Corynebacterium striatum, a bacterium that is part of the normal skin microbiota, is also an opportunistic pathogen. In recent years, reports of infections and in-hospital and nosocomial outbreaks caused by antimicrobial multidrug-resistant C. striatum strains have been increasing worldwide. However, there are no studies about the genomic determinants related to antimicrobial resistance in C. striatum. This review updates global information related to antimicrobial resistance found in C. striatum and highlights the essential genomic aspects in its persistence and dissemination. The resistome of C. striatum comprises chromosomal and acquired elements. Resistance to fluoroquinolones and daptomycin are due to mutations in chromosomal genes. Conversely, resistance to macrolides, tetracyclines, phenicols, beta-lactams, and aminoglycosides are associated with mobile genomic elements such as plasmids and transposons. The presence and diversity of insertion sequences suggest an essential role in the expression of antimicrobial resistance genes (ARGs) in genomic rearrangements and their potential to transfer these elements to other pathogens. The present study underlines that the resistome of C. striatum is dynamic; it is in evident expansion and could be acting as a reservoir for ARGs. Corynebacterium spp. as etiologic agents of various infectious processes in both immunocompromised and immunocompetent patients. In addition to the etiologic agents of classic and zoonotic diphtheria, at least 50 diphtheria toxin (DT)-producing and non-DT-producing Corynebacterium species have been recognized as medical, veterinary, or biotechnologically relevant, including members of human microbiota  s. (2018)  found neStreptomyces roseosporus. It is generally used to treat serious infections caused by Gram-positive bacteria. Daptomycin could act as a temporary ionophore in the membrane by forming a complex with phosphatidylglycerol (PG) and calcium ions, bringing about depolarization and a subsequent ion leakage. An ion leakage can have several consequences, including an osmotic imbalance and metabolite uptake deficiency. However, the bactericidal mechanisms of daptomycin are not yet entirely clear , i, iC. strl., 2019 , showed  ISAar26 C and wer. (2018)  assessedtet(W) gene product eliminates the inhibitory effect of tetracycline on protein synthesis through the non-covalent modification of the ribosomes. The gene is not transported by pTP10, suggesting another recombination event beyond pTP10 (or this plasmid acquired this gene in later events). It has been reported that IS3504 carried the tet(W) gene in isolates from the USA [C. jeikeium, and it has been transported to its plasmids and to C. urealyticum. On the other hand, IS3504 was described in the pNG2 plasmid of C. diphtheria [C. resistens [tet(W) gene was first described for corynebacteria in plasmid pJA144188 of C. resistens. The gene was described in a genomic context similar to how it presents in C. striatum, with a close IS3504 element, as the studies by Nudel et al. (2018) [erm(X) gene as was found in the studies by Wang et al., 2019 [The  the USA , but it  the USA . Accordi the USA , both ISesistens . The tet. (2018)  describel., 2019 .tet(W) and aac(3)-XI genes, and the diversity of insertion sequences. Therefore, we propose that insertion sequences play a fundamental role in the continuous formation and evolution of the resistome of C. striatum.The genomic contexts associated with transposons Tn5432, Tn5717, Tn5716, and Tn5715 suggest that pTP10 underwent past recombination events with the chromosome, constituting a portion in the chromosome open to new recombination events. New realignments of the transposons can explain this, the appearance of the tnp). IS elements are grouped according to the motives of the active site and according to their recombination mechanism. Traditionally, IS elements do not transport passenger genes, but they can move ARGs as part of a composite transposon, which is a region flanked by two copies of the same IS element  are small mobile elements; they typically contain a gene that codes for a transposase  that can form hybrid promoters, which generate a promoter derived from two different sequences [C. striatum can be explained partly by the efficiency of the promoter of its IS elements. However, further research will be required on these elements in C. striatum.There are elements in the IS3 and IS256 families  [C. striatum has the regulatory mechanism bla gene, needed for conferring beta-lactam resistance.crobials , with pe enzymes . It has /B genes . In othe-lactams . The bla. (2019) ; this susul genes found in the studies by Wang et al. (2019) [sul gene confers sulfamethoxazole resistance  [sul gene and the qacEdelta1 gene are frequently seen in class 1 integrons. Integrons are considered scarce in Gram-positive bacteria. Nevertheless, in the genus Corynebacterium, class 1 integrons have already been documented in C. glutamicum, Corynebacterium ammoniagenes and Corynebacterium casei [Corynebacterium spp. [Corynebacterium, there are class 1 integrons in C. diphtheriae, C. amycolatum, and Corynebacterium efficiens. However, class 1 integrons have not been described in C. striatum. Therefore, to evaluate the emergence or presence of integrons in C. striatum, greater epidemiological monitoring will be required, with studies focused on identifying the integrase gene Int1 and its associated genes.The resistance to sulfonamides may be due to the . (2019) . The sulsistance . Accordi. (2020) . On the um casei  and, recium spp. . Additioium spp. , in the C. striatum. Although the vanW gene, which is associated with vancomycin resistance, was found in the genome of C. striatum 2308, this isolate proved to be susceptible to vancomycin [Corynebacterium aquaticum has been reported [Corynebacterium spp. has also been documented [Fortunately, the glycopeptide vancomycin continues to show good results in the treatment of ncomycin . Howeverreported , and a ccumented .C. striatum is larger (data not shown). This finding reflects the need to explore and characterize the resistome of C. striatum involving computational approaches of prediction methods and functional characterization.Finally, based on searches of ARGs in databases like MEGARes  and ResFC. striatum should not be underestimated, given that it is causing several types of infections. Nevertheless, despite the seeming virulence factors, recent studies revealed a mortal virulence of C. striatum in Caenorhabditis elegans [The studies compiled here indicate that  elegans ,85, a ne elegans . TherefoC. striatum highlights the plasticity of this bacterium in acquiring resistance genes and propagating as a persistent and pathogenic clone. Added to this, the ability to create biofilms in medical devices makes it a potential propagator of multidrug-resistant genomic elements. Thus, C. striatum may act as a potential reservoir to propagate resistance to other pathogenic bacteria.The diversity of IS elements  associatCorynebacterium species can be detected in the clinical environment using biochemical techniques, commercial kits (such as API Coryne or Vitek 2) and recently MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) [C. striatum [Corynebacterium spp. [Corynebacterium since the clinically relevant Corynebacterium species still show susceptibility to glycopeptides, such as vancomycin and linezolid [-flight) . Biochemstriatum ,87. Whetium spp.  should binezolid ,89,90.C. striatum could cause an epidemic is a question that has not yet been answered. Since it is part of the normal human microbiota, C. striatum has only primarily caused disease to date as an opportunistic pathogen. However, a recent phylogenetic study considering Chinese strains of C. striatum suggests that a lineage of C. striatum has been extended worldwide and could be a pandemic lineage [Whether  lineage . FurtherC. striatum. This resistome comprises chromosomal determinants and mainly acquired elements. Intrinsic resistance includes fluoroquinolone and daptomycin resistance via mutations on chromosomal genes. The increasing resistance and adaptation to daptomycin tolerance deserves special attention, which requires greater monitoring of its presence and the mechanisms that produce it. Many of the studies described here point out that a large part of the C. striatum resistance presents a genomic context similar to mobile elements such as insertion sequences and transposons mainly related to pTP10 plasmid.In this review, based on the literature and searches in specialized databases, we have explored the antimicrobial resistance gene repertoire found in C. striatum. Therefore, future challenges aim to systematically study the diversity of IS and its associated genes to understand the role of IS in the maintenance and dispersal of ARGs and its impact on the generation of lineages of concern of C striatum.The transposon Tn5432 stands out, which has been associated with the MLS phenotype and has insertion sequences along with different resistance genes. The ISs that compose this transposon have been identified in other bacteria and in isolates throughout the world. Although there are some similarities in the genomic context of ISs, we can observe some gene composition diversity and rearrangements in these regions. These findings suggest that insertion sequences are potential propagators of resistance at the interspecies level and play a fundamental role in the continuous formation and evolution of the resistome of qacEdelta1, sul, bla and merA have not been associated with mobile genetic elements in C. striatum. However, considering their occurrence in other bacteria, they could be part of integrons or other genomic islands, making it necessary to carry out future studies focused on the genomic context of these genes. Moreover, although genes conferring resistance to vancomycin and linezolid have not been verified among nosocomial and community strains of C. striatum, the emergence of multidrug-resistant strains and acting as a reservoir for ARGs reveals a worrisome public health scenario. Therefore, in addition to the species-level confirmation, more significant efforts and attention should be dedicated to monitoring its occurrence and investigating its resistome.On the other hand, genes such as C. striatum in the context of epidemiological factors, the ARGs repertoire, and its genomic localization. Furthermore, it is highlighted that the resistome of C. striatum is dynamic and in evident expansion. Consequently, it is emphasized that actions such as implementing genomic surveillance programs and studies on the mechanisms of antimicrobial resistance should be carried out. These measures would help to warn of the emergence of multidrug resistance lineages and prevent future public health problems.This work uncovers the dynamics of antimicrobial resistance in


