This case report describes the chairside fabrication of a monolithic posterior crown using a multilayer super-translucent zirconia material. According to the manufacturer's information, the newly introduced multilayer zirconia (4-YTZP) offers a unique combination of fracture strength (>850 MPa with speed-sintering) and improved optical properties, thus allowing a reduced minimum material thickness and optional temporary luting. By using up-to-date components of the CEREC system, including superfast dry-milling and a speed-sintering process, the fabrication of a monolithic zirconia crown is possible within an acceptable timeframe for the chairside workflow (60-75 min). The usage of a multilayer super-translucent material allows for the individualization of the restoration, typically in a single combined stain and glaze firing. However, it should be noted that clinical data for this type of restoration are still sparse. Therefore, manufacturer recommendations regarding indication, preparation, and cementation must be followed very strictly. Since the introduction of translucent zirconia ceramic materials more than 10 years ago, these materials allow the fabrication of monolithic crowns and fixed partial dentures (FPDs) .Omission of ceramic veneering reduces the risk of fractures in the veneering ceramics to a great extent . ExistinHigh-strength glass-ceramics are the most frequently used materials in chairside processing  , 11–14. High translucency that allows for a distinct chameleon effect and good conditioning properties for adhesive luting is the main advantage of this group of materials , 17. TheMeanwhile, translucent zirconia ceramic materials are available with different modifications; the translucency is mainly caused by varying percentages of alumina and yttria. Although the first generation of zirconia with a portion of 3 mol% yttrium oxide shows high 3-point flexural strength ISO 6872-2015) in the region of 1,000 to 1,200 MPa, the translucency of this group of materials is relatively low compared to glass-ceramic materials 15 in the, 10, 21.Zirconia materials of the third generation contain 5 mol% yttrium. This allows a translucency comparable with that of high-strength glass-ceramic materials. On the other hand, in comparison with classic zirconia materials with an yttria content of 3 mol%, these materials show a reduced strength , 10, 20.The fourth generation of zirconia materials contains 4 mol% yttrium. Although this leads to a slight reduction in translucency compared to high-strength glass-ceramics, the mean flexural strength increases to 850–1,000 MPa , 20–22 .This modification results in 3 clinically relevant aspects: First of all, according to the manufacturers' information, these materials are suitable for the fabrication of anterior and molar single crowns and 3-unit FPDs, allowing for a restoration with FPDs to replace the first or second molar. Additionally, due to the increased strength of the material, the minimum material thickness can be reduced to 0.6–0.7 mm. The third clinically relevant advantage is the approval of this material group for temporary cementation , 20.Summarizing these material properties, it is apparent that especially zirconia materials of the fourth generation are a meaningful addition to high-strength glass-ceramics in the chairside workflow, as they allow for a wider range of indications while offering a more minimal-invasive preparation.Short Processing Times and Dry Processing to Avoid Drying Prior to Sintering. As zirconia materials are processed in a presintered, chalk-like state, processing times of 5-6 minutes for a single crown can be achieved when using an appropriate milling unit  [te, USA) .The sintering period should be kept as short as possible. By choosing a speed-sintering program, it can be reduced to less than 20 minutes. In vitro studies show that speed-sintering does not have a negative influence on the material properties  of appropriate zirconia materials , 9, 24In this combination, the main indications for the use of high-strength glass-ceramics are inlays, onlays, partial crowns, crowns, and veneers. Zirconia materials of the fourth generation should preferably be used for the fabrication of single crowns and 3-unit FPDs . For theIdeally, individualization of the restoration should be finished in a single stain and glaze process. This can be achieved by using multilayer zirconia blocks. Pigmented layers of varying intensities simulate the typical color gradient of a natural tooth. These multilayer zirconia blocks consist of 4–5 color layers in different pigmentations while showing similar translucencies . In compUntil now, the mechanical and optical properties, as well as the abrasion and fatigue behavior of multilayer translucent zirconia materials, have been evaluated in several in vitro studies , 26–28. The present case report demonstrates the chairside fabrication of a monolithic single-tooth restoration by using a new multilayer zirconia material of the fourth generation  with the current version of the CEREC system .During a prophylaxis appointment, an insufficient composite restoration of an endodontically treated first upper molar was detected in a 31-year-old female patient . X-ray dAfter removing the existing restoration, the tooth was prepared and treated with an adhesive build-up .The equigingival preparation limit was performed as a shoulder with rounded inner angle . The occBefore digital impression taking, a retraction cord  was applied. In order to achieve maximum lateral soft tissue displacement, one layer of hemostatic cotton was placed as top layer . In addiImmediately before intraoral scanning  of the preparation quadrant, the compression cap and hemostatic cotton were removed and the scanning area was gently air-dried. Throughout the scanning process, the retraction cord remained in situ. The application of hemostatic cotton caused a pronounced lateral extrusion of the soft tissues, allowing a very good reproduction of the preparation limit. Moreover, the construction software allows the automatic definition of the preparation limit. In the next step, data acquisition of the opposite jaw and a digital bite registration by lateral scanning were performed.After the final data acquisition, the preparation was analyzed by construction software . The occlusal reduction was evaluated as sufficient, and no undercuts were detected . The autIn the next step, the vertical position of the restoration within the CEREC MTL Zirconia block was determined. Normally, the restoration is placed in the middle of the block, thus covering the entire color gradient , 29, 30.μm (standard settings).The crown was fabricated in a dry-milling process  from a multilayer translucent zirconia block . For fabrication, the milling mode “fine” was selected and the settings for the occlusal and radial spacer were 120 The restoration was milled from a block in color A3. This selection was based on the basic shade of the buccal surfaces of the adjacent teeth.Apart from the mode “fine,” a “superfast” milling mode is available. When using the superfast mode, a zirconia restoration can be dry-milled in 5 to 6 minutes. If this mode is chosen, it is essential to set the minimum material thickness parameter from 0.6 to 0.7 mm before processing. After milling, the retention pin of the crown was removed, and the connecting area was finished with a fine-grid diamond instrument .Afterwards, the crown was densely sintered in an 18-minute speed-sintering process . The CEREC software transferred all the information required for this process directly to the sintering unit . If requFor this step, the entire external surface of the crown was covered with a transparent glazing . A layer of effect ceramic was applied to intensify the coloring of the cervical parts of the restoration  . FinallyIn most cases, an esthetically optimized restoration can be fabricated with a colorless glazing material and 3 staining colors only  in a single firing process, thus resulting in significantly shorter processing times. Afterwards, the individualized crown was tried in. An occlusal adjustment was not necessary. Only the proximal contacts were a little too tight; they were adjusted with a 2-step polishing system for high-strength ceramics. The contact point was slightly reduced with a prepolishing instrument  . In a seμm) at 0.1-0.2 MPa  , 2, 8, 9The use of a speed-sintering process is an important technical precondition. In the present case report, a special sintering oven for the CEREC system  was used , 8. ZircIt has to be considered that speed-sintering is a relatively new processing technique. Several in vitro studies have demonstrated that for suitable zirconia materials, this cost- and time-effective processing technique has no negative influence on the mechanical and optical properties such a hardness, fracture toughness, Weibull characteristics, hydrothermal aging behavior, and translucencies , 27, 28.Due to the constant development of the design software, the current version  offers practical design proposals that mainly only need minor adjustments at the occlusal and proximal contact points . This adWith the procedures and materials presented in this case report, a monolithic zirconia crown can technically be fabricated in approximately 75-90 minutes. Compared to previous fabrication processes, this is a significant shortening of chairside processing times for zirconia restorations . HoweverFor optimal performance in molar restorations, it is helpful to choose the block color (CEREC MTL Zirconia) one shade darker than the tooth color if the restoration has a wall thickness of approximately 1 mm. This is based on the clinical experience that the sintered restorations were too light when the determined target shade was used. Although color adaptation was possible, it required a second stain firing. In contrast, when choosing a block in a darker shade, the correct color match could be obtained in one combined stain and glaze , 21, 23.According to the manufacturer, the minimum material thickness of multilayer zirconia of the fourth generation can be reduced to 0.6-0.7 mm due to the comparably high flexural strength (>850 MPa) compared to glass-ceramic materials. This would present a major advantage of this new material, as the minimum required wall thickness allows for a minimum-invasive preparation. Moreover, the strength of the material offers the option of temporary cementation, which is not possible for high-strength glass-ceramics and zirconia of the third generation , 16.Nevertheless, as it happens with other restorative esthetic materials, also the flexural resistance of zirconia frameworks could be altered by several factors over time  , 34. TheMoreover, it is necessary to mention that the fabrication process presented in this report is based on comprehensive in vitro data , 20, 21,Overall, if the technical prerequisites (superfast dry-milling and speed-sintering processes) are given, the fabrication of a zirconia restoration can easily be performed in a dental practice. The use of a multilayer zirconia block of the fourth generation facilitates the process and allows for a time-saving esthetic individualization of the restoration.


