Gibco). The cells were transfected with the plasmid pVAX1 harboring a gene encoding SARS-CoV-2 RBD subunit. As the protein was designed to be extracellularly secreted, the culture medium was harvested. The presence of the RBD subunit in the medium was confirmed by Western blot. Protein concentration in the harvested medium was measured using Bradford assay (Bio-Rad).2SO4). Optical Density at the wavelength of 450 nm (OD450) was measured .ELISA microplates (Greiner bio-one) were coated with 1 nmole of synthetic peptides or 20 μg of the RBD protein (in medium) diluted in 50 μL PBS. Medium without RBD protein was used as a negative control. Following an overnight incubation at 4 °C, plates were washed 3 times with PBS containing 0.05% Tween 20 (PBST) and blocked with 100 μL PBST containing 5% FBS (5% FBS/PBST) for 1 h at room temperature (RT). Sera were diluted 1:100 in PBST containing 1% FBS (1% FBS/PBST) and added to the plates (100 μL/well) (in duplicate for monkey sera and one well for human sera) and incubated at RT for 2 h. After a 3-time wash, goat anti-monkey IgG HRP antibody (Abcam) diluted 1:10,000 in 1% FBS/PBST or rabbit anti-human IgG HRP antibody (Abcam) diluted 1:80,000 was added to the well (100 μL/well) and the plates were incubated at RT for 90 min. After washing, TMB substrate (BioLegend) was added (70 μL/well) to develop color. Following a 30-min incubation, the reaction was stopped by adding 30 μL 2 N sulfuric acid (HELISA microplates (Greiner bio-one) were coated with 4 μg/mL (50 μL/well) of recombinant human ACE2 . Following an overnight incubation at 4 °C, plate was washed 3 times with PBST and blocked with 100 μL 5% FBS/PBST for 1 h at RT. After washing, RBD protein (2.5 μg crude protein in DMEM) prepared in a final volume of 100 μL in 3 different conditions (i) RBD alone, (ii) RBD mixed with monkey serum (diluted 1:500), and (iii) RBD mixed with monkey serum and peptide, was added into each well and incubated for 1 h. The experiment was conducted in triplicate. After a 3-time wash, mouse anti-V5 antibody (Invitrogen) diluted 1:2,000 in 1% FBS/PBS was added and the plate was incubated for 1 h at RT. After washing, goat-anti mouse IgG-HRP (Abcam) diluted 1:100,000 was added to the well (100 μL/well) and incubated for one hour. The following steps followed ELISA method as described above. Percent change was calculated based on the formula: 100 x (OD of RBD with serum and peptide - OD of RBD with serum)/(OD of RBD – OD of RBD with serum).27) obtained from https://www.rcsb.org/. Epitope labeling was performed using PyMOL 2.3.4 program. Additionally, locations of the epitopes were depicted on the monomeric SARS CoV-2 S protein in both closed (PDB 6ZB527) and open (PDB 6VYB54) conformations.Surface presentation of the B cell epitopes on the SARS CoV-2 S protein was depicted on the 3-D structure of trimeric SARS CoV-2 S protein in the closed conformation . Parametric students’ t-test was used to analyze the results of ELISA testing monkey sera, while non-parametric Mann–Whitney test was used to analyze ELISA result of human sera. Supplementary Information.


Since the closure of university campuses due to COVID-19 in spring 2020 necessitated a quick transition to online courses, medical students were isolated from hospitals and universities, negatively impacting their education. During this time, medical students had no opportunity to participate in academic discussions and were also socially isolated. Furthermore, medical doctors and professors of medical schools were given additional responsibilities during the pandemic because they were the frontliners in the fight against COVID-19. As a result, they did not have enough time to contribute effectively to medical student education.This paper describes the establishment of the Cerrahpasa Neuroscience Society Journal Clubs, a group of entirely student-run online journal clubs at Cerrahpasa Faculty of Medicine, Istanbul University-Cerrahpasa.The website, mass emailing, and social media accounts were used to announce the online journal clubs. Only medical students were eligible to apply. Journal clubs included psychiatry, neuroradiology, neurosurgery, neurology, and neuroscience. Following the last journal club meeting, a questionnaire created by the society’s board was distributed to the participants. SPSS Statistics (version 26) was used for statistical analysis.Since March 15, 2021, synchronous online journal club meetings have been held every 2 weeks on a weekday using Google Meet, Microsoft Teams, or Zoom. Meetings of each journal club lasted approximately 1 hour on average. Interstudent interaction across multiple institutions was achieved since a total of 45 students from 11 different universities attended the meetings on a regular basis. Students on the society’s board served as academic mentors for the clubs. The clubs received excellent feedback from participants, with an overall contentment score of 4.32 out of 5.By establishing these clubs, we have created a venue for academic discussions, which helps to reduce the negative impact of the pandemic on education. In addition, we believe it greatly aided students in staying in touch with their peers, thereby reducing the sense of isolation. We realize that traditional journal clubs are run by faculty; however, we believe that this experience demonstrated that medical students could run a journal club on their own since the feedback from participants was excellent. Additionally, as a medical student, being a journal club academic mentor is a challenging responsibility; however, having this responsibility significantly improved our academic mentors’ leadership abilities. SARS-CoV-2, which is responsible for COVID-19, was declared a pandemic by the World Health Organization on March 11, 2020, posing new challenges for all medical students and faculty, as well as requiring remote learning by universities worldwide [Since their introduction by Sir William Osler in 1875, journal clubs have a long history in the medical sciences . TraditiMost journal clubs were still held in person prior to COVID-19. One significant problem of in-person journal clubs is that participants find it difficult to attend meetings on a regular basis, perhaps due to logistic challenges. This problem can be solved by using online platforms that allow participants to join the meeting from any location, thereby providing a flexible and feasible platform for evidence-based learning . These mAccording to Keet et al , journalThis study was conducted in compliance with the principles of the Declaration of Helsinki. Ethical approval was waived because the Cerrahpasa Neuroscience Society’s journal clubs are run independently of the university. Informed consent was obtained from all participants.Cerrahpasa Neuroscience Society is a student-led organization that was founded in 2018 at Cerrahpasa Faculty of Medicine, Istanbul University-Cerrahpasa (IUC). In our society, we established five distinct online journal clubs with regard to the subfields of neuroscience: psychiatry, neuroradiology, neurosurgery, neurology, and neuroscience journal clubs. On February 19, 2021, all of the online journal clubs were announced to all Cerrahpasa Neuroscience Society email newsletter subscribers, as well as via postings on the Cerrahpasa Neuroscience Society’s Facebook, Twitter, and Instagram accounts. Between February 22 and 28, 2021, applications were accepted online at the society’s website for each journal club. Only pregraduate medical students were eligible to apply. Although the presentation language was announced to be Turkish, all of the reviewed papers were published in English. Therefore, applicants were reminded that English proficiency would be necessary for efficacious meetings. The number of participants in each journal club was intended to be between 9 and 12. The absolute number of participants was decided after the applications were submitted. Students on the society’s board served as academic mentors for the clubs. The board members