Alr) could be used as a screening marker and develop an efficient oral vaccine against PEDV infection, a recombinant strain was constructed using Lactobacillus casei with Alr deletion (L. casei ΔAlr W56) to deliver the Alr gene and a core-neutralizing epitope (COE) antigen. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in mice. Compared to the other strains, the recombinant bacteria were able to grow without the addition of D-alanine, revealing that Alr in the plasmid could function normally in defective bacteria. This oral mucosal vaccine would provide a useful strategy to substitute the application of antibiotics in the future and induce efficient immune responses against PEDV infection.Porcine epidemic diarrhea (PED), which is caused by the porcine epidemic diarrhea virus (PEDV), has occurred worldwide and poses a serious threat to the pig industry. Intestine is the main function site of PEDV; therefore, it is important to develop an oral mucosal immunity vaccine against this virus infection. Most traditional plasmid delivery vectors use antibiotic genes as a selective marker, easily leading to antibiotic accumulation and gene contamination. In this study, to explore whether the alanine racemase gene ( Porcine epidemic diarrhea (PED), characterized by watery diarrhea, vomiting, and dehydration, infects pigs at different ages and is caused by the porcine epidemic diarrhea virus (PEDV) . In receLactic acid bacteria (LAB) are considered to be safe microorganisms with beneficial effects on the human and animal health [Lactobacillus casei W56 (Lactobacillus casei W56) is a member of the LAB group, with a powerful potential as a vaccine delivery vehicle, regulating T-helper cell response and stimulating specific secretory immunoglobulin A (SIgA) secretion for mucosal immunity [At present, the protective efficacy of traditional commercial vaccines, including transmissible gastroenteritis virus (TGEV)-PEDV dual inactivated vaccines and attenuated vaccines, has gradually decreased, while the incidence of PED has increased every year . PEDV inl health  by improl health . Lactobaimmunity ,13. SIgAimmunity . Therefoimmunity .Alr) gene deletion could result in the incomplete synthesis of the cell wall and the death of the bacteria [Alr gene deletion bacteria [Lactobacillus.The traditional biotechnology industry typically uses resistance genes as a screening marker, which is faced with the problem of antibiotic residue contamination and the spread of resistance genes . D-alanibacteria ,19,20. Dbacteria ,22. ThisLactobacillus casei with Alr gene knockout (L. casei ΔAlr W56), containing a plasmid expressing a core-neutralizing epitope (COE) antigen and the Alr gene, was constructed. Its immunogenicity as an oral vaccine was evaluated through the significant levels of anti-PEDV systemic immunoglobulin G (IgG) and mucosal IgA antibody responses in mice. Our results clearly showed that the recombinant strains were effective at inducing anti-PEDV mucosal immune responses.In this study, a genetically engineered strain, All animal experiments were approved by the Ethical Committee for Animal Experimentation of Northeast Agricultural University, Harbin, China.L. casei ΔAlr W56 was constructed in our laboratory and grown anaerobically in a de Man, Rogosa, and Sharpe (MRS) broth supplemented with D-alanine at 37 °C without shaking. PEDV LJB/15 strain was dissociated from clinical samples by our laboratory and propagated in porcine small intestinal epithelial cells (IPECs) at 37 °C with 5% CO2. The constitutive expression plasmid pPG-T7g10-PPT, with a hyper constitutive expression promoter, T7g10 enhancer, pgsA anchor, and rrnBT1T2 terminator, was constructed in our laboratory. The details of all plasmids and primers used in the study are listed in Alr was obtained from the genome of L. casei W56. The COE gene was then inserted at the Sac I and Apa I sites of the corresponding expression vector pPG-T7g10-PPT constructed by Song et al. [Alr was cloned into the recombinant vector pPG-COE, resulting in the plasmid pPG-Alr-COE. All recombinant plasmids were assessed by polymerase chain reaction (PCR) and sequencing.A schematic diagram of the recombinant plasmid construction is shown in g et al. , generatL. casei ΔAlr W56 competent cells were prepared according to an electroporation method described previously, resulting in the recombinant strains pPG-COE/ΔAlr W56, pPG-Alr-COE/ΔAlr W56, and pPG-T7g10-PPT/ΔAlr W56 [COE and Alr were identified by PCR confirmation and sequencing. These strains were constructed successfully and streaked on chloramphenicol MRS plates with or without D-alanine to detect the demand situation.To construct the recombinant strains, ΔAlr W56 . BrieflyAlr W56, pPG-T7g10-PPT/ΔAlr W56, and ΔAlr W56 were grown overnight in MRS broth with or without D-alanine, harvested by centrifugation at 10,000× g for 2 min and washed twice with sterile phosphate-buffered saline (PBS). The supernatants were lysed and centrifuged using a Mini-Beadbeater  and separated in a 12% gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the western blot assay. The proteins were then transferred to a polyvinylidene difluoride membrane . Immunoblotting was performed using a mouse anti-Flag antibody (1:1000) as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:5000) as the secondary antibody . The results were visualized using a chemiluminescent substrate reagent  according to the manufacturer’s instructions. To evaluate the expression of the protein of interest by the recombinant strains, pPG-COE/ΔAlr W56, pPG-Alr-COE/Δn = 120) were purchased from Liaoning Changsheng Biotechnology Co., Ltd.  and maintained under SPF conditions for one week with free access to a standard chow diet and water in accordance with institutional guid/elines. Prior to oral administration, the recombinant strains were cultured overnight in an MRS medium with or without D-alanine, washed with sterile PBS and resuspended in PBS at a concentration of 1010 colony forming units (CFU) CFU mL−1. At the indicated time points, three mice were randomly selected from each of the 4 groups (each of the 4 groups had 30 mice each). The mice were immunized with 200 μL of the whole volume live recombinant L. casei by oral gavage, once for 3 consecutive days  and boosted twice at 2 week intervals .All the experimental and animal management procedures were approved by the Institutional Committee of the Northeast Agricultural University for the Animal Experiments , Harbin, China. To assess the immunogenicity of recombinant strains applied as an oral vaccine, four-week-old specific-pathogen-free (SPF) female BALB/c mice (−1 phenylmethylsulfonyl fluoride (Sigma-Aldrich) and 1% bovine serum albumin, incubated at 4 °C for 16 h and centrifuged at 10,000× g for 5 min, after which the supernatants were stored at −40 °C until use.The immunized mice were randomly selected three in the different groups and collected the samples, respectively. The mice were sacrificed, and the sera and intestinal mucusal were collected from the first immunized mice on days 0, 7, 14, 21, 28, 35, 40, 42, 49, 56, 63, and 70 and stored at −40 °C. Intestinal mucusal was scraped with a glass slide for detection after rinsing with sterile PBS. Vaginal and nasal fluid were collected by aspirating and discarding repeatedly with 500 µL sterile PBS to stimulate the nose of mice for sample collection and stored at −40 °C until analysis. Fecal samples were collected at different timepoints, used for detecting the SIgA antibody and treated according to methods described previously with slight modifications . BrieflyTo determine the levels of the anti-PEDV-specific IgG antibody in the sera and the SIgA antibody in the fecal, intestinal mucusal, vaginal, and nasal fluids, the samples were collected from the immunized mice at the corresponding timepoints and measured using ELISA. The PEDV propagated on IPECs were coated on polystyrene microtiter plates overnight at 4 °C, and the culture of IPECs was used as a negative control for the antigen. After blocking with 5% skimmed milk and washing three times with 0.1% PBS Tween 20 (PBST), the collected samples were serially diluted in PBS  or skimmed milk (sera), added in triplicate and incubated at 37 °C for 2 h. Next, an HRP-conjugated goat anti-mouse IgG or IgA antibody  was added to each well  and incubated at 37 °C for 1 h. The color was detected using the o-phenylenediamine dihydrochloride substrate (Sigma-Aldrich) and measured at an absorbance of 490 nm. To detect the level of cytokines, sera samples were collected from the immunized mice and subjected to ELISA according to the manufacturer’s instructions  on 40 d post-immunization. The assays included IL-4, IFN-γ, and IL-17. The detection ranges of these cytokine detection kits were as following: IFN-γ (47–300 pg/mL), IL-4 (0.63–40 pg/mL), and IL-17 (7.8–500 pg/mL).Alr W56, 50 µL 2 × serial diluted samples were collected on 40 d post-immunization, mixed with 50 µL of PEDV passages with 200 50% tissue culture infective dose (TCID50) in IPECs and incubated at 37 °C for 1 h. The mixture was then incubated to IPECs monolayers in 96-well plates. PEDV-specific cytopathic effects (CPEs) were observed with incubation at 37 °C in a 5% CO2 atmosphere for 3 days. The Reed–Muench statistical method was applied for analyzing the data [To determine the neutralizing activity of the sera or intestinal mucusal antibodies obtained from the mice immunized with pPG-Alr-COE/Δthe data .p < 0.05 was considered statistically significant, and p < 0.01 was considered highly significant.GraphPad Prism  was used to perform the statistical analyses. Tukey’s multiple comparison test was used to analyze the differences among the control groups. All animal experiments were approved by the Ethical Committee for Animal Experimentation of Northeast Agricultural University, Harbin, China.L. casei ΔAlr W56 competent cells, then purified and identified by standard PCR and sequencing. Subsequently, the requirement for D-alanine was verified. As shown in Alr W56 was able to grow in a medium without D-alanine. These results indicated that the recombinant strains were successfully constructed, and the Alr gene in the plasmid could play a similar role to that in the L. casei W56 genome. For constructing the recombinant strains, the plasmid pPG-Alr-COE was transferred into Alr W56, pPG-T7g10-PPT/ΔAlr W56, and ΔAlr W56 were cultured overnight in MRS media with or without D-alanine, and cell lysates were analyzed by western blot assay with an anti-flag monoclonal antibody as the primary antibody  than those in the PBS, pPG-T7g10-PPT/ΔAlr W56, and ΔAlr W56 groups. No significant difference (p > 0.05) was observed in the PBS, pPG-T7g10-PPT/ΔAlr W56, and ΔAlr W56 groups pre- and post-immunization. These results showed that the recombinant bacteria could effectively induce mucosal immunity. To analyze the level of SIgA induced by oral recombinant strain pPG-Alr-COE/ΔAlr W56, sera were collected from the first immunized mice on days 0, 7, 14, 21, 28, 35, 42, 49, 63, and 70 . Expectedly, no significant elicitation of the anti-PEDV IgG antibody was observed in the control groups of mice, including PBS, pPG-T7g10-PPT/ΔAlr W56, and ΔAlr W56. Moreover, a virus neutralization assay was performed to assess the PEDV-neutralizing activity of the sera and intestinal mucosal antibodies stimulated in the immunized mice 40 d post-immunization, as shown in Alr W56 exhibited a stronger anti-PEDV-neutralizing activity than that in mice orally administered with PBS, pPG-T7g10-PPT/ΔAlr W56, and ΔAlr W56. As expected, the group pPG-Alr-COE/ΔAlr W56 expressing the COE antigen could effectively arouse an anti-PEDV immune response in vivo.To measure the IgG level induced by oral recombinant strain pPG-Alr-COE/Δ, and 70 a. Our reAlr W56 was higher than those induced by PBS, pPG-T7g10-PPT/ΔAlr W56, and ΔAlr W56 groups (p < 0.05), and among these three, no significant difference was observed (p > 0.05). The ratio of IL-4 (Th2-type response) to IFN-γ (Th1-type response) is usually applied to analyze the immune level of Th1 or Th2. In the current study, the IL-4/IFN-γ ratio was 1.12  40 d post-first immunization, to detect the cytokine IL-4 and IFN-γ, as shown in was 1.12 , revealiIn recent years, the diarrhea epidemic dominated by the PEDV has occurred in Taiwan, Italy, Germany, and America, of which the mortality reached 100%, causing severe economic losses in the pig industry ,28. In CPEDV infection mainly occurs on the mucosal surface, and the mucosal immune system is the first barrier against viral infections . In piglLactobacillus is often applied as a vaccine delivery vehicle. It owns many advantages, such as the resistance to the harsh GI environment, the powerful capacity for intestinal colonization, and the nonspecific immunoadjuvant effect [Lactobacillus as a live carrier to develop oral mucosal vaccines against pathogens owing to its safeness and nonspecific adjuvant effect [Lactobacillus live vector vaccine to improve the level of immune response and achieve a good immune protection rate [Lactobacillus to orally immunized mice, efficiently inducing anti-PEDV mucosal, humoral, and cellular immune [L. casei W56 as one of Lactobacillus could attack and colonize in intestinal mucosa and has bile tolerance and acid resistance, thus serving as a promising candidate to deliver the antigenic protein to the mucosa [L. casei as an