Wheat bread supplemented with mushroom powder from three different species of mushrooms was investigated in terms of starch characteristics  and antioxidant capacities. The decrease in total starch contents, and increase in phenolic contents of the breads, were associated with increased mushroom powder contents. Mushroom inclusion reduced the rate of reducing sugar released over 120 min in an in vitro digestion compared to the control sample, implying a lower area under the curve (AUC) value with the inclusion of mushroom powder and a potentially lower predicted glycaemic response of the bread. Mushroom powder incorporation also enhanced the DPPH radical scavenging assay and oxygen radical absorbance capacity (ORAC) compared to control bread. The action of the addition of different mushroom powders on the bread crust and crumb microstructure properties was also studied. Mushroom powder altered the internal microstructure of the bread crust and crumb by affecting the interactions between starch and the other components of the bread. Overall, this shows that mushroom powder could be added to bread to deliver health benefits to consumers. There are a wide range of bakery products available to consumers, among which bread is regarded as an important dietary food in the majority of countries worldwide. The main ingredient used for bread making is wheat flour, which is mostly composed of starch (contributing 78–88% of the dry mass of the flour) . Bread mPleurotus eryngii mushroom powder [Mushroom fruiting bodies have been used for food, in food flavouring materials and in traditional Chinese medicine for centuries. Mushrooms are a good source of high-quality digestible protein (10–40% dry mass), B vitamins, vitamin C and essential minerals ,4. Most m powder ,8,9 on bAgaricus bisporus), shiitake (Lentinula edodes), and porcini mushrooms (Boletus edulis) were incorporated into wheat bread at 5%, 10% and 15% substitution levels. The influence of such inclusions on the nutritional characteristics and microstructure of breads were determined, and included total starch content, starch gelatinisation, in vitro starch digestibility, total phenolic contents, and antioxidant capacities. The microstructure properties of both the bread crust and crumb were evaluated by scanning electron microscopy. These final novel breads may introduce more edible mushroom species to Western consumers’ diets in an acceptable way through simple preparation.In this study, white button , white sugar , iodised table salt , yeast  and butter  were obtained from the local supermarket. Dried shiitake mushroom and porcini mushroom sliced  were used in this study together with fresh white button mushroom obtained from Meadow Mushrooms .Mushroom powders were prepared according to the method reported by . The breThe dough was formed by using a mixer  to combine wheat flour, mushroom powder, dry ingredients, yeast and water at speed 1 for 5 min. The speed of the mixer was increased to 3 for another 10 min until elastic dough was formed. The dough was rested for 30 min, then hand-kneaded for 5 min and left for 15 min. Dough pieces were divided (100 g), manually rounded, rolled and put into baking pans greased with margarine and placed in a proofer  at 40 °C for 60 min. The bread was baked in an electric oven at 200 °C for 15 min. The baked bread loaves were allowed to cool for 2 h for subsequent analysis.w/w), respectively. The control reference sample was prepared using wheat flour only  content was determined in duplicate using a Total Dietary Fiber assay kit  and measurements were recorded for soluble (SDF) and insoluble fibre (IDF) composition as described by .Differential scanning calorimetry  was employed to measure the starch gelatinisation characteristics of bread samples by measuring the temperature at which different stages of the process occur. Ground samples (4–6 mg) were weighed into a hermetic DSC pan and 10 μL of water was added. The sample was hermetically sealed and allowed to equilibrate overnight at room temperature. An empty pan and lid were used as reference. Duplicate thermal scans were performed from 10 to 110 °C at a rate of 10 °C/min. Temperatures of onset (Tonset), gelatinisation (peak), endset (Tendset) and the enthalpy of the transition (ΔH) were obtained in this process .Total starch was determined according to AOAC Official Method 996.11, using Amyloglucosidase–α-Amylase Method. This method was conducted using the Megazyme Starch analysis kit .The potential amount of glucose released over 120 min was conducted in triplicate using a multi-enzyme in vitro digestion method for each bread sample as described previously .Total phenolic content (TPC) of all samples was measured using 0.2 N Folin-Ciocalteu reagent  according to the method reported by . The resThe microstructure analysis of bread crust and crumb was evaluated by scanning electron microscope (SEM) on samples. The crust and crumb samples were freeze-dried prior to the analysis, then coated with gold using a MCI000 Ion Sputter Coater . Samples were viewed using a SU8010 SEM  at 1000× magnification level.p < 0.05). Pearson’s correlations were also carried out to analyse the significant correlations at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively.Unless stated elsewhere, experiments were performed in triplicate. Statistical differences were determined by one-way analysis of variance (ANOVA) and Tukey’s comparison test . Such a phenomenon may be due to the limited water content during bread making and competitive action by all the hydrophilic components in the mushroom powder being able to retard the starch granules’ swelling. A similar observation regarding the reduced degree of gelatinisation has been recorded [Previous research has shown that variations in gelatinisation temperatures within a dough and bread sample matrix may be important factors to predict bread quality . The melrecorded . As pectrecorded .r = −0.618; p ≤ 0.001), SDF , and TDF  , whereas a negative correlation existed between ΔH and both the total starch content and AUC .≤ 0.001) . Figure ≤ 0.001) , as well≤ 0.001)  and moreThe nature of starch (granule size and amylose/amylopectin ratio) plays a principal role in the glycaemic response of breads , in thatp ≤ 0.001, 0.05 and 0.001, respectively). Interestingly, compared with SDF, both IDF and TDF played more important roles in modulating the starch digestibility of the final bread samples. This may be attributed to the fact that fibre has the capacity to protect the starch granules from human physical digestive actions inside the stomach by increasing the viscosity of the digesta and form a physical “barrier” to the digestion of starch granules [Dietary fibre is resistant to the digestion within the human small intestine and benefits the ‘good’ microfauna growth though fermentation in the human colon . Severalgranules . In addigranules ,23.As determined in our previous work , the thrThe results of our previous work show that the fat content of all three mushroom species powders were higher than those of wheat flour. It has also been shown that the proportion of polyunsaturated fatty acids in the mushroom fat can be as high as around 75%; including 68.8–84% linoleic acid; 19.2% palmitic acid and 8.3% oleic acid . Ref 2121 illustOleic and linoleic acids in the mushroom extracts have been shown to have a strong anti-α-glucosidase capability . Furtherr = 0.960 and 0.979, respectively; at p ≤ 0.001), and between DPPH and ORAC .Bread production processing, including mixing, fermentation, and baking, has been shown to influence the final phenolic contents and activity with research indicating that the antioxidant capacity of bread decreased after baking . This obp ≤ 0.05, 0.001, and 0.001, respectively), between DPPH and both SDF and TDF , and between ORAC and all IDF, SDF and TDF . This indicates a synergistic effect of dietary fiber and antioxidant activity possibly through carrier bioactive compounds.Except for the white button mushroom breads, there were no significant differences between the 10 and 15% substitution levels for the DPPH and ORAC assay results in porcini mushroom breads and in the DPPH of shiitake breads , which mr = 0.493, 0.484 and 0.551, respectively, at p ≤ 0.01), whereas negative correlations existed between AUC and ORAC , the starch content and all TPC, DPPH and ORAC . The results of the in vitro starch digestibility and the antioxidant properties of mushroom enriched breads are in agreement with the work of [It is of added interest is that there were significant and positive correlations between ΔH and all TPC, DPPH and ORAC . Furthermore, the mushroom powder disrupted the initial intact starch–protein matrix and reconstructed the bread crust and crumb microstructures. The SEM images indicate multiple interactions (synergistic or antagonistic) between the additional nutrients that the mushroom powder provided and the inherent constituents. It should be noted that in this research, we did not investigate the separate effects of bound and free phenolic compounds on the nutritional quality of the breads. Other researchers have endeavoured to determine the different effects of bound phenolic compounds and free phenolic compounds on starch and protein digestion, based on the understanding that the solubility and binding strengths of phenolic compounds affects the physical and chemical characteristics of fortified foods . For ins