of the society include Atacan Zeybek, Batuhan Davuş, Elif Kaymaz, Ferit Ulaş Özkan, Kardelen İnan, Naz Bilaloğlu, Öykü Melek Tepe, Zeynep Sude Furkan, Zeynep Özcan, Burak Berksu Özkara, Mert Karabacak, and Duygu Demet Alpaydın. The board members were selected via voting by the members of the Cerrahpasa Neuroscience Society based on their performance and contribution to the club in the 2019-2020 education year.Careful planning and the establishment of clear, defined goals are essential for a successful journal club . TherefoInitially, each academic mentor selected articles for their participants to present based on their interests in a particular topic relevant to the journal club they run. All selected articles were in English, published in a well-respected journal, and possibly appealing to the students. However, after providing educational material on how to perform a literature review, a few academic mentors chose to provide presenters with autonomy by allowing them to choose what to present, which is an idea to motivate participants based on self-determination theory (SDT). SDT explains motivational processes and inspired us to foster this element of learning . FurtherTwo authors (MK and DA) created a questionnaire that was controlled and evaluated by the first author (BO) on July 3, 2021. The questionnaire was sent via email to participants who attended more than 80% of the meetings, with a response deadline of July 9, 2021. To receive their certificate of participation, participants were required to complete the questionnaire, which resulted in a 100% response rate. The questionnaire was divided into three sections. The first section asked participants about their universities, current grades, journal clubs they attended, and the platform they used during meetings. The second section included 22 items that questioned participants’ level of contentment with our journal club. The third section included 8 items pertaining to the preferences of the participants . Questiot tests were used to investigate group differences. Pearson correlations were used to investigate bivariate relationships between items of the questionnaire. P values less than .05 were regarded as statistically significant.SPSS Statistics (version 26) was used for statistical analysis. Because the first section of the questionnaire was about the demographics and the third section considered the preferences of the attendees, all statistics were calculated based on the second section of the questionnaire. The internal consistency of the questionnaire’s second section was assessed using Cronbach α. Independent-sample A total of 13 applicants were accepted for neurology, 8 for neuroradiology, 8 for neuroscience, 9 for neurosurgery, and 12 for psychiatry, and they attended the meetings on a regular basis. Five participants, out of a total of 45, participated in two clubs. For each journal club, a member of our society’s general assembly was assigned as the academic mentor.Synchronous online journal club meetings were held every 2 weeks on a weekday since March 15, 2021, using Google Meet, Microsoft Teams, or Zoom. Each journal club’s meetings were approximately 1 hour long on average. First, an educational meeting was held to demonstrate how to present an article to participants. Following the first educational meeting, three articles were presented by three participants, who were selected 2 weeks earlier by academic mentors, at each of the subsequent meetings. In addition to presenters, each participant was expected to read the selected three articles in 2 weeks. A 10-minute presentation aided by slideshows was followed by a 10-minute discussion for each article. Academic mentors did not present any articles, but instead guided the discussions following each presentation. In some journal clubs, after the 1-hour meeting, participants and academic mentors would stay on the online platform to chat about their personal lives, which we believe contributed significantly to the motivation of participants and success of our journal club. The last meeting took place on June 29, 2021.Cronbach α was used to evaluate the internal consistency of the second section of the questionnaire in terms of reliability, which reached .84 .The journal club general contentment score was calculated by averaging all answers given by each participant to the second section of the questionnaire . The meaP=.86) between IUC students (mean 4.32) and participants from other universities (mean 4.31).Among the 45 participants, 27 (60%) were IUC students, whereas 18 (40%) were students from other universities, including Hacettepe University, Balikesir University, Hitit University, Kutahya Health Sciences University, Altinbas University, Istanbul University, Ankara University, Ataturk University, Gazi University, and Kahramanmaras Sutçu Imam University. In terms of the journal club general contentment score, there was no difference  yielded a low Pearson correlation, indicating that these three items were scored significantly lower than other items by participants.The first item with a low Pearson correlation was “Meetings were not productive because of technical issues ” with a mean score of 4 among the items with low Pearson correlation (reversed). This finding indicates that most participants were satisfied with their online connection; however, some participants encountered technical difficulties. The second item, with a mean score of 4.02 (reversed), was “It was hard to get used to meetings being online,” indicating that a few participants struggled to adapt to the online nature of the club. The last item was “I would be more anxious if the meetings were in person” with a mean score of 3.07. This finding implies that participants are unsure whether they would be more nervous in a face-to-face meeting as opposed to an online meeting.In Turkey, medical education consists of 3 years of basic medical sciences courses , 2 years of clerkships, and 1 year of internship . Participants included 38 preclinical medical students with 6 in their first year, 12 in their second year, and 20 in their third year. By contrast, there were only 7 clinical students, including 6 in their fourth year and 1 in their fifth year.t test was used to compare the responses of these two groups, including responses to the preference questions. Significant differences between the answers of these two groups were found for the scores on only two items: preference 5 (P5) and preference 6 (P6). The first item that significantly differed between preclinical (mean 2) and clinical (mean 2.86) medical students (P=.01) was “I would prefer meetings with less people.” Despite the fact that neither preclinical nor clinical students prefer meetings in smaller groups, this finding suggests that clinical medical students are more likely to prefer small-group journal clubs. The second significantly different item between preclinical and clinical medical students was “Journal clubs are more beneficial for clinical students rather than preclinical students” . This finding suggests that clinical medical students think that participation in a journal club would be better after completing preclinical studies. However, we are very pleased with the positive attitude of preclinical participants, which indicates that they think the journal club has greatly benefited them even though they are still in the early stages of their medical education. As a result, we believe that preclinical medical students should be encouraged to participate in journal clubs.The independent-sample t test was also used to compare the answers of IUC students and non-IUC students. There was no statistically significant difference in responses between IUC and non-IUC students .The independent-samples t test revealed that the overall experience for IUC and non-IUC students was the same, indicating that institutions should be more welcoming of students from other institutions. Overall contentment scores suggested that peer-to-peer journal clubs could be satisfactory