antigen delivery vehicle to immunize mice and obtained a good immune effect [L. casei as an antigen delivery vector to express Rabbit hemorrhagic disease virus (RHDV) capsid protein VP60(VP1)-fused eGFP, and the results indicated that the oral administration of this probiotic vaccine could stimulate SIgA-based mucosal and IgG-based humoral immune responses in rabbits [L. casei ΔAlr W56 to deliver the PEDV S protein-neutralizing epitope COE fragment and the Alr gene and to develop an oral mucosal vaccine against the PEDV and be used as a screen marker in the future. In traditional molecular biology, antibiotic genes are often used as a selectable marker of the plasmid. In addition, although the widespread use of antibiotics is indispensable for human health and social development, the harm has become increasingly significant. To address this, a new selective marker should be used. We have found that deleting the Alr gene may cause bacterial death; in contrast, strain growth is maintained following the exogenous addition of D-alanine [Alr is considered as a candidate to replace the use of antibiotic genes [Alr gene into the recombinant plasmid pPG-COE and electrotransformed it into L. casei ΔAlr W56. The results revealed that the recombinant bacteria are able to grow in an MRS broth without the exogenous addition of D-alanine, while other strains are not, indicating that the Alr gene in the plasmid could complement deficient strains to be applied as a selective marker in the future. In this study, we used the genetically engineered pPG-Alr-COE/ΔAlr W56 as an oral vaccine to immunized mice, eliciting a humoral immune response against PEDV. This was proved by the significant levels of virus-neutralizing antibodies and anti-PEDV serum IgG in mice orally immunized with pPG-Alr-COE/ΔAlr W56, compared to the levels in the PBS, pPG-T7g10-PPT/ΔAlr W56, and ΔAlr W56 groups, demonstrating good immunogenicity and a potential vaccine strategy against PEDV infection. t effect ,34,35. Mt effect ,37. In 2ion rate . Ma et ar immune . L. casee mucosa . Qiao ete effect . Wang et rabbits . In this-alanine ,44. Theric genes . We ligaAlr W56, inducing the mucosal and systemic responses against PEDV. An antigen-specific SIgA antibody in the feces, intestinal mucus, and vaginal and nasal fluid was detected by ELISA post-oral immunization. Our data revealed that there was a noticeable increase of the anti-PEDV-specific mucosal SIgA antibody in these samples induced in the mice orally immunized with pPG-Alr-COE/ΔAlr W56 after a second booster immunization. Thus, the genetically engineered strain pPG-Alr-COE/ΔAlr W56 as an oral vaccine can effectively induce antigen-specific mucosal response against PEDV infection.Mucosal immunity plays an important role in preventing viral diarrheal diseases, and sIgA antibodies are a good way for piglets to acquire immunoprotection . In thisAlr W56, and ΔAlr W56 groups, the levels of the cytokine oral recombinant strain pPG-Alr-COE/ΔAlr W56 significantly increased, indicating that oral recombinant strains stimulate mice to produce Th1- and Th2-cell type immunity. The IL-4/IFN-γ ratios in the experimental groups were higher than that of the control group, revealing that the cellular immunity induced by oral recombinant bacteria pPG-Alr-COE/ΔAlr W56 was mainly Th2-cell-based immunity. Therefore, oral immunization with genetically engineered pPG-Alr-COE/ΔAlr W56 may provide effective protection against PEDV infection.T-helper cells are divided into two major subsets, Th1 and Th2 cells. LAB can induce local mucosal immune responses in the intestine, and an appropriate dose can promote Th0 differentiation and boost B lymphocytes to produce SIgA . CytokinAlr W56 oral vaccine (with an Alr gene in the plasmid) exhibited excellent immunogenicity and can grow without D-alanine. It is worth mentioning that the Alr gene was used as a selective marker in L. casei ΔAlr W56 to avoid the main disadvantage of traditional plasmid expression systems that employ antibiotic resistance genes as a selective pressure for genetically engineered bacteria [Bacillus subtilis [In addition, our results showed that the recombinant pPG-Alr-COE/Δbacteria ,50. Moresubtilis ; thus, tAlr W56 was constructed in this study, providing a candidate replacement for antibiotic genes used as selection marker in the future. We confirmed that the genetically engineered strain pPG-Alr-COE/ΔAlr W56 could efficiently induce mucosal, humoral, and cellular immune responses against the PEDV, suggesting that it may be a promising strategy for vaccine development against PEDV infection. In the future, the immunization and challenge protection experiments would be conducted on the host animal pigs to further evaluate the vaccine value of recombinant strains and lay a foundation for the preparation of new vaccines.In conclusion, an oral mucosal vaccine pPG-Alr-COE/Δ


P. pastoris were constructed, one of which produced authentic recombinant human granulocyte-macrophage colony-stimulating factor (ryGM-CSF), and the other was a chimera consisting of ryGM-CSF genetically fused with mature human apolipoprotein A-I (ApoA-I) (ryGM-CSF-ApoA-I). Both forms of the cytokine were secreted into the culture medium. The proteins’ yield during cultivation in flasks was 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Both forms of recombinant GM-CSF stimulated the proliferation of human TF-1 erythroleukemia cells; however, the amount of chimera required was 10-fold that of authentic GM-CSF to induce a similar proliferative effect. RyGM-CSF exhibited a 2-fold proliferative effect on BFU-E (burst-forming units—erythroid) at a concentration 1.7 fold less than non-glycosylated E. coli-derived GM-CSF. The chimera together with authentic ryGM-CSF increased the number of both erythroid precursors and BMC granulocytes after 48 h of incubation of human bone marrow cells (BMCs). In addition, the chimeric form of ryGM-CSF was more effective at increasing the viability of the total amount of BMCs, decreasing apoptosis compared to the authentic form. ryGM-CSF-ApoA-I normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. The chimera form of GM-CSF exhibited the properties of a multilinear growth factor, modulating the activity of GM-CSF and, perhaps, it may be more suitable for the normalization of granulopoiesis.In this study, two strains of the yeast  GM-CSF is a pleiotropic cytokine that plays a key role in the proliferation and differentiation of many hematopoietic cells, especially monocytes, granulocytes, and dendritic cells . In clinGM-CSF is synthesized by a variety of cells, including lymphocytes, fibroblasts, endothelial cells, monocytes, and some malignant cells . Human GSaccharomyces cerevisiae yeast and Escherichia coli bacteria, respectively. Molgramostim contains non-glycosylated GM-CSF with an additional N-terminal methionine. Its use is accompanied by autoimmune reactions to this cytokine and a high incidence of side effects, as a result of which the drug was not approved by the FDA for use in therapy in the United States [S. cerevisiae yeast as a producer of therapeutic proteins is that they produce hyperglycosylated proteins, in which each oligosaccharide can contain more than 50 mannose residues, as a result of which the biological activity of these proteins is reduced [Currently, there are two recombinant GM-CSF preparations on the pharmaceutical market: Sargramostim and Molgramostim, which contain recombinant GM-CSF produced by d States ,21. Sarg reduced .Pichia pastoris has several advantages for the production of therapeutic proteins: namely, they carry out post-translational protein modifications similar to those in humans; do not produce exo- and endotoxins; provide a high yield of recombinant proteins; and their secretion into the culture medium greatly facilitates the purification of recombinant protein [The methylotrophic yeast  protein .Due to the severe side effects associated with its use, GM-CSF is used in the clinic much less frequently than G-CSF. One of the side effects of GM-CSF is the escalation of inflammation . In addiTo increase the clinical efficacy and reduce the side effects of recombinant GM-CSF, researchers use two main strategies for its modification: covalent binding to PEG (pegylation) ,27 and tThe fusion construct is a chimeric molecule containing rGM-CSF fused to a recombinant protein that often serves two functions. First, it has beneficial properties and in a functional sense complements the cytokine; secondly, it ensures protection of the cytokine from the action of proteases and thus prolongs the activity of the cytokine in the human body.Currently, there are many developments of this type of construction that can be widely used in clinical medicine: GM-CSF fused to IL-3 is used to stimulate hematopoiesis ; GM-CSF In this study, we have obtained recombinant human GM-CSF fused to human ApoA-I. The choice of ApoA-I was not accidental, since this protein performs many functions in the body, such as antiatherogenic , antiapoThis protein is involved in various anti-inflammatory strategies of the body, suppressing the TLR-mediated secretion of the pro-inflammatory cytokines IL-6 and TNF in monocytes . By affeTherefore, the production of a recombinant GM-CSF-ApoA-I chimera might mitigate the toxic effect of GM-CSF on target cells, both due to the anti-inflammatory properties of ApoA-I in the chimera and due to the expected more prolonged action of the chimera in the blood, consequently decreasing its dose. Additionally, it is not known whether the biological activity of the cytokine will be fully preserved. In the literature, there are examples of a decrease in the biological activity of cytokines in the development of fusion hybrid constructs ,50,51. PP. pastoris X33 strains were obtained. The biological activities of authentic and chimeric GM-CSF forms were studied using the human TF-1 erythroid line and human bone marrow cells.In connection with the previous research, it was of interest to compare the biological activities of recombinant authentic and chimeric forms of human GM-CSF. In this work, both forms of recombinant human GM-CSF produced by P. pastoris cells and designated as ryGM-CSF was synthesized by DNA2.0 (USA) and cloned into plasmid pD912 at the XhoI and SalI sites. We used the plasmid pD912/ryGM-CSF as the source of the ryGM-CSF gene to construct the recombinant plasmids pPICZα-A-rhGM-CSF and pPICZα-A/rhGM-CSF-link-ApoA-I.At the first stage of our work, we constructed recombinant plasmids encoding an authentic and chimeric form of mature human GM-CSF. The synthetic gene of mature human GM-CSF optimized for expression in E. coli Top10 cells.The recombinant plasmid pD912/ryGM-CSF was digested with restriction endonucleases either XhoI and SalI or XhoI and KpnI. The excised ryGM-CSF gene was inserted into the pPICZαA plasmid at the XhoI and SalI sites and cloned into E. coli Top10 competent cells. The scheme of the constructed recombinant plasmid pPICZαA/ryGM-CSF-link-ApoA-I is shown in In parallel, the previously constructed plasmid pPICZαA/IFN-link-ApoA-I was cleaved at the XhoI and KpnI sites, and thus, the IFN gene was excised from it. The ryGM-CSF gene was inserted into the cleaved plasmid at the XhoI and KpnI sites, and the resulting recombinant plasmid was transformed into http://raptorx.uchicago.edu/, accessed on 6 May 2021). As seen in Plasmid pPICZαA/ryGM-CSF-link-ApoA-I was designed to express the chimeric protein ryGM-CSF-ApoA-I. To determine if the ryGM-CSF function would be potentially impaired due to a possible change in the conformation of cytokines in the chimera, a hypothetical 3D model was built using the RaptorX protein structure prediction server  non-ionic detergent Tween 20 to the culture medium would affect the expression efficiency of both authentic and chimeric ryGM-CSF in our case. The addition of 0.2% (w/v) Tween 20 increased the yield of ryGM-CSF and ryGM-CSF-ApoA-I by 2.5–3 times (figures not shown). In this regard, the preparative production of recombinant cytokines was carried out by culturing the corresponding strains in the presence of 0.2% (w/v) Tween 20.Clones No # 7 and No 3, producing the highest amounts of ryGM-CSF and ryGM-CSF-ApoA-I, respectively, were selected for preparative production and purification of these cytokines. Notably, according to reports ,53, the Selected yeast clones producing the highest amounts of ryGM-CSF and ryGM-CSF-ApoA-I were grown in conical flasks with deflectors in BMGY medium on an orbital shaker at 25 °C and 250 rpm. During cultivation in the presence of the inducer, daily aliquots of the culture liquid were sampled and analyzed by electrophoresis in SDS-PAGE to assess the dynamics of synthesis and secretion of recombinant cytokines . At the The erythroid-stimulating activity of ryGM-CSF and ryGM-CSF-ApoA-I was evaluated in human TF-1 erythroleukemia cells. GM-CSF more strongly supports the proliferation and viability of myeloid progenitors due to the direct proliferative stimulus on all progenitors and precursors of the granulomonopoietic lineage. GM-CSF is also a powerful stimulator of burst forming unit-erythroid (BFU-E) and megakaryocyte colony forming unit (CFU-MK) proliferation.E. coli cells (GM-CSF), was used as a reference protein for quantitative evaluation [In this regard, the biological activity of recombinant yeast GM-CSF (ryGM-CSF) was assessed using a culture of human erythroleukemia cells TF-1, which is GM-CSF-dependent and proliferates in the presence of GM-CSF and other human cytokines. rGM-CSF, which was expressed in aluation . The expThus, ryGM-CSF is a more potent stimulator of the proliferation of erythroid explosive units (BFU-E) compared to the control rGM-CSF, exhibiting 1.7-fold greater biological activity against TF-1 cells. At the same time, ryGM-CSF-ApoA-I activated the proliferation of TF-1 cells, but it exhibited less activity compared to rGM-CSF and to ryGM-CSF bacterial preparation and to yeast GM-CSF. A 2-fold proliferative effect on TF-1 cells was achieved at a concentration of 0.62 ng/mL.Recombinant GM-CSF is most widely used in the prevention of neutropenia and neutropenic complications in patients with a reduced number of neutrophils in the blood. In this regard, the bone marrow of a person with a reduced level of neutrophils and impaired maturation was taken as a sample for the study of the recombinant growth factors.BMC were incubated with growth factors for 48 h. The effect of ryGM-CSF and ryGM-CSF-ApoA-I was analyzed by flow cytometry . Under t2M phases) −19%) compared to ryGM-CSF (12%) and the control (8%)  and ryGM-CSF-ApoA-I  I. Analysrol (8%) II.Additionally, the percentage of apoptotic cells decreased to 21% under the influence of ryGM-CSF-ApoA-I, to 34% in the case of GM-CSF, and to 38% in the control after 48 h of incubation. A more significant decrease in apoptosis and an increase in the level of proliferation in the presence of ryGM-CSF-ApoA-I provides an increase in the total number of viable cells in comparison with both the authentic form and the control .Notably, in this case, the additive effect of ApoA-I and GM-CSF was likely manifested. ApoA-I is known to increase cell viability, although in these cases, it was used in microgram amounts. During our research, both factors were used in nanogram amounts. This underlines that both recombinant proteins exhibit the properties of growth factors.For a more complete understanding of the effect of growth factors, human BMC smears were prepared and stained and the number of cells was counted: 500 per glass.Analysis of stained smears of human BMC samples showed that in the control, after 24 h of incubation, the number of cells of the granulocyte line was reduced and amounted to 27% of the total BMC population.The general decrease in the number of cells of the granulocytic series in the control was accompanied by an increase in the number of cells of the monocytic series: monocytes, macrophages and stromal cells. Both factors increased the number of granulocyte cells significantly (to 51 ± 6%) (data taken from complete myelogram). Notably, the control samples also contained an increased number of blast forms of granulocyte cells (8.8 ± 0.3%) and an increased number of hyposegmented neutrophils (26 ± 0.35%). Growth factors significantly increased the number of normally segmented neutrophils, reducing the percentage of cells with abnormal segmentation after 24 h of incubation .To visualize the dynamics of changes (24–48 h) in the cellular composition of granulocyte cells in the experimental and control samples, the cells were divided into groups:(a) blast forms of granulocytic cells ; (b) young forms ; (c) maturing cells ; (d) mature forms of granulocytes (segmented neutrophils) .As can be seen from The dynamics of cell maturation in control samples at 48 h of incubation did not change compared to 24 h. In the control at 48 h of incubation, a significant reduction in all forms of blast cells, including granulocytic cells and an increase in the level of apoptosis, was noted.The data were consistent with the results of flow cytometry, where there was an increased apoptosis, an increase in cell debris, and a low level of proliferation A of contIn the presence of ryGM-CSF, a significant increase in the number of mature granulocytes after 1 day of incubation was accompanied by a further decrease in the number of blast forms of the granulocyte on the second day of incubation. The chimeric form stimulated the growth of granulocyte cells a similar way during 24 h of incubation while maintaining the number of blast forms on the second day of incubation (increase from 2.5% to 3.2%) .This agreed with the increased level of human BMC proliferation during incubation (48 h) in the presence of the chimeric form as assessed by flow cytometry B,C. AccoThe number of erythroid blast cells under the influence of both factors became equal and amounted to (6.1 ± 0.3%) after 48 h of incubation. The authentic form demonstrated the full spectrum of multilinear differentiation; there was also a small number of megakaryoblasts. At the same time, these cells were not observed in the presence of a chimeric cytokine. The chimera well supported the viability of the reticular–stromal system of the bone marrow. This effect may have been due to the presence of ApoA-I in the chimera . TherefoRecombinant GM-CSF is used in clinical practice for the treatment of a wide range of hematopoietic diseases, including chemotherapy-induced neutropenia, and it is also being investigated for the treatment of several other diseases . A significant limitation of its use is its high toxicity and short half-life in humans. The toxicity of GM-CSF is associated with the activation of macrophages and the synthesis of pro-inflammatory cytokines.GM-CSF produced by bacterial cells is also highly toxic and immunogenic due to the presence of additional N-terminal methionine and impurities of LPS-producing cells, which are very difficult to purify. One of the possible approaches to solving the problem of GM-CSF toxicity is the creation of its potentially prolonged form by fusion with other proteins.P. pastoris methylotrophic yeast. One of the main advantages of heterologous gene expression in these microorganisms over bacterial expression systems is the inherent ability of methylotrophic yeast to secrete synthesized heterologous proteins and glycosylate them in much the same way as in humans [We have obtained authentic GM-CSF as well as its chimeric form by expression in the n humans .Glycosylated rhGM-CSF has a better pharmacokinetic profile than bacterial (non-glycosylated) rhGM-CSF, which is characterized by a relatively stable plasma concentration . Stable We have selected human apolipoprotein A-I as the fusion protein. A flexible oligopeptide linker was provided between the pair of fusion proteins, which in our case made it possible to preserve the activity inherent in GM-CSF, with modulation of its properties by ApoA-I. Notably, the creation of chimeric proteins is often accompanied by a significant decrease in the biological activity of the cytokine, despite the presence of linkers in the chimera.Preparative production of recombinant proteins was carried out on a thermostatic shaker in flasks. The protein yield during cultivation was about 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Considering that protein production took place under non-optimized conditions, it can be expected that fermentation in the bioreactor would significantly increase the yield of recombinant proteins.P. pastoris, which was studied on cytokine-dependent cells of human erythroleukemia TF-1, was higher than that of the chimeric form of ryGM-CSF-ApoA-I and the recombinant GM-CSF synthesized in E. coli.The study of the biological activity of the obtained cytokines was carried out both on a standard human erythroleukemia cell line TF-1 and on human bone marrow cells. Hematopoietic growth factor GM-CSF, which ensures the proliferation of several hematopoietic germs, is also a potent stimulator of erythroid proliferation (BFU-E activity), but it does not affect more mature erythroid cells . Our stuAnalysis of the human BMC myelogram showed four times more proerythroblasts in samples stimulated for 24 h with ryGM-CSF compared to samples stimulated with ryGM-CSF-ApoA-I. However, on the second day, the number of erythroid progenitors was equal in both BMC samples. Transformed TF-1 cells have an unlimited number of divisions, while in the bone marrow, there are fewer progenitors and more different factors to maintain their viability and proliferation are required. Under these conditions, the chimeric cytokine was sufficient to soft activate erythroid progenitors within 48 h, while authentic ryGM-CSF exhausted this potential within 24 h. Slower activation of erythroid units in the presence of a chimera can be explained by steric hindrances of its interaction with receptors on erythroid cells.Considering that ApoA-I itself, used in microgram amounts, stimulates the proliferation of granulo/monocytic progenitor cells and maintains their viability , it can Notably, the property of ApoA-I to support cell viability and proliferation was manifested in a chimeric form, which was taken in ng quantities, while ApoA-I per se did not show this activity in such a quantity. The property of maintaining proliferation, reducing apoptotic cell death, and preserving the pool of progenitors was most fully manifested at 48 h of incubation. The chimeric form better supported the viability of human BMC, promoting the maturation of cells of the granulocyte series and reducing the number of hyposegmented neutrophils more effectively than in the control or in samples treated with ryGM-CSF. The obtained chimera retained the ability of the authentic factor to support the cells of the erythroid lineage. According to the results obtained, the chimera can be considered as a new multilinear factor with GM-CSF activity, which is supplemented by the ability to maintain cell survival. The property of a chimeric protein to increase cell viability may be especially important for the stimulation of hematopoiesis after chemotherapy and radiotherapy. Cells stimulated to proliferation are highly sensitive to unfavorable environmental factors, and a decrease in apoptotic cell death is essential for therapy with growth-stimulating factors. In this case, the ability of the chimera to increase the viability of proliferating cells in adverse conditions is relevant for possible use in the therapy of myelosuppression of cancer patients.P. pastoris yeast cells: authentic ryG-CSF and ryG-CSF fused to human ApoA-I (ryG-CSF-ApoA-I). The biological activity of these cytokines was tested on rat and human bone marrow cells. Both ryG-CSF and ryG-CSF-ApoA-I stimulated the proliferation of the granulocytic series cells. In addition, the ryG-CSF-ApoA-I chimera exhibited new functions not characteristic of G-CSF; they supported the viability of the lymphocytic lineage cells, as well as macrophage-stromal cells, which, as was previously established, is characteristic of ApoA-I [We have previously obtained two other recombinant human colony stimulation factors produced by f ApoA-I . As mentf ApoA-I . Howeverf ApoA-I ,58 threef ApoA-I .These data indicate that ApoA-I