Tropomyosin (TM) is a heat-stable protein that plays a crucial role as a major pan-allergen in crustacean shellfish. Despite the high thermal stability of the TM structure, its IgG/IgE binding ability, immunodetection, and in vitro digestibility can be negatively influenced by glycation during food processing, and the underlying mechanism remains unclear. In this study, TM was subjected to glycosylation using various sugars and temperatures. The resulting effects on IgG/IgE-binding capacity, immunodetection, and in vitro digestibility were analyzed, meanwhile, the structural alterations and modifications using spectroscopic and LC-MS/MS analysis were determined. Obtained results suggested that the IgG/IgE binding capacity of glycosylated TM, immunodetection recovery, and in vitro digestibility were significantly reduced depending on the degree of glycosylation, with the greatest reduction occurring in Rib-TM. These changes may be attributable to structural alterations and modifications that occur during glycosylation processing, which could mask or shield antigenic epitopes of TM , subsequently reducing the immunodetection recognition and digestive enzyme degradation. Overall, these findings shed light on the detrimental impact of glycation on TMs potential allergenicity and digestibility immunodetection and provide insights into the structural changes and modifications induced by thermal processing. Shrimp is a vital primary source of protein to sustain the growing global population, owing to its abundance of high-quality proteins and nutrients ,2. AnnuaDespite the high prevalence of crustacean allergies, no effective therapy is currently available, leaving diet avoidance as the only recommended course of action. However, this poses a challenge for allergic consumers, as crustacean ingredients are commonly used in a wide range of food products ,3. ThereIn the thermal processing of food, in addition to generating single heat-induced protein structural changes, there are also interactions and modifications between the food matrix components and proteins, with the most important modification being glycation reactions between carbohydrates and proteins. Glycation reactions are widely present in the thermal processing of food and are one of the common interactions between proteins and sugars, also known as the Maillard reaction. In the process of glycation reactions, covalent bonds are formed between the free amino groups of proteins, primarily lysine  and arginine , and the carbonyl groups of reducing sugars through the amino acids of proteins ,7.Tropomyosin (TM) is a significant allergen that has been found in different crustaceans. It possesses high heat stability attributed to its distinctive α-helical coiled-coil structure, making it responsible for triggering a positive IgE-mediated allergic response in 72–98% of individuals with shrimp allergies. Significantly, TM in crustacean seafood is a type of protein rich in Lysine (Lys) that is prone to undergo glycation reactions with reducing sugars during the thermal processing of food. Researchers, both domestically and internationally, have extensively utilized glycation treatment on crustacean seafood TM to investigate its structural changes and allergenicity reduction. However, research findings have shown that when different sugar molecules are used to glycate TM, the allergenicity of TM can vary to different degrees . Hence, it can be inferred that glycation reactions can alter the structure of TM and lead to the destruction, masking, exposure, or generation of new antigenic epitopes, resulting in varying degrees of change in allergenicity ,8,9,10. Therefore, the present study aims to further investigate the effects of glycation treatment using different types of sugar molecules and different temperatures on shrimp TM, including its IgG/IgE binding capacity, immunodetection, in vitro digestibility, and structure. Additionally, the glycation modification sites will be further elucidated through HPLC-MS/MS analysis to clarify the molecular mechanisms underlying the reduction in immunodetection recovery of shrimp TM caused by various glycation treatments.6, 1.28 × 106, and 4.096 × 106 in rat antisera, rabbit antisera, and mAb, respectively. Horseradish peroxidase (HRP) labeled rabbit-anti-rat and goat-anti-rabbit IgG were purchased from Bai Aotong Experimental Materials Center . Horseradish peroxidase (HRP) conjugated goat anti-human IgE, and goat anti-rabbit IgG was purchased from Zhongshan Jinqiao Biotechnology Co., Ltd. . All chemicals used in the study were of analytical grade.Purified raw TM with at least 95% purity was obtained, as described in our recent