Ipomoea batatas) is one of the largest food crops in the world. Due to its abundance of starch, sweet potato is a valuable ingredient in food derivatives, dietary supplements, and industrial raw materials. In addition, due to its ability to adapt to a wide range of harsh climate and soil conditions, sweet potato is a crop that copes well with the environmental stresses caused by climate change. However, due to the complexity of the sweet potato genome and the long breeding cycle, our ability to modify sweet potato starch is limited. In this review, we cover the recent development in sweet potato breeding, understanding of starch properties, and the progress in sweet potato genomics. We describe the applicational values of sweet potato starch in food, industrial products, and biofuel, in addition to the effects of starch properties in different industrial applications. We also explore the possibility of manipulating starch properties through biotechnological means, such as the CRISPR/Cas-based genome editing. The ability to target the genome with precision provides new opportunities for reducing breeding time, increasing yield, and optimizing the starch properties of sweet potatoes. Sweet potato ( Ipomoea batatas) is one of the largest food crops in the world (Sweet potato (he world a. Althouhe world b. Sweet Starch biosynthesis requires four classes of core enzymes: ADP-glucose (Glc) pyrophosphorylase (AGPase), starch synthases , starch branching enzymes (SBEs), and starch debranching enzymes   Figure . AGPase AGPase AGPase  induces the sugar responsive kinase gene IbSnRK1. Overexpression of IbSnRK1 in sweet potato not only enhances photosynthesis and carbohydrate accumulation, but also increases nitrogen uptake efficiency [Lc (leaf color) gene, involved in flavonoid biosynthesis, suppresses sweet potato storage root expansion and results in increased lignin synthesis and decreased starch accumulation in storage roots at the initiation stage [Proteomic studies provide a list of proteins that function in the regulation of starch and sugar metabolism during cassava storage root tuberization . Carbon ficiency . Fertilificiency . These ron stage . A naturon stage , althougon stage  will be The genetic regulation of starch metabolism in sweet potatoes and other crops determines the starch properties by affecting the amylose/amylopectin ratio, chain length, and distribution of amylose and amylopectin . BecausePasting properties are characterized by the viscosity developed from a programmed heating and cooling cycle with a constant shearing force ,72. PastAmylose content is one of the most important parameters to be considered for starch properties and industrial applications ,78. DuriThe amylose content reaches 65.5% in a transgenic sweet potato . AmyloseThe sizes and distributions of starch granules are important factors in determining the physicochemical properties of starch for different applications . The staThe transgenic waxy sweet potato with high amylose have larger starch granule sizes (70 and 90 µm) compared with the wild type (WT) . ReductiCLDs dictate the primary structure of starch . CLDs an2 emission in bioethanol production [Based on size-exclusion chromatography (SEC), structures of amylose and amylopectin of sweet potato starch are at DP ≥ 100 and DP < 100, respectively . Two peaoduction . Recentloduction . The higStarch phosphorylation is a naturally occurring chemical modification; however, its physiological function is not known. Phosphorylated creamy starch, such as that from potato tubers, is easy to hydrate, producing a clear and sticky paste. Similar functionalities can be achieved though industrial chemical treatments. Detailed descriptions of starch phosphorylation have been nicely reviewed ,89,90,91GWD1 expression altered the starch phosphate content in potato tubers, indicating a direct link between GWD1 expression and starch phosphorylation levels [SP, SSII, SSIII, and SBEII was affected by the starch phosphate content [Thus far, two kinases, the glucan, water dikinase (GWD1) ,93 and pn levels . Althoug content . These r content ; however content ,98,99. T content .Sweet potato starch is used in various food and industrial applications. Sweet potato starch is particularly valued as an important ingredient in the manufacturing of starch-based food products, such as noodles, vermicelli, jellies, steamed bread, cakes, alcoholic beverages, soup, flavoring agents, sweeteners, and other consumables . Sweet pOne promising attempt was made to fill a thermoplastic starch matrix with nanofillers, because the nanoscale particles of starch showed different crystallization kinetics, resulting in varied crystalline morphology and size . CompareStarch-based nanoparticles are conventionally produced by acid hydrolysis or high-pressure homogenization, although enzymatic hydrolysis is more effective and environmentally friendly. Starch-based nanoparticles with desired sizes can be produced using amylases or/and debranching enzymes to digest the α-1,4-glucosidic or α-1,6-glucosidic bonds in starch. Enzymatic hydrolysis is likely to yield homogeneous nanoparticles when the starch contains more A and B type short chains with an average DP of 14–18 . Sun et The genome-editing technology CRISPR/Cas has emerged to be the tool of choice to manipulate genomes . After tSBE alleles using CRIPSR/Cas9 technology, a novel potato starch with no detectable branches was produced [IbGBSSI and IbSBEII, in the starch-type cultivar Xushu22 and the carotenoid-rich cultivar Taizhong6. The mutation efficiency was 62–92% with multi-allelic mutations in both cultivars [In addition, genome editing has proven to be efficient for multiple unlinked-loci mutagenesis and plants with complex genomes, e.g., the hexaploid sweet potato with 90 chromosomes, in addition to the polyploid wheat and potato ,126. By produced . The firultivars , providiThe use of well-established biotechnological tools to improve tuberous crops is effective. For example, silencing the vacuolar invertase inhibitor gene by RNAi improves the resistance to cold-induced sweetening of potato without affecting tuber quality when stored in cold conditions . HoweverAlthough genome editing is superior in many aspects compared to other biotechnological platforms or chemical mutagenesis in conventional crop breeding, possible off-targeting (mutating a non-targeted genomic site), due to low sgRNA specificity and the lack of genomic information for some plant species, can hinder its wide applications. Nevertheless, the second generation of CRISPR technology has significantly reduced off-targeting ,131. TheStarch from sweet potato possesses certain unique chemical and physical characteristics compared to that from other sources. The existence of many germplasms with varied starch contents and characteristics establishes a genetic base for novel sweet potato crops that are suitable for many food and industrial applications. CRISPR/Cas-based genome editing has been successfully demonstrated in sweet potato and will expand the diversity of natural starch without the need of physical and chemical treatments.Genetic manipulation of starch properties has advantages over physical or chemical methods, which are often associated with environmental pollution, expensive equipment, and special design and optimization in production ,136. ForIn contrast, genetic engineering methods are more environmental and cost friendly. Genetic manipulation of genes, using gene overexpression, RNAi, and genome editing, has been shown to be effective in the production of sweet potato starch with altered functionality. However, difficulties remain in genetic engineering of sweet potato. Starch properties are generally controlled by multiple genes, and simultaneously targeting multiple genes is an engineering challenge. Due to the complexity and insufficient annotation of the sweet potato genome, it is currently difficult to manipulate multiple loci by upregulation, knockout, or their combination. Due to the rapid advancement in gene sequencing and genomic analyses, these issues will be overcome in the near future. In addition, the generation of transgenic plants is time consuming, especially for crop plants, such as sweet potato, that are known to be difficult to regenerate in tissue culture. Nevertheless, we are now able to overexpress genes, knockdown gene expression, and knockout genes in sweet potato. These abilities allow us to generate libraries of sweet potato lines with varied starch properties. These libraries will be the basis for designer starch production through crossing or secondary gene transformation.CRISPR/Cas genome editing technology has fundamentally changed agricultural breeding. Since the invention of the first CRISRP/Cas9 system, novel tools, such as CRISPR/Cas-mediated chromosome engineering, and Cas13 a, have been developed, and the development of more genome-editing tools is underway ,141. TheThe ability to naturally modify sweet potato starch significantly enhances the economic value of the crop. In this review, we provide an overview of sweet potato starch metabolism, describe the direct and indirect links between starch properties and genes involved in sweet potato starch metabolism, and associate different starch properties with final industrial products. The connections among genes, starch properties, and industrial products are the basis for modification and improvement of sweet potato starch through techniques such as genome editing. Gene knockout by CRISPR/Cas technology has another advantage compared to many other gene-silencing technologies, namely, the T-DNA carrying the CRISPR/Cas can be removed from the genome through segregation, thus significantly reducing the burdens in the governmental regulatory process. In summary, the maturation of the enabling technologies described here has opened the door to a new era of precision molecular breeding of sweet potato ,143.