in the composition of the chimera endows the cytokines fused with it with additional new properties that can be useful for the treatment of a number of diseases. It is possible that the additive effect of the ApoA-I protein and the growth factor GM-CSF in the chimera will manifest itself in models of alveolar macrophages. It has recently been suggested that GM-CSF is required for the clearance of cholesterol from alveolar macrophages, with a decrease in this clearance being a major macrophage defect leading to pulmonary alveolar proteinosis . The impThe biological properties of the obtained ryGM-CSF-ApoA-I have yet to be investigated in more detail. Comparison of the pharmacokinetic profiles of authentic recombinant ryGM-CSF and its chimeric form is of great interest. The presence of the ApoA-I protein and glycosylated ryGM-CSF in the chimera likely provides increased stability of the chimeric protein in plasma.E. coli and with bioactivity equivalent to that of commercial rhGM-CSF was kindly provided by Dr. Gileva I.P. Restriction endonucleases were purchased from SibEnzyme . T4 DNA ligase and Phusion DNA-polymerase were purchased from Thermo Fisher Scientific Inc. . Oligonucleotides were purchased from LTD “Biosynthesis” . Yeast extract, bacto peptone, and triptone were purchased from Thermo Fisher Scientific Inc.  and were used for preparations of Luria–Bertani (LB) medium for the growing of E. coli. Yeast culture media  were prepared as specified in the manufacturer’s protocol “EasySelectTMPichia Expression Kit” . RPMI media, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Thermo Fisher Scientific Inc. ; XTT and Propidium Iodide were purchased from Sigma-Aldrich . Sephadex G25, DEAE-Sepharose FF, and SP Sepharose FF ion-exchange resins were purchased from GE Healthcare Bioscience . PVDF transfer membrane were purchased from Thermo Fisher Scientific Inc, . The water used in this study was deionized and autoclaved.The non-glycosylated human GM-CSF with a methionine residue at the N-terminus produced in Escherichia coli str. TOP10, Pichia pastoris str. X33, and the pPICZα-A were purchased from Invitrogen Inc. The recombinant pPICZα-A/hIFN-link-ApoA-I plasmid was constructed earlier in our laboratory. E. coli str. TOP10 was used for the cloning and propagation of synthetic human GM-CSF in pPICZa-A and pPICZα-A/hIFN-link-ApoA-I plasmids. Pichia pastoris str. X33 was used for cloning pPICZa-A/hGM-CSF and pPICZa-A/hGM-CSF-link-ApoA-I plasmids and expression and production of the authentic and chimeric forms of recombinant human GM-CSF.P. pastoris was carried out using the Gene Designer software . The synthetic gene designated as ryGM-CSF contained a XhoI restriction site and a nucleotide sequence encoding a Kex2 proteolysis site and two Ste13 proteolysis sites at the 5’-terminus, and a KpnI restriction site, a stop codon, and restriction sites EcoRI and SalI at the 3’-terminus. The optimized gene was synthesized by ATUM and cloned into plasmid pD912 at the XhoI and SalI sites. The synthesized and cloned sequence of the ryGM-CSF gene was confirmed by sequencing. The recombinant plasmid pD912/GM-CSF was used as a synthetic gene source for mature human GM-CSF.The construction and optimization of the nucleotide sequence encoding the synthetic gene of mature human GM-CSF to ensure its efficient expression in The recombinant plasmid pD912/ryGM-CSF was digested with restriction endonucleases XhoI and SalI and analyzed by electrophoresis in 0.8% agarose gel. A fragment containing the ryGM-CSF gene was eluted from the gel and ligated with T4 DNA ligase with the plasmid pPICZαA, which had preliminarily been cleaved at the XhoI and SalI sites.The pPICZαA/IFN-link-ApoA-I plasmid, constructed previously in our laboratory earlier, was used as the initial vector. The plasmid contained a chimeric gene containing in the 5’-end region the human interferon-α2b (IFN) gene sequence flanked by the XhoI and KpnI restriction sites and fused to a linker encoding the GSSGSGSSGSGSGSSGGSG sequence. The linker was attached with the mature human ApoA-I gene, flanked by EcoRI and SalI restriction sites and located in the 3’-terminal region of the chimera. The IFN gene was removed from the plasmid by digestion with restriction enzymes XhoI and KpnI and replaced with the synthetic ryGM-CSF gene inserted into the same restriction sites using T4 DNA ligase to form the recombinant plasmid pPICZαA/ryGM-CSF-link-ApoA-I.Escherichia coli str. TOP10 electrocompetent cells were transformed with recombinant plasmid pPICZαA/ryGM-CSF or pPICZαA/ryGM-CSF-link-ApoA-I using the electroporator MicroPulser  as described previously Aliquots of 50 and 100 µL of LB medium, containing transformed E. coli str. TOP10 cells were spread onto low salt Luria–Bertani agar plates comprised of 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, 1.8% (w/v) bactoagar and 50 μg/mL of zeocin. Clones grown on agar medium were checked by colony PCR for the presence of recombinant plasmids containing inserts of the synthetic gene ryGM-CSF and the chimeric gene ryGM-CSF-ApoA-I. PCR was performed using the DNA Thermal Cycler BIS  in the presence of forward AOX1-F 5’-GACTGGTTCCAATTGACAAGC-3’ and reverse AOX1-R 5’-CAAATGGCATTCTGACATCC-3’ primers used for the detection and sequencing of genes cloned in pPICZαA plasmid. The sizes of the amplicons were determined by electrophoresis in 0.8% agarose gel stained with ethidium bromide. Selected clones containing the pPICZαA/ryGM-CSF or pPICZαA/ryGM-CSF-link-ApoA-I plasmid were used for their preparative isolation and subsequent cloning in P. pastoris X33 cells.P. pastoris X33 cells using a MicroPulser electroporator . Transformants were grown on YPD agar plates containing 2000 μg/mL zeocin in an incubator at 30 °C for 3–5 days. Then, zeocin-resistant transformants were evaluated for their ability to synthesize and secrete the recombinant cytokines ryGM-CSF or ryGM-CSF-ApoA-I by culturing the clones in 96-well plates (2 mL square wells). The cultivation was carried out in BMGY  medium on an orbital shaker at 300 rpm for 60 h at 28°C. Then, 250 μL of BMM2 (same as BMGY with 1% methanol instead of glycerol) was added to each well. Over the next three days, 50 μL BMM10 (same as BMGY with 5% methanol instead of glycerol) was added to the wells.Plasmids pPICZαA/ryGM-CSF and pPICZαA/ryGM-CSF-link-ApoA-I were digested with restriction endonuclease BstXI, and their linearized forms were used to transform electrocompetent On the 4th day of induction, cells from the cultures of each well were pelleted by centrifugation. Proteins from selected supernatants were precipitated with 10% TCA and analyzed for the presence of the target protein by SDS-PAGE electrophoresis. Clones producing the largest number of proteins with a molecular weight close to the molecular weight of the target proteins ryGM-CSF and ryGM-CSF-ApoA-I were used for subsequent studies. The antigenic nature of the ryGM-CSF-ApoA-I chimera was determined by Western blotting using rabbit anti-ApoA-I IgG .w/v) Tween 20 was added to the culture medium, and 1% methanol was added daily On the 4th day after induction, the cells were pelleted by centrifugation; then, ammonium sulfate was added to the supernatant containing the target protein to a concentration of 75% saturation and incubated at 4 °C overnight. Protein precipitates were harvested by centrifugation at 39,000 g using a JA20 rotor in a J2–21 centrifuge  for 30 min at 4 °C.All procedures for growing yeast clones were performed as described previously. Selected clones were grown in 50 mL of BMGY medium for two days on an orbital shaker Unimax 2010  at 250 rpm and 28 °C. Then, the culture was transferred to 100 mL of BMGY medium in 500 mL conical baffled flask and grown in an orbital shaker at 25 °C and 250 rpm. Then, 0.2%  was loaded onto an SP Sepharose FF column equilibrated with the same buffer. The column was washed with buffer A, and the protein was eluted with a NaCl gradient (0–0.5 M). Fractions were analyzed by electrophoresis on 12% PAGE. Fractions containing the highest amount of purified target protein were pooled and dialyzed against buffer B, containing 10 mM sodium phosphate and 1 mM EDTA, (pH 7.5). The dialyzed solution was loaded to a DEAE-Sepharose column equilibrated with buffer B. The column was washed with buffer B and the protein was eluted with a NaCl gradient (0–0.25 M). All fractions were analyzed using SDS-PAGE (12%). The fractions containing the highest amount of the target protein were pooled and stored in 100 μL aliquots at −70 °C until use.The pellet, containing mainly the ryGM-CSF-ApoA-I chimera, was dissolved in buffer A. The solution was desalted on a Sephadex G25 column. The demineralized chimera solution was loaded onto a DEAE-Sepharose column equilibrated with the buffer B. Unbound to resin material was collected and loaded to an SP Sepharose FF column equilibrated with the buffer B. Then, the column was washed with buffer B and the protein was eluted with a NaCl gradient (0–0.5 M). Aliquots of the eluted fractions were analyzed by electrophoresis on 12% PAGE. Fractions containing the highest amount of chimeric protein were pooled and stored in 100 μL aliquots at −70 °C until use.The concentrations of ryGM-CSF and ryGM-CSF-ApoA-I were measured spectrophotometrically by UV absorption at a wavelength of 280 nm, considering their molar extinction coefficients as well as densitometry of cytokine bands stained with Coomassie brilliant blue in a gel after SDS-PAGE versus standard human serum albumin (HSA) titration bands using analytical imaging software Gel-Pro analyzer .E. coli cells was used [4 cells/well in the culture medium with the investigated amounts of the cytokines and incubated for 72 h at 37 °C, 5% CO2, and 85% humidity. Cells incubated without added cytokines served as negative controls. The optical density was determined on a SpectraCount plate spectrophotometer (Packard) OP 490/620. The stimulation of proliferation of TF-1 cells was calculated as a percentage relative to the control. The number of living cells in the negative control was taken as 100%. All calculations were performed using the LabView software.The biological activity of ryGM-CSF and ryGM-CSF-ApoA-I was evaluated by the level of stimulation of human erythroleukemia cells TF-1 proliferation using 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilid (XTT) (Sigma-Aldrich). To calibrate the activity of both forms of recombinant human yeast GM-CSF (ryGM-CSF and ryGM-CSF-ApoA-I), a recombinant GM-CSF preparation obtained by expression in was used . We prep2, 20% O2 at humidified atmosphere). BMCs were cultivated in 24-well culture plates at a density of 2 × 106 cells/mL in the presence of the ryGM-CSF or ryGM-CSF-ApoA-I (50 ng/mL) for 24 and 48 h. Cells cultured under the same conditions, but without additives of cytokines, were used as a control.In this study, we used human bone marrow cells (BMC) taken from the culture bank of Research Institute of Clinical and Experimental Lymphology—Branch of the ICG SB RAS (RICEL–branch ICG SB RAS). Cells were cultured in cell culture medium RPMI (Thermo Fisher Scientific Inc.) with 10% fetal bovine serum (FBS) under standard cultivation condition  as described previously . Cells tBone marrow smears were prepared and stained with May-Grunwald and Giemsa dyes. Stained cells were assessed microscopically ), and 500 BMCs per smear were counted.P. pastoris strains that efficiently produce both authentic and chimeric forms of human GM-CSF were obtained. The chimera ryGM-CSF-ApoA-I exhibited the properties of a growth factor, demonstrating the activity of GM-CSF, increasing the number of cells of the granulocyte lineage on BMC and BFU-E.At the same time, ryGM-CSF-ApoA-I increased the viability of the total amount of BMC and reduced apoptotic cell death more efficiently than ryGM-CSF. The chimeric form normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. These results indicate the modulating effect of ApoA-I in the composition of the chimera on the biological activity of GM-CSF and the advisability of a more in-depth study of the properties of the chimeric cytokine.