study . PorcineAll clinically shrimp-allergenic patient sera (n = 6) and 2 normal human sera were collected from the Affiliated Hospital of Qingdao University  and tested using an ImmunoCAP system  . Writtenw/w) in a glycosylation sample buffer  at a concentration of 2 mg/mL. The mixtures were thoroughly mixed and filled into 2 mL centrifuge tubes, which were subjected to vacuum freeze-drying to obtain different sugar–TM mixtures. Subsequently, the dried samples were subjected to glycosylation in a desiccator containing anhydrous magnesium nitrate. For the preparation of glycosylated samples with the same sugars at different temperatures, the samples were subjected to glycosylation at 60 °C for 8 h. For the preparation of glycosylated samples with the same sugar (Rib) at different temperatures, Rib was used as the research target, and the samples were separately subjected to glycosylation at 25 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, and 100 °C for 8 h. The control groups consisted of samples without added sugar and samples treated with heat. After the glycosylation reaction, the samples were dissolved in ultrapure water and dialyzed using PBS (3 kDa) to remove unreacted sugar components. Subsequently, the dialyzed glycosylated samples were placed into 2 mL centrifuge tubes and subjected to vacuum freeze-drying to obtain different sugar–TMs and different temperature–TM glycosylated samples, which were stored at −20 °C for future use.Preparations of glycosylated samples of shrimp TM with different sugars at the same temperature and with the same sugar at different temperatures were prepared with slight modifications to the method described by Zhang et al.  Glucose The glycosylated samples were separated using gel electrophoresis using 5% stacking gels and 15% resolving gel, as described by Zhao et al. [The free amino group content of each glycosylated sample was determined using the o-Phthalaldehyde (OPA) method . For eacTM gel transfer stacks . After blocking with 1% (w/v) BSA, the membrane was incubated with a diluted solution of rat anti-TM sera , rabbit anti-TM sera , and TM monoclonal antibodies (1:5000 in PBST) for 2 h in an incubator. For IgE immunoblotting, the membrane was reacted with pooled shrimp patients’ sera (diluted 1:4) overnight at 4 °C using an incubator. Subsequently, HRP-labeled goat anti-rabbit IgG  and HRP-labeled goat anti-human IgE (1:2000) were added at room temperature for a 1 h incubation. After washing the membrane three times with PBST  for 10 min each, the results were analyzed using a BIO RAD Universal Hood II gel imaging system  with multiple exposures and ECL solutions to obtain an optimal signal.Immunoblotting was performed following the method described by Zhao et al. . In brie2CO3-NaHCO3 (CBS) buffer (pH 9.6) to 5 μg/mL and coated in a Corning-Costar ELISA microplate  with 100 μL per well at 4 °C overnight. Following this, the plates were blocked with 1% (w/v) BSA (200 μL/well) at 37 °C for 2 h. These plates were then incubated with rat antisera , rabbit antisera , and TM monoclonal antibodies  against TM for 1 h. In addition, polled shrimp-allergic patients’ sera diluted 10 with PBST at 37 °C for 1.5 h diluted 10-fold with PBST were added and incubated at 37 °C for 1.5 h. These plates were subsequently washed and incubated with HRP-labeled rabbit anti-rat IgG , HRP-labeled goat anti-rabbit IgG , HRP-labeled rabbit anti-mouse IgG  or HRP-labeled goat anti-human IgE  for 1 h at 37 °C. Afterward, the enzyme reaction was initiated upon adding the TMB solution and stopped after adding sulfuric acid. The absorbance was measured at 450 nm using a MultiscanMK3 microplate reader .In indirect ELISA, the glycosylated samples were diluted using 50 mM Naw/v) BSA solution, as previously described. Next, the assay was conducted by adding TM standards or differently treated samples, all at an equal concentration (100 μL/well). The plates were incubated for 20 min at 37 °C. Afterward, the wells were washed three times with PBST . Subsequently, 100 μL/well of rabbit anti-TM sera, diluted 1:10,000 with PBST, were added and incubated for 60 min at 37 °C. Following another round of washing, the plates were incubated with HRP-labeled anti-TM antibodies, diluted 1:10,000 in PBST, for 60 min at 37 °C. Finally, the subsequent determination was performed according to the aforementioned protocol.The immunodetection recovery of the glycosylated samples was determined using PcAb and mAb sELISA analysis following the method previously reported by Zhao et al. . To summ2CO3 solution  was immediately added. Similarly, in the simulated intestinal digestion process, glycosylated samples