to participants and should be encouraged when the senior doctors or professors are unavailable to supervise.This innovation widened students’ exposure to journal clubs, as many participants did not have this available at their university. Participation in our journal clubs is a valuable experience for our students, since involvement in a journal club was shown to enhance academic reading habits . ParticiThe COVID-19 pandemic has disrupted education, which, when combined with physical distance, has resulted in challenges in disseminating valid education for medical students, as well as social isolation. We present a student-led online synchronous journal club format that promotes education of medical students while also fostering peer interaction. During a time of societal stress, our journal club is unique in that it is entirely student-run, with participants consisting entirely of medical students, and it brings together a large number of medical students from all over Turkey, creating a venue for medical students, who are one of the most affected groups by self-quarantine .Our survey provided valuable information on the essence of an online synchronous journal club for medical students. To obtain these valuable data, filling out the survey was required to obtain an online journal club certificate, which resulted in a 100% response rate. Some authors in the literature mentioned low response rates, which we believe could be resolved by making survey responses mandatory to receive a certificate ,5,12.Interstudent interaction across multiple institutions was generally accomplished through face-to-face conferences and courses, which were all completely cancelled during the pandemic. Our journal club had 45 participants from 11 universities, demonstrating that online events can partially compensate for the lack of in-person events for interinstitutional peer interaction. We believe that interinstitutional peer interaction is important not only for medical education but also for motivation and psychological well-being during this time of crisis. Furthermore, because we were aware that an online journal club was not available at other Turkey-based universities, we accepted non-IUC students to support these students, who did not have the same opportunity as IUC students. The mean score for the item “I would like to see journal clubs in other medical schools” among non-IUC participants was 4.56, indicating that such organizations are needed in other universities that participants attend. The multi-institutional nature of our journal clubs demonstrates that our online journal clubs were truly nationwide, with students from all over Turkey participating.We believe that the small number of participants significantly contributed to the success of our journal club, because it is more difficult for participants to focus in large groups and for us to see how many participants are actively listening . FurtherSince our journal club was entirely run by students, academic mentors in each journal club were members of the society’s board. Even though running a journal club was a huge responsibility for a pregraduate medical student, we believe that our mentors did a great job since overall participant contentment was very high. Another finding that supports our academic mentors’ success is that the item “I would prefer meetings be chaired by faculty members” received a mean score of 2.84. This finding suggests that our participants benefited sufficiently from their journal club experience while being supervised by student academic mentors. Furthermore, we believe that having this responsibility as a pregraduate medical student markedly improves leadership abilities. In addition, to handle this level of responsibility, our academic mentors had to thoroughly read the literature and internalize the particular topic, which significantly contributed to their medical and academic knowledge.Learning necessitates an understanding of the subject matter, a willingness to put forth effort in studying, and the ability to control one’s education . AccordiOur study is not without limitations. First, the number of participants is insufficient to draw general conclusions. Perhaps beginning advertising earlier and more broadly could be beneficial in increasing the number of participants. However, as aforementioned, we do not intend to increase the number of participants in a particular journal club since participants were comfortable with the existing number. Instead, to accommodate more participants, the number of journal clubs should be increased. Second, since we do not have participant data to compare before and after implementation of the journal club, the survey only questioned participants’ perspectives rather than providing objective results. To overcome this obstacle in the future, participants should be evaluated in terms of academic knowledge such as literature searching and database usage, as well as the presentation of an academic paper before the first meeting and after the last meeting.As a society, we intend to continue our journal club for years to come. Even though medical education in Turkey is scheduled to be held face-to-face for the upcoming academic year, we intend to organize our journal club virtually for the upcoming academic year to minimize the COVID-19 risk and maintain the multi-institutional nature of our journal club. For this reason, to assess participants’ attitudes toward “online” journal clubs for the next year, one of the Likert-scale items was “I would prefer online journal club meetings when the medical education becomes in person again,” which resulted in a mean score of 3.96, indicating that the majority of our participants were pleased with the online format.Given the short time between the announcement and the application deadline, there were more applicants than we expected. We intend to begin advertising for the upcoming academic year much earlier and increase the number of journal clubs, allowing us to contribute to the education of many more medical students whose institutions do not offer journal clubs.Asynchronous Twitter journal clubs, in which participants contribute via tweets over a set period, have existed for some time, creating a diverse global forum for discussion ,18. We pIn a time of crisis that isolated medical students from hospitals and universities, our journal club facilitated continued interaction between medical students by providing a platform for academic discussions. During the pandemic, students from all over Turkey regularly attended our club, which reduced social isolation and increased cross-institutional interaction. Our survey revealed that our participants were pleased with our journal club, which makes us very proud given that the club is entirely student-run. This experience, we believe, demonstrated that medical students can run a journal club on their own, and we hope that this paper serves as a guide for other organizations as they plan their journal clubs.