MAPK activated protein kinase 3 (MAPKAPK3/MK3) is a serine/threonine protein kinase regulating various cellular responses and gene expression. However, the role of MK3 in tumor progress, prognosis, and immunity for glioma remains unclear. Here, we determined the expression and prognostic values of MK3. We further analyzed the correlation of MK3 expression with immune infiltrations by using the biochemical methods and bioinformatic approaches with available databases. We find that MK3 is aberrantly upregulated in glioma. In addition, the higher MK3 expression is closely linked to the poor clinicopathologic features of glioma patients. Importantly, MK3 expression is negatively correlated with the prognosis of patients with glioma. Mechanistically, we demonstrated that the correlated genes of MK3 were mainly enriched in pathways that regulate tumor immune responses. The MK3 level was significantly associated with tumor-infiltrating immune cells and positively correlated with the majority of tumor immunoinhibitors, chemokines, and chemokine receptors in glioma. Thus, these findings suggest the novel prognostic roles of MK3 and define MK3 as a promising target for glioma immunotherapy.Glioma is the most common primary brain tumor that causes significant morbidity and mortality.  O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, histone code, and chromatin organization have been used for classification of glioma and treatment decisions  was first reported in 1996, located on human chromosome band 3p21.2 -activated protein. MK3 shares a highly similar sequence to MAPK activated protein kinase 2 (MAPKAPK2/MK2), possessing 72% nucleotide and 75% amino acid identity .2.Human GBM cell lines , human normal brain glial cells (HEB), and normal human astrocyte (NHA) were originally obtained from the American Type Culture Collection (ATCC). All these cell lines were cultured with Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  at 37°C within a humid atmosphere containing 5% COMK3 expression with the “Single Gene Analysis” module of Gene Expression Profiling Interactive Analysis (GEPIA) web (http://gepia.cancer-pku.cn/) and observed the differential expression of MK3 between tumor and adjacent normal tissues for the different tumors or specific tumor subtypes of The Cancer Genome Atlas (TCGA). RNA-sequencing (RNA-seq) data of 689 GBM and low-grade glioma (LGG) tissues from TCGA dataset  and 1,157 normal tissues from Genotype-Tissue Expression (GTEx) (https://www.genome.gov/Funded-Programs-Projects/Genotype-Tissue-Expression-Project) were used for MK3 expression analyses. RNA-seq data of 413 glioma tissues (batch I) and 273 glioma tissues (batch II) datasets after deletion of incomplete data from the Chinese Glioma Genome Atlas (CGGA) dataset were also utilized for the expression analyses. In addition, we analyzed the expression of MK3 in glioma by using the Gene Expression Omnibus (GEO) database, GSE4290, and GSE7696 (https://www.ncbi.nlm.nih.gov/geo/). t-Test was used for the expression analyses by using R package “ggplot2.”We analyzed the MK3  and GAPDH  at 4°C overnight. Then the membrane was washed three times using 0.1% TBST buffer and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature, followed by three times TBST buffer washing. Finally, the signal was detected with enhanced chemiluminescence reagent.Cells were lysed with radioimmunoprecipitation assay (RIPA) extraction reagent  containing the protease inhibitors . Total protein was separated using 10% polyacrylamide gel electrophoresis and transferred to NC membrane . The membrane was blocked with 5% bovine serum albumin (BSA) in TBST for 1 h at room temperature and then incubated with the primary antibodies against MK3  at 4°C overnight. Then the sections were incubated with secondary antibody  for 1 h and DAB for 2–5 min at room temperature, followed by hematoxylin staining. The images were acquired by Olympus microscopy and scored by two experienced pathologists without knowing the patients’ characteristics. Scores were calculated based on the intensity and percentage of positive tumor cells within the whole tissue, which were evaluated by using the German semiquantitative scoring method. The intensity score of cytoplasmic staining of each specimen was defined as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The percentage of positive cells was evaluated with 0.0% staining; 1, 1%–24 % staining; 2, 25%–49% staining; 3, 50%–74% staining; and 4, 75%–100% staining. The final immunoreactive score was calculated by the multiplication of the intensity scores and proportion scores.The 4-μm-thick glioma sections were stained with a specific primary antibody against We obtained the survival and clinical phenotype data of each sample from TCGA and CGGA datasets. The Kaplan–Meier (KM) curve analyses were conducted by the R package “survival” and “survminer.” Cox analysis was used for survival analyses by using the R package “survival.” The receiver operating characteristic (ROC) analysis was performed with the R package “pROC.”MK3 expression were included in these analyses.Univariate and multivariate Cox analyses were conducted by using the R package “survival.” The WHO grade, IDH status, 1p/19q codeletion, primary therapy outcome, gender, age, and MK3 with the query of protein name (“MK3”) and organism (“Homo sapiens”) on the STRING website (https://string-db.org/). The basic setting parameters were the network type (“full STRING network”), meaning of network edges (“evidence”), active interaction sources (“experiments”), minimum required interaction score (“low confidence (0.150)”), and max number of interactors to show (“no more than 50 interactors in 1st shell”).We searched the MK3 were screened with Pearson’s correlation coefficients (|r| >0.4 and p < 0.001) with R package “deseq2.” Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analysis were conducted on MK3 correlated genes with R package “clusterProfiler” to identify the possible biological functions and signaling pathways affected by MK3. The biological process (BP), cellular component (CC), and molecular function (MF) were applied in GO analysis.Gene expression data of GBM and LGG in HTSeq-Counts were downloaded from TCGA website for further analysis. The correlated genes of p. adjust value and false discovery rate (FDR) q value both less than 0.05 were considered as significantly enriched.The Gene Set Enrichment Analysis (GSEA) was performed to dissect the cancer-related pathways by using the MSigDB Collection (c2.cp.v7.2.symbols.gmt) of the clusterProfiler R package. The gene sets with normal BETA PORTAL website (https://singlecell.broadinstitute.org/single_cell/study/SCP393/single-cell-rna-seq-of-adult-and-pediatric-glioblastoma#study-download), , and    to exploMK3 expression and immunoinhibitors, chemokines, and chemokine receptors across human cancers was determined on the Tumor-Immune System Interaction Database (TISIDB) website (http://cis.hku.hk/TISIDB/index.php). The correlation analysis between the expression of MK3 and immunoinhibitors, chemokines, and chemokine receptor genes of the glioma was evaluated by using the R-package “ggplot2.”The immune cell infiltration levels from tumors were quantified by the single-sample GSEA (ssGSEA) with the R package “GSVA” . The corMK3 expression with immune checkpoint expression from glioma was analyzed on the Tumor IMmune Estimation Resource (TIMER) website (https://cistrome.shinyapps.io/timer/) and adjusted by the tumor purity.The correlation of 2) test, Pearson’s correlation, or Spearman’s correlation analysis. Student’s t-test was used to determine the statistical significance of differences between groups, and the differences between more than two groups were analyzed by one-way ANOVA or the Kruskal–Wallis test. A two-sided Mann–Whitney test was used for non-parametric data. p-Value < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001.All gene expression data were normalized with log2 transformation. Statistical