were mixed with equal volumes of simulated intestinal fluids (SIF) containing trypsin (100 U/mL of digestate). The mixture was incubated for the same time intervals as in the gastric digestion process. After each time interval, aliquots of the digested solution were taken out, and the digestion process was halted by adding Pefabloc®  for further analysis. Finally, all the digestion samples were subjected to SDS-PAGE analysis.In vitro simulated gastrointestinal digestion of the glycosylated samples was performed as previously described by Zhao et al. . During CD spectrum data of the glycosylated samples between 190 and 260 nm were collected using a Jasco-810 spectropolarimeter  at 25 °C. The samples, each at a concentration of 0.2 mg/mL, were scanned three times at a scanning speed of 120 nm/min using a 1 mm pathlength quartz cuvette. Finally, the secondary structure contents were determined using the CDNN Program .UV spectrum data of the glycosylated samples (0.2 mg/mL) were recorded from 200 to 320 nm at a wavelength interval of 0.5 nm, using a UV-vis spectrophotometer  at 25 °C. PBS was used as the blank.The intrinsic and extrinsic fluorescence spectra of the glycosylated samples (0.2 mg/mL) were determined following the method described by Zhao et al.  using anw/w). The enzymatic digestion was performed overnight at 37 °C. The digested samples were then subjected to ultrafiltration using 10 kDa cutoff filters to remove undigested proteins, and the filtrate was collected. Subsequently, all samples were desalted using ZipTip C18 columns. The specific protocol is as follows: each sample was washed five times with pure acetonitrile washing solution, activated with a 50% acetonitrile aqueous solution (v/v), equilibrated with 0.1% formic acid aqueous solution (v/v), and then repeatedly aspirated and expelled 15–20 times for sample adsorption. After that, the ZipTip C18 columns were washed three times with 0.1% formic acid aqueous solution (v/v) for desalting. Gradient elution was performed using different elution solutions: 20 μL of 0.1% formic acid aqueous solution and 20% acetonitrile aqueous solution (v/v), 20 μL of 0.1% formic acid aqueous solution and 50% acetonitrile aqueous solution (v/v), and 20 μL of 0.1% formic acid aqueous solution and 70% acetonitrile aqueous solution (v/v). The eluates were collected in small vials, and 100 μL of 0.1% formic acid was added for subsequent HPLC-MS/MS analysis.Preparation of enzymatically digested peptides from glycosylated TM samples: first, samples with different glycosylation patterns were subjected to enzymatic digestion using trypsin at a substrate-to-enzyme ratio of 1:50 (v/v) and 0.1% formic acid–water solution (v/v), respectively. The adsorbed peptides were dynamically eluted and separated according to the following gradient program: 0–1 min, 5%A, and 95%B; 1–2.5 min, 5–10%A; 2.5–12.5 min, 10–25%A; 12.5–20 min, 25–52.5%A; 20–22 min, 52.5–90%A; 22–24 min, 90–5%A; 24–30 min, 5%A and 95%B.HPLC conditions: the chromatographic column used was a Hypersil gold C18 column . The flow rate was set at 0.2 mL/min, the injection volume was 20 μL, and the column temperature was maintained at 40 °C. The mobile phases A and B comprised 0.1% formic acid–acetonitrile solution (2) flow rate was 35 arb, and the auxiliary gas flow rate was 10 arb. The scan mode was set as full scan + data-dependent MS2 mode, with a scanning range of m/z 200–200, a resolution of 70,000, a normalized collision energy of 30, and a resolution of 17,500.MS/MS analysis parameters: the analysis was performed in positive ion mode using electrospray ionization (ESI+). The spray voltage was set at 3.6 kV, the capillary temperature was 300 °C, the sheath gas . The parameters were set as follows: a peptide precursor ion tolerance of 10 ppm, MS/MS matching tolerance of 0.5 Da, allowance of up to 3 missed cleavage sites, high peptide confidence level, and trypsin was used as the protease. The fixed modification was carboxymethylation, and the variable modifications included oxidation, carbamylation, and deamidation.p < 0.05 considered indicative of a statistically significant difference.Experimental data were analyzed and plotted using SPSS 25.0 and Origin 2022b. Data were presented as mean ± standard deviation (n = 3). Statistical significance analysis was conducted using the ANOVA method, with SDS-PAGE analysis was performed to analyze the molecular weight changes in the glycosylated TM samples under different types of sugars and temperatures. The results are shown in Furthermore, the effect of different reaction temperatures on the degree of TM glycosylation was investigated using the Rib−TM glycosylation