Bacillussubtilis interact with the resistant starch fibers of chickpea through preferentially inducing biofilm formation pathway. The successfully colonized starch fibers enable enhanced survivability of the cells during different environmental stresses.Cells of probiotic bacterium  Bacillus subtilis cells with the resistant starch fibresof chickpea milk (CPM), herein CPM fibres, along with the production of a reddish‐pink pigment. Genetic analysis identified the pigment as pulcherrimin, and also revealed the involvement of Spo0A/SinI pathway in modulating the observed phenotypes. Besides, through successful colonization of the CPM fibres, the wild‐type cells of B. subtilis displayed enhanced survivability and resilience to environmental stress, such as heat and in vitro gastrointestinal treatments. In total, we infer that the biofilm formation on CPM fibres is an adaptation response of B. subtilis for strategic survival.Beneficial biofilms may confer effective adaptation to food matrices that assist bacteria in enduring hostile environmental conditions. The matrices, for instance, dietary fibres of various food products, might serve as a natural scaffold for bacterial cells to adhere and grow as biofilms. Here, we report on a unique interaction of  Chickpea seeds are rich in healthy nutrients, minerals, proteins, carbohydrates and dietary fibres that are known to favour healthy gut microbiota  and to understand the survival tactics espoused by the bacteria. Through investigating the mechanism of interaction of B. subtilis cells with the CPM starch fibres, we discovered intriguing morphological changes enabling the extraordinary adaptability of the cells to growth within CPM.A variety of plant‐based milk is gradually gaining attention due to its copious nutritional values  of the CPM medium revealed the presence of insoluble components that was categorized as non‐fluorescent  or autofluorescent fibres . Interestingly, the non‐fluorescent fibres of CPM resembled the starch granules  in consistent with previous reports  in CPM and assessed its ability to interact with these starch fibres (Appendix S1). Interestingly, we found that B. subtilis YC161 selectively colonize the autoflouresent starch fibres apparently through tight interactions . Z‐stack analysis by confocal laser scanning microscope (CLSM) confirmed that this intriguing bacterial‐fibre interaction was rigid and dynamic (data not shown). We assume that the observed bacterial interactions with colloidal components of CPM might have immense biological and technological importance for the food industry.Next, it was investigated whether these starch fibres might serve as a natural scaffold for bacteria to grow as biofilms. To test this, we initially grew fluorescently tagged ons Fig. . These iB. subtilis biofilm relies on extracellular polymeric substance matrix, we assessed the growth of strains (ΔtasA and ΔepsH) that harboured mutants in matrix operons. None of them adhered to the autoflouresent fibres nor formed biofilm bundles in CPM  cells formed robust pellicle and colony‐type biofilm in CPM (Appendix S1). As expected, matrix mutants  could not form either type of biofilm in CPM , and that the pulcherrimin biosynthesis pathway in B. subtilis was well established  synthase) and CypX (Pulcherriminic acid synthase), that convert two tRNA‐ molecules of leucine to pulcherriminic acid (PA), which in turn binds to iron (Fe3+) to form pulcherrimin. The mutants failed to produce the pigment in solid and liquid CPM  that might be responsible for the biofilm or pigment formation. Our going on observations indicate that the soluble fibre, pectin and no other tested tested sugars  are involved in biofilm and pigment formation .Next, pulcherrimin was further purified as previously described , auto‐fluorescent starch fibers that are ruptured due to heat during autoclaving (B), scale bar: 20 µm, and comparative analysis of natural autofluorescence and propidium iodide staining (C). Propidium iodide stains the starch fibers that are ruptured, while not the intact starch granules as the dye is membrane impermeable. For analysis, unvarying minimal auto‐exposure (20 ms) was used which sinks out the natural autoflouresence of CPM, scale bar: 100 µm.Fig. S2. Lugol's stain (potassium iodide and iodine) confirms that most of the CPM is starch. KI stained CPM (A), and microscopic visualization shows insoluble fibers as blue color  under light microscope (B). Yellow arrow denotes the auto‐fluorescent starch fibers while the black arrow and all that shows faint blue is the non‐fluorescent starch granules.Fig. S3. Alkaline pH (by addition of KOH to CPM) solubilizes the starch fibers as well as quenches the autofluorescence, thus confirming them as the resistant starch fibers.Fig. S4. Extensive chaining in CPM by WT strains. GFP expression was monitored in case of the fluorescently tagged B. subtilis (YC161), while the untagged NCIB3610 strain was stained with SYTOTM 9 dye.Fig. S5. Sequential biochemical procedure for extraction of pulcherrimin from CPM.Fig. S6. CPM polysaccharides act as an environmental signal for biofilm formation. Most of the carbon sources tested formed fragile pellicle in LB supplemented with 0.1 mM manganese (A), while pectin (the only soluble dietary fiber in chickpea) formed induced biofilm (as pellicle as well as colony type biofilm) at 0.1% and 0.5% concentration (B and C).Fig. S7. Survivability of B. subtilis following in vitro gastro‐intestinal digestion. Quantitation of B. subtilis survival was done based on colony forming units (CFU) of WT cells grown in either LB or CPM at 30 °C (with shaking at 25 rpm for 24 h), following to the either gastric or intestinal phases of the in vitro gastro‐intestinal digestion.Fig. S8. Diagrammatic representation illustrating the detailed experimental procedures for analysis of the CPM fiber‐attached bacteria following pasteurization and in vitro‐digestion system.Appendix S1. Experimental procedureClick here for additional data file.


Ischemic stroke is the most common cerebrovascular disease and is caused by interruption of blood supply to the brain. To date, recombinant tissue plasminogen activator (rtPA) has been the main pharmacological treatment in the acute phase. However, this treatment has some drawbacks, such as a short half-life, low reperfusion rate, risk of hemorrhagic transformations, and neurotoxic effects. To overcome the limitations of rtPA and improve its effectiveness, we recently designed sonosensitive sub-micrometric capsules (SCs) loaded with rtPA with a size of approximately 600 nm, synthesized using the layer-by-layer (LbL) technique, and coated with gelatine for clot targeting. In this study, we evaluated the rtPA release of ultrasound (US)-responsive SCs in healthy mice and the therapeutic effect in a thromboembolic stroke model.In healthy mice, SCs loaded with rtPA 1 mg/kg responded properly to external US exposure, extending the half-life of the drug in the blood stream more than the group treated with free rtPA solution. The gelatine coating also contributed to stabilizing the encapsulation and maintaining the response to US. When the same particles were administered in the stroke model, these SCs appeared to aggregate in the ischemic brain region, probably generating secondary embolisms and limiting the thrombolytic effect of rtPA. Despite the promising results of these thrombolytic particles, at least under the dose and size conditions used in this study, the administration of these capsules represents a risk factor for stroke.This is the first study to report the aggregation risk of a drug carrier in neurological pathologies such as stroke. Biocompatibility analysis related to the use of nano-and microparticles should be deeply studied to anticipate the limitations and orientate the design of new nanoparticles for translation to humans.The online version contains supplementary material available at 10.1186/s12951-022-01252-9. Stroke is a cerebrovascular disease and one of the main causes of death and disability in developed countries. It occurs as a result of disruption of cerebral blood flow (CBF), which induces metabolic and cellular changes, leading to cell death and alteration of brain function. Ischemic stroke is caused by an obstruction in the vessel, often due to the presence of a clot, and constitutes approximately 85% of all stroke events. Protective interventions focused on the prevention of cell death after an ischemic event have not yet demonstrated benefits in clinical practice, and recovery therapies are still far from becoming a treatment strategy for stroke patients , 2.Reperfusion therapies, based on the use of pharmacological thrombolysis and/or mechanical recanalization, focus on restoring CBF in the first hours after stroke onset . PharmacNanotechnology has emerged as a promising strategy for overcoming the limitations of rtPA and improving its efficacy. These nanomedical approaches focus on targeting the drug to the arterial clot-region and inducing a controlled drug release, with