analyses were conducted by R (3.6.3). The survival data from the CGGA database were acquired by KM. The correlation analysis was evaluated by chi-square  and LGG (n = 518) tissues compared with the normal brain tissues (n = 207) that had a low level of MK3 mRNA expression among different normal human organs (MK3 expression levels in glioma tissues by immunohistochemistry (IHC) staining. The staining results showed a similar MK3 expression pattern from the tumor tissues when compared with the para-tumor tissues , HEB, and NHA cell lines by Western blotting analysis. We observed that the protein levels of MK3 were also higher in the majority of the glioma cell lines , and disease-specific survival (DSS) event of GBM and LGG, and the results showed that the expression of MK3 was lower from alive patients  was 0.966, which indicated a high diagnostic value of MK3 in glioma  = 3.037 (2.349–3.297), p < 0.001). Moreover, WHO grade, IDH status, 1p/19q codeletion, primary therapy outcome, age, and MK3 expression were all significantly correlated with poor OS  and 1,229 negatively correlated genes (Pearson’s correlation coefficient r < −0.4) of MK3 from TCGA transcriptome dataset. The top 15 positively correlated genes and negatively correlated genes of MK3 were plotted in the heatmap  , CD8 T cells, T gamma delta (Tgd), T central memory (Tcm), NK CD56bright cells, and plasmacytoid DC (pDC) cells and was positively correlated with infiltrating levels of macrophages, neutrophils, eosinophils, activated dendritic cells (aDC), immature DC (iDC), T, Th17, cytotoxic cells, NK CD56dim, Th2, NK, T helper, Th1, B cells, and DC , programmed cell death 1 ligand 1 (PD-L1/CD274), and cytotoxic T lymphocyte antigen 4 (CTLA-4) are vital immune checkpoints that play important roles in tumor immune escape. And they also served as predictive markers for the therapeutic efficacy of immune checkpoint inhibitors (ICIs). Thus, we especially assessed the relationship of MK3 with PD-1, PD-L1, and CTLA-4 in glioma by using TIMER tool. The results from TIMER website illustrated that there was a significant positive correlation between MK3 expression and PD-1, PD-L1, and CTLA-4 in glioma, which was adjusted by tumor purity , PD-L1 , and CTLA-4  in glioma , National Natural Science Foundation of China (82002516), the Natural Science Foundation of Jiangsu Province in China (Grant No. BK20190984), and the Natural Science Fund for Colleges and Universities in Jiangsu Province (19KJB310023).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The presence of resistant starch resulted in increased entrapment of bromelain, improved swelling properties with sustained release behavior, and improved gastric stability than pectin hydrogels alone. The swelling of hydrogel beads was higher at pH 7.4 than pH 1.2. Optimized batch of hybrid pectin/resistant starch exhibited a spherical shape. Optical and scanning electron microscopy showed a more packed and spherical shape from the pectin/resistant starch hydrogel bead network. Fourier transformation infrared spectroscopy was also used to confirm the presence of bromelain in the hydrogel beads. The encapsulated bromelain in the pectin/hi-maize starch beads produced at a pectin/hi-maize ratio of 4.5:1.5  obtained the highest relative bromelain activity in all heat treatments including at 95°C, whereas the highest activity of free bromelain was found only at 30°C. Bromelain encapsulated in hydrogels released at a faster rate at simulated intestinal fluid  than at simulated gastrointestinal fluid .Hybrid pectin and resistant starch–based hydrogel beads loaded with bromelain using the extrusion gelation method were prepared and evaluated to enhance the activity of bromelain during gastrointestinal passage and thermal processing. The solutions of pectin–resistant starch with bromelain were dropped into the gelation bath containing calcium chloride (0.2 M) solution to develop various types of hydrogel beads. The physicochemical characteristics of the synthesized hydrogel beads were evaluated. The ratio (4.5:1.5 w/w) of pectin and resistant starch concentration significantly ( Pterophylla beltrani (Bolivar & Bolivar), peptides from leatherjacket (Meuchenia sp.), protein hydrolysates, and bioactive proteolytic enzymes derived from pineapple  as wall materials to protect it from higher processing temperature and its controlled passage and release in the simulated gastrointestinal tract (GIT). This study thus aims to evaluate the effects of resistant starch (Hi-maize) on the activity and controlled delivery of bromelain microencapsulated with the pectin in the simulated digestive system and higher processing temperatures. Hi-maize starch, a modified resistant starch, is considered along with pectin which remains as macromolecular aggregates in the upper portions of GIT. The application of these two biomolecules forms a densely packed matrix (superior protection and tightly packed hydrogel beads) in which the active compounds such as bromelain can be embedded for protection against GIT and thermal conditions.Low methoxyl pectin  from citrus peel was obtained from Ingredient Flavours Co., Ltd., Thailand. Hi-maize starch was procured from National Starch and Chemical Co., Ltd., Bangkok, Thailand. Bromelain from pineapple was purchased from Sisco Research Laboratories Pvt. Ltd., India. Pancreatin enzyme was purchased from Sigma-Aldrich, United States, and Pepsin from Acros, Denmark. All other chemicals used in the study were of analytical grade.Pectin/hi-maize starch hydrogel beads were prepared by gelation method adopting the protocol described by The mixed solution  was dropped through an 18 gauge hypodermic needle  at a controlled pump speed of 6 rpm using a peristaltic pump  into coagulation bath containing 0.2 M calcium chloride solution (50 ml) using a hydrogel bead making unit , designeThe particle size was determined with a micrometer  and expressed as the average of 100 hydrogel beads.The bromelain content in the hydrogel beads was analyzed by following the method described by t and Wo are the weight of swollen hydrogel beads at a fixed time interval and the initial weight of the hydrogel beads, respectively.The swelling ratio of hydrogel beads was determined by following the methods described by A scanning electron microscope (SEM)  was used to evaluate the morphology and surface appearance of hydrogel beads. The samples were mounted to a specimen stub with double-sided tape and coated with a thin layer of gold on an ion sputter coater to improve their visibility and sufficient resolution. Additionally, the physical appearance of the hydrogel beads was observed through a digital camera .−1, and spectra of all samples were collected and analyzed using the system software (OMNIC software).Infrared spectra of the pectin, hi-maize starch, bromelain, and encapsulated bromelain in pectin/hi-maize starch were measured using FTIR spectrophotometer . The pellets were scanned in the range of 4,000–400 cmThe disintegration of the hydrogel beads in SIF (pH 7.4) was studied by following the method described by In vitro release of entrapped bromelain was performed using a conical flask (125 ml) by following the methods described by The thermal stability test was conducted by following the method described by −1 ml−1 was described as one unit of enzyme activity, which can be expressed as Units/ml. The enzyme solution (1 ml) was mixed with casein  in 0.05 M potassium phosphate buffer . The reaction was performed at 37°C in a water bath  for 10 min. Subsequently, the reaction was terminated by the addition of trichloroacetic acid . After that, the mixture was then filtered through a syringe filter (0.45 µm diameter) . The filtrate (2 ml) was mixed with 5 ml of 0.5 M sodium carbonate solution and 1 ml of 20% (v/v) Folin Ciocalteu’s phenol reagent and incubated for 30 min in a water bath at 37°C. Then the solution was filtered through a syringe filter (0.45 µm diameter) , and the absorbance of the mixture was measured