Perineal ectopic testis is a rare congenital abnormality mostly diagnosed during childhood period. The diagnosis can be easily reached by physical examination. The treatment of choice is either orchidopexy or orchiectomy through scrotal or inguinal approach.We report a case of 30 year old male with painful perineal mass and empty right hemiscrotum. Ultrasound of the mass was done prior to operative procedure and the ectopic testis was the working diagnosis ever since. At exploration there was no abnormality found on the testis; thus, orchidopexy was done with uneventful postoperative period.Ectopic testis is rare congenital anomaly with perineal ectopic testis accounting for only 1% of all cases. Most cases of ectopic testis are diagnosed during childhood period. Our case presented at the age of 30 years, the reason could be either late diagnosis by physician or lack of insight of parents/care takers or both. The late presentation might pose a diagnostic challenge but also the testis may be atrophic or undergo malignant changes. In this case the testis was normal and therefore it was successfully relocated surgically through trans-inguinal approach.It is suggested to make perineal ectopic testis one of the differential diagnoses on painful perineal swelling without ipsilateral testis on any man. •Perineal ectopic testis is a very rare anomaly that represents a true ectopic location of the tests.•It is more rare in adults and young boys.•Diagnosis depends on level of suspicious index as it is mostly clinical.•Any man presenting with painful perineal swelling in the setting of absent testis should be regarded as having perineal ectopic testis until proved other wise.•Trans-inguinal orchidopex offers a safe way of managing perineal ectopic testis as the testis is usually of normal size and the spermatic cord is of adequate length even in adults. Later on when he was 15 years, he noticed a perineal swelling that was progressively increase in size. The swelling was initially painless but started to be painful when sitting and it was progressively increasing in severity with time for about one year. However, there was no history of increase in size or pain on standing position, fevers or nausea/vomiting. The patient reported to have no voiding or sexual function problems. He further denied history of any surgery or being on any medication regularly as well as history of similar problem in the family. Upon examination he was noted to have hypoplastic right hemiscrotum  while th3Testicular descent is a complex process that is influenced by several factors including hormonal and mechanical factors Abnormality in the process of decent can result into abnormally located testis along the normal part of descent referring to as undescended testis or location of the testis outside its normal path of descent which is called ectopic testis In children, perineal ectopic testis may be found on the lower part of the scrotum Delay in diagnosis and treatment may lead into testicular atrophy, trauma, cancer, torsion and infertility in cases of bilateral ectopia 4Perineal testis is very rare congenital anomaly of boys. The diagnosis is clinical based on the absence of testis in the scrotum and perineal location of oval shaped structure. Orchidopexy is the treatment of choice in cases of normal testicular size and consistence that gives good immediate postoperative outcome. Any man presenting with painful perineal swelling in the absence of testis in the scrotum should be examined thoroughly including the perineum to exclude possibility of perineal ectopic testis.There was no any financial support received from individual or company.Ethical standards were reviewed and approved by urology team.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.N/ABartholomeo Nicholaus.BNN, JSM, FB, BTM and KAM reviewed the patient and planned the treatment. BNN, MM and EL operated the patient and followed him post operatively. BNN and OJM conceptualized and prepared the first manuscript. All authors read and approved the final manuscript.All authors declare that they have no conflict of interest regarding the information presented in this case report.


Dear Editor,The brain-derived beta-trace protein (BTP), which is synthesized mainly in the epithelial cells of the choroid plexus, is a low molecular weight  monomeric glycoprotein of the lipocalin protein family  in acute and chronic kidney disease  is a reliable and widely accepted surrogate marker of the blood CSF-barrier integrity . Howeveter, 2001). NevertTherefore, the present study aimed to analyze serum/CSF concentrations of BTP and albumin in patients with diagnostic lumbar punctures. Possible associations between serum and CSF BTP concentrations were assessed in individuals sub-grouped by the presence or absence of acute-phase protein and blood-CSF barrier dysfunction.A total of 253 consecutive medical records of patients, who received diagnostic lumbar puncture with assessment for CSF and serum BTP concentrations, blood-CSF barrier function and acute-phase reaction at the Institute of Clinical Chemistry and Laboratory Medicine of the General Hospital Hochsteiermark , were retrospectively examined. Patients < 18 years of age and individuals with acute or chronic kidney diseases were excluded. All patients provided informed consent. The ethical approval of this study was obtained from the Ethical Committee of the Medical University Graz . The study was carried out in accordance with the current version of the Declaration of Helsinki.®  Z Serum Clot activator tubes and 2 mL VACUETTE®  Z No Additive tubes . Venous blood and CSF samples were collected simultaneously in sterile 5 mL VACUETTE® NEPH 630 system . The intra- and inter-day coefficients of variation (CVs) ranged between 2.4 - 6.5 and 2.4 - 6.1 %. The serum (reference range: 35 - 52 g/L) and CSF (reference range: ≤ 350 mg/L) concentrations of albumin were also determined by nephelometric method on the Atellica® NEPH 630 system . The intra- and inter-day CVs were between 2.7 - 3.1 and 1.7 - 3.5 %. To assess the function of the blood-CSF barrier, the QALB was calculated   and serum (reference range: ≤ 0.7 mg/L), the nephelometric N Latex BTP assay  was performed on the Atellicaal., 2001). The up® 8000 c701 analyzer . The estimated glomerular filtration rate (GFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation  and the creatinine (Jaffe) were measured on a cobasal., 2009).Normal distribution of the data was examined with the Kolmogorov-Smirnov test. As continuous variables were not normally distributed, they were expressed as medians with interquartile ranges (Q1 - Q3). Categorical variables were expressed as percentages. Spearman's rho (ρ) was calculated to assess possible positive or negative correlations between study parameters. Linear regression models were performed to assess the association between variables. The exact Mann-Whitney U test was calculated for subgroup comparisons of not normally distributed metric variables. A p-value < 0.05 was considered statistically significant. The statistical analyses were performed using Analyse-it® software version 4.92 .Table 1ALB . There was a negative correlation between serum BTP concentrations and the CKD-EPI-GFR .There was a positive correlation between serum BTP and CSF BTP , between serum BTP and age , and between serum BTP and creatinine . Serum BTP concentrations correlated also with the QALB had a statistically relevant influence on the serum BTP concentration .The univariate regression models between serum and CSF BTP are presented in Figure 1As demonstrated in Figure 1DIn the present study, serum and CSF BTP concentrations were assessed at the blood-CSF barrier in 253 CSF/serum pairs obtained from clinical routine. The serum BTP levels were positively correlated with the CSF BTP levels and in the linear regression model CSF BTP showed a significant influence on serum BTP. Patients with acute-phase reaction and blood-CSF barrier were observed with a much stronger association between serum and CSF BTP compared to individuals without acute-phase reaction and normal blood-CSF barrier function. ALB) and age. Albumin- and age-related increase of CSF BTP concentrations were also reported by Tumani et al.  and 0.57 (0.49 - 0.66) mg/L. These results are in line with a previous study by Tumani et al., who measured mean CSF and serum BTP concentrations of 14.6 ± 4.6 and 0.46 ± 0.13 mg/L in 40 adult control patients . In theal., 1998). A posiHere, we observed higher serum BTP and CSF BTP levels in individuals with an acute-phase reaction compared to subjects without acute-phase reaction. These results indicate that inflammation may influence the BTP status in the brain and in the blood circulation. A previously published study reported that the choroid plexus, which synthesizes the BTP, is a relevant mediator of immune signals between the peripheral blood and the brain . An acuAn acute-phase reaction may not only influence the BTP production in the choroid plexus but also the CSF flow rate . CSF BTA previous study, which investigated CSF and serum BTP concentrations in 35 patients before and after elective knee replacements, could not found any significant BTP changes during and after the surgery  . RecentThe strength of this study is the high number of consecutive serum/CSF BTP pairs obtained from neurological patients out of daily clinical routine. Nevertheless, some limitations of this work should be addressed: the data do not allow to draw conclusions about possible links between CSF or serum BTP concentrations and neurological conditions. Because of CSF dynamics of brain-derived proteins, the exact relationship of the lumbar BTP, where CSF samples are collected, and the site of BTP synthesis in the brain is not fully possible to evaluate .