a final goal of increasing the number of patients that can safely benefit from rtPA .3 application on the cerebral artery. However, these models are characterized by the generation of a platelet-rich thrombus when rtPA-mediated thrombolysis is mainly active on fibrin clots [Due to the limitations involving the encapsulation of a large (~ 70 kDa) and sensitive enzyme, such as rtPA, specific nanomedical carriers have been developed to encapsulate and optimize the efficacy and safety of rtPA for stroke, such as liposomes, polymeric nanoparticles, magnetic nanoparticles, microbubbles, and echogenic liposomes, all with different levels of success . For manin clots –13. For in clots , 15. TheIn recent years, sonosensitive sub-micrometric capsules synthesized by the layer-by-layer (LbL) technique have been described as a promising strategy for drug delivery for many reasons: (i) versatility of polymeric composition; (ii) loading capabilities of sensitive molecules of different sizes, from small molecules  to large macromolecules ; (iii) mild conditions for core removal, which ensures the activity of bioactive molecules; and (iv) stabilization and protection from natural inhibitors of the cargo –18. TherFor this purpose, we recently designed sonosensitive SCs loaded with rtPA with a size of approximately 600 nm and synthesized by the LbL technique  for subsequent ultrasound (US)-controlled delivery . Based oIn the present study, the biocompatibility and therapeutic efficacy of our newly designed thrombolytic carriers in ischemic stroke conditions was tested for the first time, using a fibrin-rich thromboembolic stroke model. This model, induced by an in situ injection of thrombin in the middle cerebral artery (MCA), appears to be pathophysiologically closer to human stroke, and is widely accepted to evaluate the recanalization effects of rtPA , 22.We validated that our SCs responded to external US exposure by releasing rtPA and that the gelatine coating helps to stabilize the encapsulation and to maintain the response to US in vivo. However, when the therapeutic effect of these SCs was evaluated in the selected stroke model, we observed, by in vivo magnetic resonance imaging (MRI), that these SCs tended to aggregate in the ischemic region, leading to an increase in infarct size, independent of the gelatine coating or US application.Different batches of SCs loaded with rtPA, with or without gelatine, were synthesized following the same synthesis protocol described in our previous study . SCs wit®). However, in preclinical mouse models, since the fibrinolytic system is ten-fold less sensitive to rtPA than the human system, most preclinical studies were performed with 10 mg/kg instead of 0.9 mg/kg rtPA [The in vivo triggering of rtPA release from SCs was initially evaluated in healthy animals. The following experimental groups were included in the analysis: (1) control or vehicle group , (2) free solution of rtPA , (3) SC@rtPA , and (4) SC@rtPA-G . The four groups were combined with or without US application. In clinical practice, for the treatment of acute ischemic stroke, rtPA is administered as 0.9–1 mg/kg, 10% of volume as loading bolus and followed by 90% as infusion  for 40 min in the MCA territory did not damage the blood–brain barrier (BBB) integrity determined by Evans Blue (EB) extravasation  produced a significant reduction in infarct volume compared with the control group (average of 18 mm3 vs. 35 mm3 at 24 h)  confirmed that the recanalization efficacy was very similar in these three groups treated with free solution of rtPA, thus, neither US or the protocol administration interferes with drug activity.The correlation between the reperfusion rate and ischemic infarct lesion was subsequently evaluated in the same animals by MRI at 24 h Fig. A and 3 dThese findings are clinically relevant, as they show that the rtPA infusion protocol is not necessarily required to achieve a beneficial effect. In addition, although we used 10 mg/kg rtPA, as is recommended for rodents, a dose as low as 1 mg/kg is sufficient to produce successful recanalization. Therefore, our data supports previous evidence  that the3, 43 mm3, and 60 mm3, respectively, vs. 35 mm3, which is an increment of approximately 1.5, 1.2 and 1.7 times, respectively). Only the group treated with SC@rtPA-G and US, with partial recanalization, showed a lower infarct, but without a therapeutic effect, as the infarct size was similar to that of the vehicle group (31 mm3vs. 35 mm3). Infarct volumes determined 3 days after ischemia showed the same trend observed at 24 h, as indicated in Additional file On the other hand, analysis of ischemic lesions in animals treated with SCs@rtPA, SCs@rtPA + US, and SCs@rtPA-G showed a higher infarct volume compared to the control group with permanent arterial occlusion  by analyzing the iron content present as a result of SCs incorporation into the ioNPs. The data included in Fig. We recently reported the synthesis and characterization of a new thrombolytic carrier based on polymeric LbL submicroparticles coated with gelatine. These SCs showed the capacity to maintain rtPA by protection from its main blood inhibitor and to release rtPA by US-triggering. Furthermore, analysis of renal and hepatic markers and MRI studies have demonstrated that these SCs do not induce toxicity or brain damage in healthy animals . The preIn the first approach, we observed that only the SC@rtPA-G 1 mg/kg group responded to US exposure, allowing extension of the half-life of rtPA in blood compared with the group treated with the same dose of 1 mg/kg rtPA-free solution administered as a bolus. Meanwhile, the group treated with SC@rtPA without gelatine showed continuous rtPA release immediately after administration, independent of US application. Although gelatine was incorporated to the SCs to target the clot regions, these findings demonstrated the critical role of this protein in stabilizing encapsulation and maintaining the rtPA release response to US. This pharmacokinetic profile could be due to the presence of the arginine-glycine-aspartic acid sequence, which allows SCs to have low antigenicity, prolong their circulatory time, and evade the reticuloendothelial system , 30. EncInteresting results were observed for the four groups treated with SCs. Independent of the rtPA release response, none were able to induce a therapeutic effect. Thus, the group treated with SC@rtPA-G + US presented a similar infarct as the ischemic control group, while the ischemic lesion was even higher in the other three groups. T2*-weighted images, used to detect the distribution of SCs in the brain, revealed the accumulation and aggregation of SCs, mainly in the ischemic regions. This particle aggregation indicates that SCs administration might produce secondary embolisms in the arteries, abolishing the thrombolytic effect of rtPA and increasing the ischemic lesion.The evolution and distribution of T2*-weighted signals up to 3 days after ischemia confirm that the hypo-signals observed in the brain (black spots) are caused by the accumulation of SCs and eliminate the possibility of petechial haemorrhages , 28. Dis9) to reach the desired rtPA dose (1 mg/kg rtPA); (2) the properties of the SCs, such as their size (~ 600 nm) combined with poor elasticity, can induce reduced mobility through capillaries that contribute to aggregation [Different explanations could explain this particle accumulation in the ischemic region: (1) the number of capsules administered  have been confirmed safe in rodent models [The aggregation response could be due to the gelatine coating or US application on the ischemic region. However, this effect was independent of the gelatine coating, as the accumulation of SCs and increase in infarct volume were observed in both the SC@rtPA and SC@rtPA-G groups. Other studies that used gelatine complexes for rtPA encapsulation did not report aggregation in a heart ischemic model , which st models  and humat models . In contt models , 42. In In line with these