at 660 nm with a UV-vis spectrophotometer . The bromelain activity was calculated as The proteolytic activity of the solution was determined using casein and L-tyrosine as a substrate and a standard, respectively, following the method described by p < 0.05) among mean observations.The experiments were performed in three replicates, and the data were presented as mean with standard deviation. A statistical analysis was performed using a commercial statistical package  in which ANOVA and Tukey’s HSD test were conducted to evaluate the significant differences (2+ ions and the negatively charged carboxylate (-COO-) on the pectin backbone was used to create hydrogels. Divalent metals promote polyanion–cation–polyanion interactions between pairs of carboxylate groups on neighboring helices of the pectin structure (p < 0.05) in the presence of different pectin and hi-maize starch ratios.In this study, bromelain was encapsulated in a pectin/hi-maize starch hydrogel using an extrusion method. Intermolecular cross-linking between Catructure . Pectin tructure . The sizAdditionally, previous studies indicated that the size of the hydrogel beads varied depending on the material used . It can The encapsulation efficiency  was deteThe formulation P4 had a maximum encapsulation efficiency of 80.53 ± 1.51%, which can be attributed to the high amount of modified starch interacting with pectin, which generates stability between the two polysaccharides to retain the highest bioactive content in the polymer matrix of the microcapsule formed .Lactobacillus fermentum CECT5716Lb Casei cells with increased concentration of starch in alginate–starch–calcium chloride–based hydrogel beads. Similarly, The obtained values showed a high range of encapsulation efficiency values compared to earlier reports for bromelain encapsulation in materials such as chitosan nanoparticles, chitosan nanofibers, and katira gum as carriers . Martin 2+ or Sr2+, promoting the gel network structure by intermolecular cross-links between the calcium ions (Ca2+) and the negatively charged carboxyl groups (COO−). The ratio and composition of ionic and non-ionic functional groups in hydrogels determine their swelling potential  by pectin and hi-maize starch concentrations. A combination of 1.5% hi-maize starch (P4) played an important role in percentage swelling at 85.92%. Carboxylic acid groups on pectin chains convert to negatively charged carboxylate ions in basic and neutral environments. Consequently, electrostatic repulsion between the deprotonated carboxyl groups causes the network to expand and swell , as shown in nd swell . The comnd swell .The shape and sizes of pectin hydrogel beads  show sig−1 are attributed to the characteristic band for the stretching vibration of the -OH group. The bands at 1700–1,600 and 1,210–1,050 cm−1 are attributed to the intramolecular hydrogen bond and C-O stretching, respectively. The vibrational absorption peaks of the C-H bond of monosaccharide are observed at 1,000–850 cm−1 . The characteristic of C-N stretching vibration frequencies are assigned to IR peaks at 1,513, 1,224, and 1,102 cm−1. The spectra of bromelain show a peak at 3,400–3,300 cmBromelain-loaded hydrogel beads exhibited the characteristic peaks of bromelain, pectin, and hi-maize starch. Similar peaks with a broad range were observed in the bromelain hydrogel bead, which represents the encapsulation of bromelain in the pectin–starch matrix−1 (COO-Ca) corresponding to pectin (without any evident interaction . The slio pectin . Previouo pectin .The disintegration of hydrogel beads was investigated in SIF (pH 7.4). The term “hydrogel bead disintegration” refers to the loss of the bead-like structure as observed under a microscope . The timIn vitro release experiments were conducted to identify variations in the release profiles of hydrogel beads. The release of the bromelain from the pectin/hi-maize starch is strongly affected by the pH of the medium. Bromelain is a water-soluble polymer that readily diffuses out  disintegrated and lost all remaining bromelain. A statistical analysis of the release after 3 h in SIF reveals that increasing the content of hi-maize starch considerably decreases the release of bromelain. As shown in The addition of hi-maize starch to the hydrogel beads prolonged the period of release. The slower erosion and consequently more prolonged release of bromelain from pectin/hi-maize starch hydrogel beads most certainly affect the beads strengthening by starch. The hydrogel network becomes denser as the concentration of starch in the hydrogel matrix increases, which may decrease the rate of solution diffusion through the hydrogel.2+ at neutral pH (Bromelain encapsulated in hydrogels released at a faster rate at pH 7.4 (SIF) than at pH 1.2 (SGF). The carboxylic acid group on the backbone of pectin transforms into negatively charged carboxylate ions at higher pH values (pH 7.4); as a result of electrostatic repulsion between carboxylate ions, the network expands and bromelain diffusion from the pectin/starch hydrogels increases. Under acidic conditions, the anionic carboxylate (pKa∼4.5) on the pectin backbone is transformed to neutral carboxylic acid, resulting in a hydrogen-bonded network . Similarutral pH . Previouutral pH .The presence of resistant starch in the alginate matrix slowed the release of nisin, resulting in a higher time required value for alginate/hi-maize microparticles . In addiEncapsulation of enzymes in hydrogel beads provides some protection against temperature variations, which results in increased stability as compared to free enzyme. Improving this property allows the systems for manufacturing applications in the operating temperature ranges . An EnzyL. plantarum cells in mango juice demonstrated effective resistance against thermal treatment (72°C for 90 s). Moreover, probiotic bacteria encapsulated in chitosan–alginate beads and alginate–fish gelatin protein were reported to be more resistant to stress conditions associated with food processing and high temperatures related to pasteurization, while retaining their functional characteristics  at all temperatures. On the other hand, encapsulated bromelain significantly increased bromelain activity in pectin/hi-maize starch hydrogel beads. Increasing the content of hi-maize starch at a different ratio of pectin content  also enhanced the bromelain activity. However, the encapsulated bromelain in the pectin/hi-maize starch hydrogel beads produced at a pectin/hi-maize ratio of 4.5:1.5 percent w/w (formulation P4) obtained the highest relative bromelain activity in all heat treatments. The pectin and hi-maize starch hydrogel beads at optimal concentration have reduced porosity and a thicker structure, which results in the hydrogel bead limiting solution entry . Furthereristics .Encapsulated beads of bromelain were successfully obtained by the extrusion gelation method of various ratios of two different biopolymers, pectin and resistant starch with different ratios into the gelation solution. The encapsulated bromelain in hydrogel beads developed with the combination of these polyelectrolytes can protect bromelain from harsh gastric conditions (lower pH) and higher processing temperatures. The presence of hi-maize starch at a concentration of 1.5% w/w in pectin 4.5% w/w formulation resulted in a higher percentage of encapsulation efficiency compared to the formula of pectin alone. The application of resistant starch blending had a significant influence on the extended and gradual release as well as activity of bromelain. Furthermore, the combination of resistant starch with pectin biopolymer significantly protected bromelain from the extreme environments of high temperature processing. Due to high efficiency in protection and controlled release carrier for bromelain, bromelain-loaded pectin and resistant starch hydrogel beads can be applied for bromelain delivery applications in food and pharmaceutical products development for controlled intestinal release.