The use of surface electromyography (sEMG) is rapidly spreading, from robotic prostheses and muscle computer interfaces to rehabilitation devices controlled by residual muscular activities. In this context, sEMG-based gesture recognition plays an enabling role in controlling prosthetics and devices in real-life settings. Our work aimed at developing a low-cost, print-and-play platform to acquire and analyse sEMG signals that can be arranged in a fully customized way, depending on the application and the users’ needs. We produced 8-channel sEMG matrices to measure the muscular activity of the forearm using innovative nanoparticle-based inks to print the sensors embedded into each matrix using a commercial inkjet printer. Then, we acquired the multi-channel sEMG data from 12 participants while repeatedly performing twelve standard finger movements (six extensions and six flexions). Our results showed that inkjet printing-based sEMG signals ensured significant similarity values across repetitions in every participant, a large enough difference between movements (dissimilarity index above 0.2), and an overall classification accuracy of 93–95% for flexion and extension, respectively. Silver nanoparticle (AgNP) based inks are the most abundant in the market and easy to produce, and for these reasons, they have been extensively used for rapid prototyping of sEMG electrodes7. Many other nanomaterials emerged in the last years with very promising electrical properties, for instance, graphene, silver nanowires, and carbon nanotubes. However, when it comes to fabrication, they all show significant limitations like lack of batch-to-batch reproducibility, limited conductivity, instability, and typically high costs. Commercial AgNPs inks conversely are very stable and reproducible, guaranteeing higher performances. Although this technology has opened new possibilities for neuroscience to measure sEMG during real-life behaviour8, its employment is still limited due to complicated fabrication procedures, expensive equipment materials, and, lastly, strong preparation and expertise9. Excellent results have recently been obtained with this approach, for example, using sEMG matrices for human–machine interface in patients with loss of voice; these applications are of interest for voice synthesis and virtual interaction10. However, as demonstrated by that one and several other works two main points has to be considered for human machine interface. First, the virtual/electronic/mechanic actuation of the classification results is not the bottleneck of the human–machine interface (as we detail in the outlook)13. Second, the transduction of the signal is still critical, requiring complicated and expensive fabrication methods, often based on clean-room equipment and long protocols, and still not directly implementable/customizable by the clinician.Surface electromyography (sEMG) represents a classical and non-invasive approach to investigate the integrity and the functionality of muscular activity. sEMG is typically acquired using two electrodes placed over the skin, measuring the voltage difference between the two locations above the target muscle. sEMG can acquire muscular activity from any muscle during motor tasks, i.e., grasping an object, limb movements, and even gait. Its usage is rapidly evolving, especially for bioengineering applications, including robotic prostheses, interfaces for muscle computer interface (MCI), and rehabilitation devices controlled by residual muscular activities. Combined with the advantages offered by printed sensors and electronics, i.e., the ultra-short design-to-prototype time and the customization, sEMG is now rapidly evolving from a laboratory setting to wearable applications14, we investigated the reliability of inkjet-printed electrodes for sEMG compared with Ag/AgCl standard electrodes. We analyzed the fatigue induced by the repeated lifting of loads with variable weights by measuring the individual maximal voluntary isometric contraction (MVIC) value in six participants. In line with sEMG clinical guidelines, MVIC, i.e., the maximal muscular activation exerted by a subject in a specific motor task, was measured from each individual and used to normalize his/her sEMG activity to compare fatigue across different subjects fairly. Results showed that inkjet-printed electrodes were reliable for recording sEMG activity with accuracy and signal quality comparable with standard electrodes.Here we propose a paradigm shift in the prototyping and production of sEMG printable sensors introducing a “print and play” fabrication technology. With this approach, any laboratory can exploit the ultra-rapid and low-cost fabrication of customizable systems for electrophysiological measurements, even in the absence of specific expertise. The idea is to provide clinicians with a fabrication platform constituted by a set of easy-to-use tools. This platform will allow users to focus on the applications and be independent of the fabrication of the sensors. With this aim, we are able to optimize every step of the process to keep the costs low and all the materials commercially available . In a preliminary study15  a similarity index across signals of different repetitions of the same gesture in every subject, (ii) a dissimilarity metric between different gestures in every subject, and (iii) the classification performance of three different classifiers that were trained to recognize gesture-specific sEMG patterns across subjects.Based on these findings, in this study, we used the same printed sensors while subjects performed several standard gestures, i.e., extension and flexion of the fingers, as in a previous workFor both extension and flexion tasks, we found high similarity values across repetitions for most of the gestures and the majority of our subjects; moreover, the dissimilarity between gestures was large enough to distinguish them. Finally, these findings were confirmed by very high classification accuracies , as well as high values of precision  and recall  for all gestures. Therefore, we proved the robustness of our inkjet-based sEMG measurements, thus paving the road to a new, more flexible way to acquire sEMG for movement control and gesture recognition applications.This section will report the qualitative observations on the quality of the inkjet-printed sEMG signals, the outcomes from the inter-trials and inter-subjects variability, and the most relevant classification performance.18.First, we evaluated the quality of raw data by visual inspection of the bubble plots, i.e., a brand-new representation showing for each participant and movement the average and standard deviation of the root mean square (RMS) values across repetitions. RMS was chosen among the available features since it is often used as a signal activity indexFigure We compared the signal amplitude differences during the task and rest phase for each repetition of the movements for each participant. Amplitude differences of at least one order of magnitude were found (not reported here for space constraints).We observed a shift toward low frequency in the rest condition completely lost after 100 Hz, mainly due to membrane potential effects, DC offset, sensor movements, and temperature. A more powerful PSD was noticed between 50 and 150 Hz during the task. In addition to spectral analysis, we assessed the average power in the signal estimating the integral of the PSD over the frequency band 15–250 Hz, evaluating power values along with the repetitions, and comparing them with the rest phases. We detected consistent amplitude differences between the task and the rest phase from spectral analysis and average power.Figure The statistical analysis carried out confirmed the significance of the similarity across the repetitions with a p-value < 0.05, for each subject and each task, except for the subject 11 task 4 (little finger flexion) for the flexion tasks  and the subject 6 task 4 (little finger extension), for the extension task .The analysis of the intra-task variability, i.e., the similarity with which different subjects performed a specific task, showed good consistency across subjects for task 3 (ring finger flexion) and 5 (thumb adduction) of the flexion tasks, with a coefficient of similarity higher than 0.75. For the extensor, tasks 2 (index extension), 3 (middle extension), and 6 (thumb abduction) verified the same condition.Figure Overall, for both extension and flexion sessions, the subset of tasks 4, 5, 6 is distinguishable from subset 1, 2, 3, while the dissimilarity coefficient between tasks 5 and 6 (both including the thumb) is below the established threshold for almost all subjects.The confusion matrices obtained during the multi-class classification problems, i.e., for extension and flexion sessions, separately, are shown in Fig. Figure 22. In particular, in one of these works, the authors acquired sEMG using an 8-channels MYO band and classified five finger movements and rest periods using several classifiers, including kNN and SVM, reaching a maximal accuracy of 95.8%22. They also reported accuracies in the range 66–92% from previous related literature. We found relatively low SNR values as in Shaabana et al. 201923. Nevertheless, we were able to find satisfactory classification performance. Accuracy reaches satisfactory levels, but precision and recall were also found above 90% for most fingers with this type of classifier. This also is in line with Shaabana et al., where precision and recall were found in the range 64–95%.For the extension session, we obtained an average accuracy value of 95% for DA, 90% for SVM, and 72% for kNN. We also computed the average precision for every classifier, ranging from 86.8% to 100% for DA, 81.8% to 100% for the SVM, and 59.5% to 96.6% for kNN, depending on the specific gesture. Similarly, the average recall ranges from 84.8% to 100% for DA, 81.8% to 100% for SVM, and 63.6% to 84.8% for kNN, depending on the specific gesture. Therefore, the classification of the five different fingers reaches accuracies above 90%, comparable with the most recent literatureWe have proved the feasibility of a fatigue detection study using the same inkjet-printed technology. Interestingly, we could classify with good levels of accuracy the same gestures across different subjects without having assessed their MVC. This could be useful in the case of patients that cannot exert MVC or in case real-time acquisition is performed without preliminary calibration on MVC.28, in this research work, we introduced a detailed investigation of the quality of the sEMG signals we acquired via inkjet-printing. In the most recent studies34, however, more attention has been dedicated to evaluating the repeatability of a sEMG pattern during the same task. In Pizzolato et al.34, the signal amplitude was analyzed across days and compared through six different sEMG setups. This analysis could favour the design of personalized inkjet-printed matrices for specific motor control applications. Furthermore, the combination of this technology with the recent advancements in Finite Element Models (FEM) simulations35 may permit obtaining unified algorithms for the automatic design of optimal distributed electrodes’ matrix with respect to the peculiar characteristics of each patient.Compared with previous literature38 and a commercial inkjet printer to obtain an 8-channel surface EMG matrix. In a previous study, we investigated the reliability of inkjet-printed electrodes for a single EMG sensor, compared to Ag/AgCl standard electrodes, and implemented a robust fatigue study with six subjects performing repetitions of a weight-lifting task14. Here, we extended our preliminary study and designed a matrix of inkjet-printed sEMG sensors. Then, we investigated and discussed in detail the quality of the sEMG signals acquired from 12 subjects during six different flexion and extension finger movements. We also assessed the robustness of the sEMG patterns across repetitions and subjects and performed gesture recognition classification .This paper exploited a low-cost sensor printing system based on silver nanoparticle (AgNP) inksOur results showed the reliability of our sEMG setup, as we could obtain high classification accuracy (93–95%), precision, and recall values, both in the flexion and extension finger movements. Moreover, we found a significant similarity level (measured by Spearman’s correlation-based similarity index) across repetitions of the same movement in every subject and dissimilarity (measured by Spearman’s correlation-based dissimilarity index) large enough to discriminate between different movements in every subject. Therefore, we have proved that our inkjet-printed sEMG matrix provides signal quality comparable to current research-purpose sEMG devices and allows effective gesture recognition.Beyond that, its ultra-short design-to-prototype time (“print and play”), its scalability towards a higher number of channels, and its customization possibilities (in terms of geometry and size of the electrodes) make it suitable for any clinical settings, avoiding complicated fabrication procedures, regardless of the availability of expensive equipment materials and without the need of advanced technical expertise.40, exosuits41, haptic