findings, risk of cerebral lesions after therapeutic interventions has been reported in stroke animal models after intra-arterial administration of mesenchymal stem  which deWhile the benefits of nanotechnology for stroke pathology have been widely reported, adverse effects are limited to the study of the toxicity of materials, inflammation response, or risk of tumoral formation . Other sWe evaluated the in vivo biocompatibility and therapeutic effect of thrombolytic sub-microparticles in a thromboembolic stroke model that reproduces the recanalization of rtPA treatment, as observed in humans. Despite promising results in healthy animals, at least under the dose and size conditions used in this study, the administration of these SCs is a risky therapeutic tool for stroke. Adverse effects related to the use of nanoparticles in vivo should be thoroughly studied to anticipate the limitations and orientate the design of new nanoparticles for translation to humans –47. Futu3 cores were synthesized by mixing CaCl2, Na2CO3, and rtPA  labelled with fluorescein-5-isotiocyanate  for subsequent fluorescence quantification. LbL assembly was then carried out using poly (sodium 4-styrenesulfonate)  as a negatively charged polymer and poly  as a positively charged polymer. Iron oxide nanoparticles (ioNPs) were added to make these SCs suitable for MRI and ICP-MS analysis. Gelatine  was incorporated as the final layer of SCs for VWF targeting. Finally, the CaCO3 cores were dissolved and eliminated by chelator treatment under mildly acidic conditions  . Additional information on the synthesis and characterization is provided in Additional file Synthesis and characterization of SCs were performed as described in our previous study . BrieflyMale Swiss mice  weighing 25–30 g were used. Mice were kept in separate rooms under controlled temperature (22 ± 1 °C) and humidity (60 ± 5%) with a 12/12 h light/dark cycle for a week prior to surgery and up to 7 days after surgery. The animals had access to food and water ad libitum. All procedures were performed under anesthesia. Anesthesia was induced by inhalation of 5% sevoflurane in a nitrous oxide/oxygen mixture (70/30). The rectal temperature was monitored and maintained at 37 ± 0.5 °C using a feedback-controlled heating system. At the end of the procedure, the mice were sacrificed under deep anesthesia (8% sevoflurane). The experimental protocol was approved by the Animal Experimental Committee of the University of Santiago de Compostela, Spain. The animal experiments were conducted under the procedure number: 15010/2019/004 according to the Spanish and EU rules .2) was continuously applied using a transcranial Doppler  in the abdominal region for a period of 40 min after treatment administration.To validate rtPA encapsulation in the SCs and US-induced drug release with and without gelatine , treatments were administered first to healthy animals (n = 3/group). All treatments were administrated as a volume of 0.2 mL through the femoral vein. Blood rtPA plasmatic activity was determined  in basal conditions (before treatment administration) and at 1, 5, 15 and 40 min after administration. Blood was sampled by inserting a cannula into the carotid artery. In these groups, external US radiation  of healthy animals were later evaluated under ischemic conditions (n = 5/group). The thromboembolic stroke model was induced by injection into the MCA of mice, as originally described by Orset et al. . The mic2 during 40 min after stroke induction), but US were applied on the ischemic side of the animal head.CBF was monitored using a Periflux 5000 laser Doppler perfusion monitor  by placing the probe  in the parietal territory of the MCA. Basal CBF was measured for at least 5 min throughout the experiment. Artery occlusion was considered successful when the CBF decreased by more than 80% relative to the basal flow and reperfusion when at least 40% of the basal CBF was recovered. Thirty minutes after thrombin occlusion, different treatments were administered intravenously into the tail vein. For this intervention, a small incision was made in the tail of the animal, the skin was excised, and the tail vein was exposed. A 30 G needle  was used for every treatment, and the puncture was rapidly closed to prevent bleeding. In the animals in which the treatment was administered as an infusion, the 30 G needle was placed in a catheter  and the injection rate was controlled using a micro-pump (4.5 µL/min). In the groups treated in combination with US, the setup was similar to the one used in healthy animals  have been confirmed to be safe in rodent models [2, 2 MHz) in our animal model, we analyzed BBB disruption after US application using the EB technique under healthy and ischemic conditions (n = 5 per group). This assay is based on the ability of EB to bind serum albumin. Extravasation of albumin to the cerebral parenchyma is correlated with BBB disruption [The selected US parameters , and the absorbance due to the EB present in the supernatant was measured with a plate reader (630 nm). The extravasated dye content was normalized to the brain weight (µg/g).In healthy animals, EB was injected prior to US application. In the ischemic group, EB was injected 30 min after thrombin injection and immediately after US application. In all the experimental groups, US was applied for 40 min. The mice were euthanized 24 h after EB injection. The brains were removed, divided into ipsilateral and contralateral hemispheres, and weighed. The hemispheres were frozen immediately in liquid nitrogen and stored at − 80 °C. For analysis, brain samples were placed in a stove at 55 °C in 1 mL of MRI was performed at 24 h and 3 days after ischemic induction to evaluate the infarct size and fate of SCs.MRI studies were conducted using a 9.4 T horizontal bore magnet  with 12 cm wide actively shielded gradient coils (440 mT/m). Radiofrequency transmission was achieved with a birdcage volume resonator, and the signal was detected using a two-element arrayed surface coil , positioned over the head of the animal, which was fixed with a tooth bar, earplugs, and adhesive tape. The respiratory frequency and body temperature were monitored during the experiments. The transmission and reception coils are actively decoupled from each other. Gradient-echo pilot scans were performed at the beginning of each imaging session to accurately position the animals inside the magnet bore.2 field of view (FOV) with saturation bands to suppress the signal outside this FOV, a matrix size of 256 × 256 (isotropic in-plane resolution of 75 μm/pixel × 75 μm/pixel), and implemented without fat suppression. The acquisition time was 23 min.The progression of ischemic lesions and infarct volumes were determined from T2-maps calculated from the T2-weighted images. Ischemic lesions were determined by counting pixels with apparent T2-map values above the threshold in the ipsilateral brain hemisphere. In healthy mice, the T2-map values of the brain are over 50 ms. In the ipsilateral ischemic hemisphere, hyperintensity on the T2-map determined the analysis of ischemic damage  [2 FOV with saturation bands to suppress signal outside this FOV, and a matrix size of 256 × 256 (isotropic in-plane resolution of 75 μm/pixel × 75 μm/pixel) and implemented with the fat suppression option. The acquisition time was 10 min.The distribution of SCs in the brain was evaluated by T2*-weighted images. T2*-weighted images were acquired using a multi-gradient-echo sequence (MGE) with a 5 ms TE, 1.2 s TR, 8 echoes with 4.5 ms echo spacing, 100 kHz spectral bandwidth, FA of 20°, 16 slices of 0.55 mm, 2 averages, 19.2 × 19.2 mmThe hypo-signals in the T2*-weighted images were quantified using two different methods: mean grey value and area fraction . ImageJ 3  under constant agitation in 50 mL Falcon tubes. Digestion took place over 48 h at 21 °C until no organic residues remained. This liquid solution was then further digested using aqua regia consisting of three parts concentrated ultra-pure (35 wt %) HCl  and one part of ultra-pure (67 wt %) HNO3. For this final digestion, 100 µL of a well-mixed sample solution was added to 300 µL of aqua regia and agitated for at least 2 h . The samples had to be diluted tenfold using a low matrix of 2% (w/v) HCL, which was adapted to the material, to use on the ICP-MS instrument.Biodistribution of SCs in different organs  was determined by analyzing the iron concentration using ICP-MS, as described elsewhere . Brieflyt-test or one-way analysis of variance (ANOVA) followed by post-hoc Brown-Forsythe evaluation was used to detect significant differences between groups. Statistical significance was set at P < 0.05. Statistical analysis and graphical representation were performed using GraphPad Prism 8.0.All data are presented as the mean and SD of the mean (mean ± SD). The data were first examined to assess the distribution using D’Agostino and Pearson omnibus normality tests. A Additional file 1: Scheme 1. Schematic representation of the layer-by-layer process. Figure S1: SEM micrographs of the samples after drop casting on Si substrates. Figure S2: TEM micrographs of the samples after drop casting on top of a copper grid coated with a layer of carbon. Figure S3: DLS graphs A) intensity, B) number and C) ζ-potential of core@rtPA (gray), SC@rtPA (blue) and SC@rtPA-G (pink) measured in Milli Q water. Table S1: Mean average hydrodynamic diameters and ζ-potential values. Figure S4: rtPA-FITC calibration curve. Figure S5: SCs dispersion analysis by flow cytometry. Figure S6A) Ischemic lesion (white brain region) determined by T2-weighted at 3 days after the treatment administration in the 9 experimental groups: vehicle, vehicle with ultrasound (US), rtPA (1 mg/kg as bolus), rtPA with US (1 mg/kg as bolus), rtPAi (rtPA 10 mg/kg as bolus and infusion), SC@rtPA (1 mg/kg as bolus), SC@rtPA with US (1 mg/kg as bolus), SC@rtPA-G (1 mg/kg as bolus) and SC@rtPA-G with US (1 mg/kg as bolus). B) Analysis of the infarct volume at 24 hours after treatment administration. Figure S7A Distribution of the microcapsules in brain analyzed by T2*-weighted in the different experimental groups at 3 days after the treatment administration. SCs can be identified as hypo-signals (indicated with yellow arrows), mainly, in then ischemic region. B Quantification of the SCs accumulation determined by grey value relative to the vehicle group. In each animal three independent measurements were performed. C Quantification of the SCs accumulation by area fraction. D MRI representation of a animals treated with saline (control) and the groups treated with the SCs, in which the hypo-signals could be observed as black spots  Figure S8. Methods to quantify the hypo-signals in T2*-weighted images.


Wheat starch is one of the most important components in wheat grain and is extensively used as the main source in bread, noodles, and cookies. The wheat endosperm is composed of about 70% starch, so differences in the quality and quantity of starch affect the flour processing characteristics. Investigations on starch composition, structure, morphology, molecular markers, and transformations are providing new and efficient techniques that can improve the quality of bread wheat. Additionally, wheat starch composition and quality are varied due to genetics and environmental factors. Starch is more sensitive to heat and drought stress compared to storage proteins. These stresses also have a great influence on the grain filling period and anthesis, and, consequently, a negative effect on starch synthesis. Sucrose metabolizing and starch synthesis enzymes are suppressed under heat and drought stress during the grain filling period. Therefore, it is important to illustrate starch and sucrose mechanisms during plant responses in the grain filling period. In recent years, most of these quality traits have been investigated through genetic modification studies. This is an attractive approach to improve functional properties in wheat starch. The new information collected from hybrid and transgenic plants is expected to help develop novel starch for understanding wheat starch biosynthesis and commercial use. Wheat transformation research using plant genetic engineering technology is the main purpose of continuously controlling and analyzing the properties of wheat starch. The aim of this paper is to review the structure, biosynthesis mechanism, quality, and response to heat and drought stress of wheat starch. Additionally, molecular markers and transformation studies are reviewed to elucidate starch quality in wheat. Triticum aestivum is used to make bread and noodles, and T. durum for spaghetti and macaroni. T. monococcum, T. dicoccum, and T. spelta, customarily referred as einkorn, emmer, and spelt, respectively, are some of the ancient species used in grain berries, farro, and salads . Whe. Whe1]. d salads .Wheat starch is an important by-product of gluten production . Wheat eWheat starch is a major storage carbohydrate and contains about 60~75% grain and 70~80% flour . Starch Starch plays a significant role in the texture of many kinds of food and serves as a major source of energy for humans . Native The waxy starch used in the food industry is typically chemically modified . Cross-l5~1 × 106 [7–1 × 109 [Starch granules are composed of two types of α-glucan, amylose and amylopectin, and make up about 98-99% of the dry weight . The pro~1 × 106 . Amylope–1 × 109 ,18 and i–1 × 109 , followe–1 × 109 . The whe–1 × 109 . Each ty–1 × 109 . Recentl–1 × 109 .Starch is the main storage compound in plants, present in both production and storage organs. As starch biosynthesis is a complex process ,17, highSucrose produced by photosynthesis moves to the amyloplast and is metabolized to hexose phosphate. These hexose phosphates act as a substrate for starch, protein, and oil biosynthesis. When the endosperm develops, most of the hexose phosphate is used for starch biosynthesis. In order to induce such an energy-intensive reaction, phosphorylation and ATP production are required. Starch synthase enzymes separate glucose residues from ADP-glucose and bind them to the ends of amylose and amylopectin to elongate polymer polysaccharide chains. In the polysaccharide chain constituting amylose, the OH groups of carbon 1 and carbon 4 of glucose inside the chain are continuously connected. Amylopectin shows a regular branch shape by connecting the OH groups of carbon 1 and 6 in addition to the polysaccharide chain of amylose. The formation of these branches involves the starch branching enzyme (SBE) . These tWhen the endosperm of wheat, corn, barley, and rice is developed, the cytosolic isoform of AGPase accounts for 65 to 95% of the total AGPase activity . In highWe consume grains, pasta, and potatoes in our daily lives, and most of these carbohydrates are starch. Starch quality is largely determined by the ratio of amylose to amylopectin . Normal In 1982, during in vitro analysis of non-starch polysaccharides, Englyst and workers discovered that some starch remained after enzyme hydrolysis ,37,38. ARS1 is starch that is physically inaccessible to digestion, such as that in whole grains or tubers. RS2 is a native starch granule that is protected from digestion by the conformation or structure of the granule and is found in raw potatoes and green bananas. RS3 is retrograded starch formed when starchy foods  are cooked and then cooled. Cooling allows the amylose and linear parts of amylopectin to form crystalline structure that reduces digestibility. RS4 is chemically modified starch formed by crosslinking, etherization, or esterification and it is found in foods containing modified starches such as some bread and cakes. RS5 is a starch wherein the amylose component forms complexes with lipids (amylose–lipid complex). The amylose–lipid complex is generally found in native starch granules and processed starch. This complex also entangles amylopectin molecules, restricting the swelling of starch granules and enzyme hydrolysis ,48. The