Pseudomonas aeruginosa. Meticulous debridement of the wound and management by negative pressure wound therapy with continuous irrigation was performed. However, the infection of the prosthetic graft could not be controlled. Extra-anatomical bypass was performed between the left common carotid artery and right subclavian artery via the right common carotid artery. Then, the infected graft was removed. After the resolution of infection, the mediastinal wound was reconstructed with a pedicled latissimus dorsi myocutaneous flap, which was harvested from the right dorsum. No recurrence of infection occurred in the nine-month follow-up period. Debridement and removal of exposed artificial graft are considered the gold standard for treating wound infection. In situ replacement of infected aortic grafts carries a risk of re-infection due to residual bacterial contamination of the periprosthetic tissue. Extra-anatomical bypass would be a useful option for reducing the risk of re-infection in patients with intractable PSM and prosthetic aortic graft infection. Further studies are warranted to evaluate the risks and benefits of this operative method.Extra-anatomical bypass grafting is a surgical method used to remove an infected aortic graft and promote revascularization with a new graft in the non-infected area. Here, we report a case of intractable post-sternotomy mediastinitis (PSM) with aortic graft infection which was treated with extra-anatomical bypass grafting. A 56-year-old woman with a history of multiple aortic dissection and prosthetic graft replacement in the thoracoabdominal area developed PSM with aortic arch graft infection. Bacterial culture of the exposed prosthetic graft tissue yielded multidrug-resistant  Post-sternotomy mediastinitis (PSM) is one of the most serious complications that can arise after open heart surgery . InfectiExtra-anatomical bypass grafting involves removal of the infected graft and revascularization using a new graft via a root away from the normal anatomy. In 1977, Brown et al. introduced this method for the treatment of infective endocarditis with prosthetic aortic valve infection . This inPseudomonas aeruginosa. Three weeks after the operation, re-sternotomy and debridement of the necrotic tissues were performed. However, she suffered from uncontrollable sepsis with multidrug-resistant P. aeruginosa and was transferred to our hospital.A 56-year-old woman with Marfanoid habitus underwent multiple prosthetic graft replacement operations for thoracoabdominal aortic dissection. Two months before admission to our hospital, she was diagnosed with an impending rupture of the brachiocephalic aneurysm. Removal of the aneurysm and prosthetic graft replacement via median sternotomy was performed at another hospital Figure . She devP. aeruginosa. Pulsed wound lavage with 10 L of normal saline using PulsaVAC®  was performed under general anesthesia. The wound was managed by negative pressure wound therapy with continuous irrigation (NPWTci) [At the initial presentation, we confirmed that the sternal bone was completely debrided, and the prosthetic graft was exposed to the mediastinal wound Figure . Bacteri(NPWTci) , which cP. aeruginosa. Removal of the infected prosthetic graft and re-replacement of the graft were considered to control the infection. However,in situ replacement of infected prosthetic grafts has a high risk of bacterial contamination as the periprosthetic infectious tissues can contaminate the new graft. Therefore, extra-anatomical bypass grafting has been proposed as an alternative treatment.The foam dressing change and wound lavage using PulsaVAC® were performed twice a week in the operating room. Thirty-five days after the initiation of NPWTci, healthy granulation tissue formation was confirmed in the wound. However, the wound culture results continued to be positive for The infected graft, which was exposed to the mediastinal wound, was removed Figure . MicroscP. aeruginosa. The pedicled latissimus dorsi myocutaneous flap, which was perfused by the right thoracodorsal artery, was elevated (Figure NPWTci was used for wound management until flap coverage. Red granulation tissue growth in the mediastinal wound was confirmed in the area of the infected graft was removed Figure . Seven dd Figure . The flad Figure .The gold standard in the treatment of periprosthetic tissue infection is debridement and removal of the infected prosthesis. However, removal of the infected graft can be challenging in patients with prosthetic graft infection after cardiovascular surgery. High perioperative mortality has been reported after the removal and replacement of infected grafts. Furthermore, the procedure has a high physical burden on patients .Therefore, local infection control and coverage with a well-vascularized flap are often attempted to avoid graft replacement . NPWTci Despite these measures, replacement operations are still required in some patients with prolonged positive bacterial cultures of periprosthetic tissue.In situ replacement of the infected graft is associated with a high risk of infection of the new graft due to the persistence of infection in the surrounding tissue of the infected graft. Patients with re-infection of the replaced graft develop a severe clinical course, including septic shock and multiple organ failure. Therefore, extra-anatomical bypass grafting is presented as a last resort in cases of intractable graft infections .Several researchers have reported the usefulness of removing the infected graft followed by revascularization with extra-anatomical bypass grafting in PSM patients with prolonged prosthetic graft infections ,8. SeegeLimited studies have been conducted regarding extra-anatomical bypass for total arch graft infections. Inoue et al. reported a case of extra-anatomical bypass grafting for an infective aortic arch aneurysm . The aneAlthough the combination of extra-anatomical bypass grafting and flap coverage could be an alternative treatment option for the PSM patients with total arch graft infection, not all patients were subjected to this aggressive treatment. We need to consider the indication of this treatment from the aspect of severe operative invasiveness and possible postoperative unfavorable result. In other words, hemodynamically unstable patients are not subjected to this aggressive surgery. The reason why we performed this surgery was that our patient had a capacity for surgical invasion.Extra-anatomical bypass grafting causes hemodynamic changes in the distal bypass area. The latissimus dorsi myocutaneous flap is perfused by the thoracodorsal artery, which bifurcates from the axial artery. In our case, the right axial artery received blood supply from the left CCA via the bypass graft. Despite the circulation pathway change in the proximal area of the vascular pedicle, the flap completely survived without ischemia. The extra-anatomical bypass grafting did not interfere with reconstruction using the pedicled latissimus dorsi myocutaneous flap.We have reported a case of intractable PSM with a prosthetic aortic graft infection that was successfully managed by extra-anatomical bypass grafting. Instead of in situ replacement of the infected graft, extra-anatomical bypass offered a new route outside the infected wound. Extra-anatomical bypass grafting would be a useful option for patients at high risk of re-infection with in situ replacement of the infected graft. Further studies are warranted to evaluate the risks and benefits of this operative method.


The purpose of this noninterventional, register-based study was to report the outcomes and wound healing of surgically treated chronic acromioclavicular (AC) dislocations using a tendon graft and knot-hiding titanium implants.Thirty-two cases with chronic AC separation underwent an arthroscopic coracoclavicular (CC) ligament reconstruction and an open AC ligament reconstruction using knot-hiding titanium implants. The wound healing was assessed 2 months after the operation. The Nottingham Clavicle score, Constant score, and Simple Shoulder Test were obtained postoperatively and at a minimum of one-year postsurgery. The radiographic change in distance between the clavicular and coracoid cortices and clavicular tunnel diameter was measured. General patient satisfaction with the outcome  was also assessed 1 year postoperatively.P ≤ .05). The Constant score increased from a preoperative mean of 44.66 ± 12.54 to 93.59 ± 7.01 (P ≤ .05). The Simple Shoulder Test score increased from a preoperative mean of 7.00 ± 2.14 to 11.84 ± .63 (P ≤ .05). The coracoclavicular distance increased from 11.32 ± 3.71 to 13.48 ± 3.79 mm (P ≤ .05). The clavicular drill hole diameter increased from 6 mm to a mean of 6 to a mean of 8.13 ± 1.12 mm. Twenty-three (71.9%) patients reported an excellent outcome, and nine (28.1%) reported a good outcome. One clavicular fracture occurred but no coracoid fractures. There was one reconstruction failure leading to a reoperation.The mean Nottingham Clavicle score increased from a preoperative mean of 41.66 ± 9.86 to 96.831 ± 5.86 (In this series, combining the arthroscopic CC ligament reconstruction to an open reconstruction of the AC joint with a tendon graft proved to be a stable solution. The knot-hiding titanium implant effectively eliminated the problems related to the clavicular wound healing.Level IV, therapeutic case series. The hypothesis was that the arthroscopic CC ligament reconstruction combined with open AC ligament reconstruction would be associated with favorable clinical and radiographic results, and the knot-hiding clavicular implant would enhance clavicular wound healing.,Patients with chronic Rockwood grades III and V AC dislocations, which were treated using two different modifications of the surgical technique as described by Ranne et al., were identified in a patient registry.The technique involved an arthroscopic double-bundle reconstruction of the CC ligament complex and an open reconstruction of the AC ligaments using a semitendinosus (ST) autograft. The synthetic polyurethane-urea tendon graft  was used in cases in which no semitendinosus autograft was available. In the CC reconstruction, the anterior graft limb projected superiorly and replaced the trapezoid ligament, while the dorsal limb of the graft was wrapped around the dorsal edge of the clavicle and reconstructed the conoid ligament. The dorsal end of the tendon graft was taken over the AC joint to reconstruct the superior AC ligament and capsule . These pThe patient was placed in the beach chair position. A standard 30° arthroscope was used. The technique included five aspects . Arthroscopy was initiated by inserting the arthroscope into the joint through the posterior portal. The lateral portal was opened by inserting a needle in the front of the long-head biceps tendon aimed at the proximal coracoid. The coracoid