feedback11, vocal synthesis10, the control of robots and drones, virtual and augmented reality interaction10. Conversely, the EMG signal transduction remains a critical part of the system, particularly for applications requiring customized electrodes geometries adapting to the specific patient needs.As proved by previous works, many machine interfaces are available in presence of a good signal transduction and appropriate and accurate classification method. These include prostheticsOur work contributes to the research efforts focused on developing tools to evolve from a laboratory setting to wearable applications, i.e., towards scalable solutions to assist people, both patients, and caregivers, to improve or recovery from neuromotor diseases or injuries. Further customization could be investigated by using FEM simulations to personalize the sEMG acquisition setup to make it suitable for different purposes, depending on the needs, e.g., less obtrusive for continuous monitoring during daily living or a denser setup for very fine clinical tests during motor training.38 and SEMG electrodes14. The only variation was the production of the insulating layer with patterned adhesive sheets, detailed below.The design and fabrication of the devices were performed following the method and protocols previously defined and optimized for the production of inkjet-printed impedance-based sensorsAll the device layouts were designed in Autodesk AutoCAD 2018 with black representing the silver nanoparticles ink areas and using yellow (cartridge loaded with standard Epson black ink) for text and cut lines. The layouts were printed directly from the software by a consumer printer  were patterned with a CraftROBO C330 Fig. c with a Thanks to previously printed registration marks, the biadhesive layer was aligned to the AgNP printed matrix and stuck in place Fig. e. Right The geometrical characteristics of the electrodes were investigated to define the inter-electrodes distance and the electrode’s diameter resulting in the higher sEMG signal quality. With this purpose, matrices of 3 electrodes, respectively positive ( +), ground (GND), and negative (-) were printed with electrode diameters of 5 mm and 7.5 mm, and inter-electrodes distances (center-to-center) of 10 mm, 20 mm and 50 mm. All the matrices were applied on the forearm of the same participant and tested while performing the repeated lifting of a 5 kg weight for 3 s. Adequate resting periods were performed to ensure the avoidance of any fatigue mechanism to influence the acquisitions.In order to test the reliability of the newly fabricated electrodes, we recruited 12 healthy participants (28.42 ± 4.36 years old). During acquisitions, a matrix of sensors with 8 bipolar channels was arranged on their forearm. The experimental protocol included two sessions: in session 1, the matrix was placed on the anterior side of the forearm; in session 2, it was placed on the posterior side of the forearm. The specific location for attaching the inkjet-printed matrix varied according to anatomical characteristics and it was identified by palpation of flexor/extensor muscles during index finger flexion/extension. A landmark for each session and each subject could then be defined. The matrix’s channels 1 and 5 were positioned on the anatomical landmark. A ground standard electrode was placed at the wrist of the non-dominant hand.42. Every participant performed two sessions: in one of them, the sEMG matrix was placed corresponding to of the extensor muscles and participants performed 6 individual finger extensions and the 2 hand movements (common to both sessions and used as control movements); in the other protocol, they performed 6 individual finger flexions and the 2 hand movements. All, but the thumb, were involved once in the movements. In contrast, two movements involved the use of the thumb. The resting position was held by keeping the hand open , with the wrist held over a rigid object at the height of the elbow (90 degrees arm flexion). The session order was randomized across participants. Within a session, the same order of movements was performed, with 5 repetitions per movement. At each repetition, an audio file instructed the participant when to start the movement and, when to stop it. At the very beginning, four short acoustic cues were produced, followed by a short silent pause. Then, a different warning cue allowed the participant to start the movement. Finally, another acoustic cue required him/her to go back to the resting position. Each extension or flexion position was kept for 5 s. An interval of 5 s was interleaved between two consecutive repetitions, in order to make the participant relax. Additionally, 1 min of resting was scheduled between two different movements to avoid muscle fatigue. At the end of each cycle, the matrix was removed and replaced with a new one. Figure Participants were introduced to the experimental task with detailed instructions and a training simulation. The experimental protocol involved the execution of 12 individual finger movements and two hand movements. All of them were selected in the DB-1 Ninapro databaseThis study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Santa Lucia Foundation IRCCS, Italy (Prot. CE/PROG.804). Informed consent was obtained from all the participants and all the experiments were performed in accordance with the relevant guidelines and regulations.The inkjet-printed sEMG matrix was positioned corresponding to either the flexor, or the extensor, muscles of the participants’ forearm. The eight bipolar channels of the matrix were aligned over the Flexor carpi radialis, the Palmaris longus, the Flexor digitorium superficialis and also a part of the Flexor carpi ulnaris muscles Fig. h. The fiEach matrix was connected to the acquisition board by a couple of shielded flat wires which had been inkjet printed with the same protocols used for the matrix fabrication and then insulated with biadhesive tape and PET. The acquisition board, allowed the tripolar channels of the amplifiers to be connected to the matrix, and collected all the amplifiers channels ground to a singular electrode placed on the wrist of the participant, over the bone, to avoid any interference. Finally, the acquisition board was equipped with a switch connected to the amplifiers triggers to synchronize the acquisitions.The amplifiers used were two Biopac MP35  and all the SEMG signals were acquired with sampling frequency of Fs = 2 kHz and resolution of 12 bit.All sEMG signals were acquired with a sampling frequency of FS = 2 kHz and filtered with a band-pass filter  in the frequency band 10—380 Hz. Power-line interference was removed with a notch filter (band-stop filter between 49 and 51 Hz using a fourth-order Butterworth filter). Systematic and random reaction times variations caused some misalignment between the audio stimulus and the actual movement performed by the participants, so we chose to extract EMG segments corresponding to the periods of time where the real EMG contraction was exerted: therefore, five 3 s-long segments were obtained from the task session. For the sake of visualization, rectification and smoothing were applied to the collected data using a moving average filter with a fixed 100 ms window.44. The feature matrix was composed of feature vectors extracted from bioelectric signals recorded during the execution of movement of the individual fingers using 1 s non-overlapping specific time windows, so from each 3 s repetition of the finger movement we collected 3 feature vectors. Each feature vector was composed of 88 observations obtained extracting 11 features for each channel. The feature matrix was used in order to assess the possibility of classifying the movement of individual fingers both for flexion and extension.Well-established time- and frequency-domain features were extracted from the pre-processed datasetMAV, Eq. (MAX = max{x}); the minimum value (MIN = min{x}); the range (RANGE = MAX–MIN); the root mean square  of the signal during task and rest segments, separately, was estimated via Welch’s periodogram. A 1 s Hamming sliding window (overlap rate of 50%) was used to obtain the power spectral decomposition with a frequency resolution of 0,9766 Hz in the frequency range 20–300 Hz. Additionally, we computed the signal-to-noise ratio /Pnoise])46.Specifically, we computed the mean amplitude value ; the maRMS, Eq. ); the vaVAR, Eq. ); the stSTD, Eq. ); the ze that coWFL, Eq. ) that prMNF, Eq. ) that isMDF, Eq. ) that isFirst, we visually inspected the raw data and the bubble plots to qualitatively evaluate any clear differences among the different movements and different subjects. Time and frequency domain inspections were carried out, both in the rest and task periods.Second, we evaluated the variability of our dataset through the statistical description of every feature extracted from the raw data (not reported here for space constraints). Then, we investigated the intra-subject and inter-subject variability in the different movements, through feature representation of the sEMG signal samples. Finally, we classified the 6 individual finger movements using three different classifiers.In order to assess the robustness of our inkjet-based sEMG measurements across the same participant, we performed a correlation analysis across the repetitions of the same movement in the same participant. As already mentioned, the RMS feature is frequently used as signal activity metrics in sEMG, thus we used it to evaluate the repeatability of a movement across repetitions and participants. Also, we limited this analysis to the 6 individual finger movements of each session, thus excluding the hand movements. The extension and flexion sessions were analysed separately.From each repetition, as explained in the features extraction section, we extracted three 8-dimensional RMS values , and from each movement we obtained fifteen 8-dimensional RMS values. The latter were normalized over their maximum value across repetitions and channels.i = 1, …, 12 and each kind of movement m = 1,…, 6, we defined a single normalized 8-dimensional RMS value as a pattern and we represented it by xi,m(k) = , with k = 1, .., 15 the observation index (corresponding to a window in a repetition of movement m of participant i).Then, for each participant xi,m(k) and xi,m(q), with k ≠ q.To evaluate the similarity between two different movement repetitions, we calculated the Spearman’s correlation coefficient between ri,m values (one for each different movement and each participant). To assess the significance of each Spearman correlation coefficient, a statistical test was used. Specifically, t-tests were applied to the average similarity coefficients across repetitions to evaluate if for each subject there is a significant repeatability for a specific task.We then aggregated the similarity values by averaging them across the repetitions, leading to 72 i = 1, …, 12 and every kind of movement m = 1, 2, …, 6, we obtained a normalized 8-dimensional vector, averaging across observations. We represented it by xi(m) = .Similarly, we evaluated the dissimilarity between two different movements. To do this, for every participant 1-ri , between xi(n) and xi(m), with m ≠ n and m,n = 1, 2, …, 6, so we obtained a dissimilarity value for every pair of movement  with m ≠ n and for each participant i.The dissimilarity coefficient was calculated using the metrics From each repetition, movement, and participant, as explained in the feature extraction section, we extracted three 11-dimensional samples . Thus, for each movement, we collected 180 11-dimensional samples (15 samples × 12 participants), and this formed the feature matrix used as the classifier’s input.Then, we performed a six-class classification to assess the possibility to distinguish the movement of individual fingers from the inkjet-printed sEMG measurements. Flexion and extension sessions were considered as separate classification problems.27.To evaluate the classification performance in a reliable way, we split the entire dataset into a training and a test set (independent from the training one). The training set included repetitions 1, 2, 4, and 5 of each movement from all participants. The test set contained the third repetition. This choice can be justified by considering that repetition 3 was the one where the participants might not be fully used to the task, yet, and at the same time they might not have already experienced fatigue from multiple repetitions of the same task. Incidentally, previous literature adopted a similar choiceThree different classifiers were selected to solve the two classification problems: a support vector machine (SVM), a k-nearest neighbour (k-NN) and a discriminant analysis (DA). We used Matlab to implement all of them. The hyperparameters of each model were optimized via a tenfold cross-validation on the training set . Particularly, for the SVM, the best kernel type and its hyperparameters were optimized by choosing the combination of values providing the best accuracy in the tenfold CV. For the k-NN, the k had to be tuned; and, for DA, linear or quadratic fit were tested.47:The classification performance was computed in terms of averaged accuracy Eq.  precisioSupplementary Information.