http://www.fao.org). Starch synthesis was observed, that this mechanism was strongly relevant during the stages of anthesis and maturity [Wheat development stages include “germination, emergence, tillering, floral initiation, terminal spikelet, stem elongation, spike emergence, anthesis and maturity” . To overcome these problems, early ripening wheat cultivars through crossbreeding (Jokyung (accession no. 102005000184), Jopum (102000200523), Joeun (102001000044) etc.) were released from the 1990s to 2010s in Korea. Additionally, transcriptome analysis under heat stress was performed on Korean wheat cultivars during the ripening period [Wheat plants are frequently subjected to varying degrees of heat and drought stress during their growth stage . Heat stg period .Starch is more sensitive than storage protein to heat stress . AlthougHigh temperatures of 20 to 23°C at night reduced the grain-filling period by 3 to 7 days . There hHeat stress decreases cereal starch content while increasing protein content during grain filling . It did During heat stress, carbohydrate availability is a significant physiological feature linked to heat stress resistance . SurvivaStarch metabolism enzymes consist of sucrose phosphate synthase (SPS), sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (AGPase), glucokinase, soluble starch synthase (SSS), and starch branching enzyme (SBE) . Heat stHeat stress reduced the activities of SPS and SuSy, resulting in lower sucrose levels during the grain filling period  and incrIn the case of India and China, waterlogging limited wheat production . Total rPre-harvest sprouting (PHS) is the premature germination of grain before harvest in wheat. PHS occurs in several wheat-growing regions . When geOne of the environmental factors that limits crop development and agricultural output is salt stress. High salinity has been shown to have an impact on carbohydrate metabolism. Salt stress induced GBSS expression that was highly controlled at the transcriptional level . Salt stIn the past, breeding research has relied on measuring only the characteristics of interest to select the superior lines. For example, it is easy to choose simple morphological properties such as plant height to select large amounts of offspring. Of course, the size and yield of each grain can also be considered. However, most of these characteristics require laboratory analysis or bioassay. Many characteristics are difficult to measure , so the resources available to breeders impose significant constraints on the speed and scale of their choice. In such cases, the use of markers is of great value to wheat breeders who indirectly represent the characteristics of interest and are relatively easy to score . MarkersIn order to develop an efficient breeding program in common wheat, four techniques  were compared to evaluate the suitability . Of thesAdditionally, the application of functional markers for the identification of LMW-GS in wheat germplasm of various types has been reported . Functiowbm gene on the 7AL chromosome and the overexpressed glutenin Bx7OE  gene [Molecular marker technology has provided a new and efficient tool to improve the quality of bread wheat. To improve and support bread-making quality, high-throughput Kompetitive Allele-Specific PCR (KASP) analysis was performed and verified for key genes including the al) gene . These hal) gene ,145,146.al) gene .An important factor in determining the amylose content of grain starch is the 59 kDa granule bound starch synthase (GBSS) protein ,149. In T. aestivum L.), barley (Hordeum vulgare L.), corn (Zea mays L.), and rice (Oryza sativa L.) [ Two types of GBSS genes, GBSSI and GBSSII, are present in wheat (tiva L.) . The GBStiva L.) . Waxy (Gtiva L.) . Each GBtiva L.)  and SDS-tiva L.) . In wheativa L.) . Meanwhitiva L.) . These PWith the development of Next-Generation Sequencing (NGS), NGS-based genotyping techniques were applied to the development of molecular markers for grain starch or quality . A genomGrain is the part harvested from wheat, and its nutrition and properties are determined by its biochemical composition. In wheat seeds, starch accounts for 55 to 75% of the total dry grain weight and contains 10 to 15% of the storage protein. In addition, starch and protein have a significant impact on the quality of products made from flour. Optimal starch and protein, and the right levels of essentials  are indispensable elements in healthy wheat products. Most of these quality traits in recent years have been developed for research using genetic modification interventions.The starch portion, which comprises about 70% of the total dry matter of wheat grains , can havWheat starch granules accumulate at least three types of starch synthase (SS) with molecular masses of about “60, 77, 100 to 115 kDa” . Most SSTemporary starch produced in photosynthetic tissue may not be fully applicable to starch build-up in sink parts, but the same enzymatic function is also believed to be involved in starch biosynthesis in both localities . Genes eTraditional breeding techniques or genetic modification can be used to produce novel starch with modified properties . Using gCKX2) gene in the transgenic line [TaCKX2.4 was negatively correlated with the number of grains per spike, and the number of grains per spike was increased in wheat with decreased TaCKX2.4 expression. RNA silencing of the waxy gene by the RNAi strategy confirmed a decrease in amylose levels in transgenic wheat seeds [RNA interference (RNAi) is a powerful tool for functional gene analysis and engineering of novel phenotypes, which is a common regulatory mechanism for gene expression in eukaryotic cells. This technique directs gene silencing after transcription in a sequence-specific manner based on the expression of antisense or hairpin RNAi constructs, or other forms of short interfering RNA molecules. The application of RNAi contributed to the manipulation of wheat particle size ,186 and nic line . That isat seeds . Accordiat seeds . The silSBEIIa gene increased the amylose content in durum wheat [SBEIIa gene causes changes in granule morphology and starch composition, resulting in high-amylose wheat. High-amylose durum wheat was produced through mutagenesis of starch synthase II (SSIIa or SGP-1) [T. aestivum) was developed, and candidate genes responsible for the amylose mutation were identified [Silencing of the um wheat . The star SGP-1) . Therefoentified .Oryza sativa L.) endosperm, and provide new insights into improving wheat yield (citation). TaMTL was edited using an optimized Agrobacterium-mediated CRISPR system to efficiently induce haploid plants in wheat [TaWaxy and TaMTL, were edited with high efficiency by the optimized SpCas9 system, and the highest efficiency (80.5%) was achieved when targeting TaWaxy using TaU3 and two sgRNAs.Starch composition, structure, and properties were modified through editing of TaSBEIIa in both winter and spring wheat varieties using CRISPR/Cas9 . TaSBEIIin wheat . Two endAfter genetically modified organisms (GMOs), the era of ’gene-edited crops’ is coming. The United States, Canada, Israel, Japan, and Australia have already begun to approve the production of gene-edited crops. Targeted gene editing, especially CRISPR/Cas9, is a tool with significant potential for plant development and breeding ,191. GenT. aestivum L.), is primarily regulated by the Pina and Pinb genes. Mutation or deletion of Pina or Pinb increases kernel hardness, resulting in hard wheat kernels. Transformation of Pinb-D1x into soft wheat using bombardment technology produces a hard wheat kernel texture [Pinb-D1x into the soft mill significantly increased the kernel hardness and changed the internal structure of the kernel. The low molecular weight glutenin subunit LMW-N13 improved the dough quality of transgenic wheat using Agrobacterium-mediated technology [Kernel hardness, a quality characteristic of common wheat , a type of starch, proved that it is not easily decomposed by digestive enzymes in the body . Resista