neck was exposed, and for that area, the interval was opened. After good access was established to the coracoid neck area, the arthroscope was moved to the lateral portal. The primary camera position during the actual reconstruction was in the lateral portal. The anterolateral and anterior portals were opened using a needle pointing to the coracoid neck. Debridement and sufficient exposure were conducted around the coracoid and clavicle. A longitudinal incision, 2.5 cm medially from the acromioclavicular joint, was made over the clavicle. The superior surface of the clavicle was exposed for drilling. A blunt tissue passageway was made behind the clavicle from the same opening for subsequent passing of the graft.A 2.4-mm guide pin was driven through the clavicle and the coracoid using a drill guide after which a 4.5-mm drill hole was made through the clavicle and coracoid. The perfect position of the coracoid drill hole essentially dictated the position of the clavicular drill hole. The clavicular drill hole was centrally located on the clavicle at ∼2.5 cm proximal to the acromioclavicular joint. The clavicular drill hole was then widened to 5.5 mm. The sutures were then passed through the drill holes using the blunt Lasso Guide . The tendon graft was first pulled through the clavicular and coracoid drill holes after which the suture for the interconnecting no. 5 suture was pulled through the clavicular and coracoid drill holes.A 2.4-mm guide pin was driven through the clavicle and the coracoid, after which only the clavicular drill hole was widened, to 5.5 mm. The suture for the tendon graft was passed through the clavicular drill hole and then medially under the coracoid using the Curved Lasso Guide  positioned in front of the clavicle and medial to the coracoid. Next, the proximal end of the lasso wire was picked up through the clavicular drill hole using a suture passer for subsequent transfer of the suture to the tendon graft. The tendon graft was then pulled through the clavicular drill hole and around the coracoid.The rest of the surgical procedure was identical in both groups. The distal graft limb end was pulled into the clavicular wound behind the clavicle. The interconnecting sutures were passed through the drill holes and pulled out through the anterolateral portal. The Subcoracoid Clip  was attached to the no. 5 suture loop and then pulled into position underneath the coracoid. The other end of the no. 5 suture and the anterior graft limb were slipped through the Clavicular Clip eyelet. The end of the dorsal graft limb was taken over the dorsal edge of the clavicle and tied to the anterior graft limb. The dorsal graft limb was kept longer than the anterior one.After the actual CC reconstruction was complete, the clavicular wound was extended over the AC joint. The overstretched AC joint capsule was opened along its fibers. The distal end of the clavicle was resected with an oscillating saw, and the distal clavicle was freed from possible soft tissue attachments and scar tissue. With the entire reconstruction in place, the clavicle was repositioned. The repositioning was visually controlled and checked. The interconnecting suture and tendon graft of the CC reconstruction were tensioned, and the interconnecting sutures were tied on the CC-Clip loop using a knot pusher. Once the sutures were tied, the loop of the Clavicula Clip was allowed to sink into the clavicular drill hole hiding the suture knot. The anterior graft limb end was then tied to the posterior graft limb and secured with no. 2 nonresorbable sutures. The superior AC ligament was reconstructed with the dorsal end of the tendon graft. The graft end was sutured over the AC joint, and the AC capsule was plicated tightly over it with interrupted no. 1 resorbable sutures. The arthroscopic portals were closed with interrupted sutures, while the clavicular wound was closed in layers .Fig 2(A)The patients were discharged the same day as the surgery, and each wore an arm sling for six weeks postsurgery. They were allowed to perform light rotatory movements and passive arm lifting within the limits of pain. Although the sling was removed after 6 weeks, active rehabilitation did not begin until 8 weeks after surgery, to provide enough time for recovery. The patients were allowed to resume heavy labor 3 to 4 months after surgery and overhead activity and sports at 6 months.t-tests, and graphs. Welch’s t-test was used to determine whether preoperative and postoperative groups were different from each other. For all t-tests, the null hypothesis stated that the difference between preoperative and postoperative groups was 0. For the clavicle drill hole diameter, significance could not be calculated because the perioperative diameter was not measured. Instead, the nominal drill diameter was used. However, in this case, a postoperative 95% CI of the mean was visually compared to the perioperative drill diameter to verify a statistically significant increase in the drill hole diameter. Regarding variables other than the drill hole diameter, statistical significance was declared when P < .05.Statistical analyses were calculated using the Minitab software, version 18.1 . This software includes descriptive univariate statistics , 95% confidence intervals (CI), A total of 32 consecutive patients were included. Nine (28.1%) of the cases were graded III and 23 (71.8%) were graded V, of which three had a lateral clavicular fracture. The time from original trauma to surgery varied from 4 weeks to 3 years. Twenty-nine (90.6%) operations were primary cases, while three (9.4%) were revision cases that followed previous failed surgeries. Twenty-nine operations (90.6%) were conducted using a semitendinosus tendon graft, while three (9.4%) were conducted with a synthetic polyurethane urea graft.Nineteen (59.4%) of the cases underwent surgery with the tendon graft going through the coracoid drill hole (Group 1), while 13 (40.6%) were treated with the graft loop going under the coracoid (Group 2). Thirty (93.8%) cases were male, whereas two were (6.3%) female. The mean age of the patients was 41.16 ± 12.93 years. In nineteen of the cases (58.3%), the dominant side underwent surgery.Indications for surgery included pain, distal clavicle instability, and scapular issues. Injuries older than 2 weeks were considered chronic.No wound infections related to clavicular wounds were found. No complaints about protruding knots on the clavicle or any sensation of foreign material underneath the skin were reported.There was one unsuccessful reconstruction, which was reoperated 6 months after the original operation. There was one clavicular fracture through the clavicular drill hole. The patient, an ice-hockey player, was rammed against the rink wall affecting the operated shoulder 8 months after the operation. The fracture was in an exact position, and the ligament reconstruction remained unharmed. The situation was treated conservatively, and the fracture healed well.  One AC jNo coracoid fractures were found. The obvious risk for a coracoid fracture is transfer of the tendon graft through the 4.5-mm coracoid drill hole. Otherwise, the techniques can be considered similar. Because no coracoid fractures occurred, the groups were handled as one in terms of the postoperative statistics. In seven of the cases, a sudden brief sensation of pain was reported 3–6 months after surgery. It was reported that the distal clavicle felt a little more mobile afterward. The pain disappeared soon, and the clavicle worked normally after that. No other complications occurred in the series. One case was lost from the radiographic follow-up because the patient refused to undergo follow-up radiographs. The reoperated original case was not included in the statistics.P < .000; P < .000; P < .000; P < .027; Among the 32 cases followed for a minimum of 1 year, the mean Nottingham Clavicle score increased from a preoperative mean of 41.66 ± 9.86 to 96.831 ± 5.86  Determining the size of a coracoid drill hole proved to be even more difficult. Therefore, tunnel widening of the coracoid drill hole was not measured in this study. The average CC distance increased during the follow-up. The increase was moderate and did not affect the stability of the distal clavicle or the procedural outcomes. The biggest increase was detected in the three revision cases. The reason for this increase may have been the pronounced instability and weaker support for the clavicle by the surrounding muscle attachments. The outcome scores were also slightly inferior. However, all three patients reported good subjective outcomes. The use of the synthetic tendon graft did not seem to affect the outcomes in this series.The Nottingham clavicle and Constant scores and Simple Shoulder Test improved significantly after surgery. In most cases, the patient satisfaction level mirrored the shoulder score levels. CC ligament reconstructions have been considered as difficult procedures plagued by complications.An arthroscopic approach in CC ligament reconstruction is a practical method, while the AC ligament reconstruction must be an open procedure. A technical error is usually the most obvious reason for less-than-perfect outcomes. With regard to the surgical technique, it is very important to position the drill holes correctly. The clavicular drill hole must be centrally located. The coracoid drill hole must be located both centrally and as proximally as possible. The distal end of the clavicle must be released from soft tissue interpositions, such as parts of the trapezius muscle, and the distal clavicle repositioning should be tension-free. Distal clavicle resection is recommended in chronic cases. Infection and fracture control and reconstruction stability are the key reconstruction elements. The typical surgical time, including harvesting and preparing the ST tendon, is 1.5–2 hours. It is extremely important that the recovery time is long enough to ensure a successful surgery. Postoperatively, an arm sling is worn for 6 weeks. Physiotherapy is initiated only 8 weeks postsurgery. The tendon graft must heal properly before rehabilitation is started. Therefore, the first 2 months postoperatively are critical. After 2 months, it is unlikely for the reconstruction to fail, excluding new external trauma.This study is not without limitations. First, we included revision surgeries, which may produce different results than only primary reconstructions. Also, the number of subjects in each group was different. This is because the graft countering the coracoid was used more often, which may also affect the results.In this series, combining the arthroscopic CC ligament reconstruction to an open reconstruction of the AC joint with a tendon graft proved to be a stable solution. The knot-hiding titanium implant effectively eliminated the problems related to the clavicular wound healing.