reaction system. As shown in Patinopecten yessoensis) when using different types of sugars . Zhang et al. [Exopalaemon modestus and sugars of different molecular weights  and observed a significant decrease in the content of free amino groups under different sugar systems. Within a certain range of molecular weights, smaller sugar molecules were found to more readily undergo glycation reactions with TM, primarily due to the influence of steric hindrance by sugar molecules. The effect of different reaction temperatures on the content of free amino groups in Rib-TM glycation samples is shown in Glycation reactions are widely present in the thermal processing of food, and the glycation of proteins is mainly due to non-enzymatic reactions between free amino groups on the protein surface and sugar molecules. Therefore, the content of free amino groups in TM glycation samples can be used as an important indicator to evaluate the degree of TM glycation ,7. TM isg et al.  performeHidrobacter spp.  by WB. Bai et al. [Chlamys nobilis) TM and found that the WB analysis of glycosylated TMs showed a similar decrease in IgE blotting intensity. The intensity of IgG immunoblotting was significantly reduced in all glycosylated TM samples, with the lowest IgG immunoblotting intensity in the pentacosylated xylose-TM glycosylated samples. The increase in the IgG/IgE immunoblot intensity of a glycosylated TM indicates a significant decrease in its IgG/IgE binding capacity, which may be attributable to the structural alterations and modifications of TM caused by glycosylation, resulting in the masking or destruction of its IgG/IgE epitopes, making it less recognizable by IgG/IgE [The antibodies used to construct the PcAb and mAb sELISA methods and human sera from shrimp allergic patients were used to assess the effect of different glycosylation treatments on TM IgG/IgE binding capacity by WB analysis, and the results are shown in i et al.  used ara IgG/IgE ,14,17.p ≥ 0.05), except for Rib−TM. However, there was a slight decrease in the IgE binding ability of Glu and Lac glycosylated TM (p < 0.05), with reductions of approximately 27.3%, 25.8%, 50.6%, and 68.0% respectively.The indirect ELISA analysis was further conducted using TM rat and rabbit polyclonal antisera, mAb, and human serum from shrimp allergic patients to evaluate the IgG/IgE binding ability to untreated and different glycosylated forms of TM, as shown in lated TM . On the p ≥ 0.05) in the IgG/IgE binding values of TM compared with untreated TM in the glycosylation reaction system below 50 °C (p < 0.05) in the IgG/IgE binding ability of TM. The trend in IgG/IgE variation for glycosylated TMs under different temperature reaction systems was also consistent with its degree of glycosylation; as the degree of glycosylation increased, there was a significant decrease in the IgG/IgE binding ability of TM. This is mainly due to the structural changes and modifications of TM antigenic epitopes caused by glycosylation, making it less recognizable by IgG/IgE antibodies [p > 0.05)  . This map < 0.05). The highest reduction was observed in Rib-TM, with decreases of 85.0% and 95.5% in PcAb and mAb sELISA analyses, respectively. This was followed by Glu-glycosylated TM (30.6% and 57.2%), Chi-glycosylated TM (33.2% and 44.9%), Lac-glycosylated TM (20.7% and 42.8%), and Tre-glycosylated TM (20.8% and 35.5%). As shown in p < 0.05). In particular, Rib-glycosylated TM showed a nearly 100% reduction in the detection recovery rate in the glycosylation reaction system above 80 °C. The decreasing trend in immunodetection recovery of different glycosylated TMs was consistent with their degree of glycosylation. As the degree of glycosylation increased, the detection recovery rate for glycosylated TMs significantly decreased. This can be attributed mainly to the modification and destruction of TM antigenic epitopes caused by glycosylation treatment, leading to a significant reduction in the content of the “antibody–antigen–antibody” complex formation of TM in sELISA evaluation [The immunodetection recovery of TM with different glycosylation treatments was determined using the PcAb and mAb sELISA. As shown in aluation .p < 0.05). This may be attributable to the higher sensitivity of the sELISA detection system compared with the indirect ELISA evaluation system that only requires one antibody. Additionally, non-glycosylated Tre-glycosylated TM also exhibited a similar decrease in detection recovery rate, which may be due to the structural changes of TM during glycosylation and the non-covalent interaction between Tre and TM resulting in the masking of TM antigenic epitopes [Furthermore, the immunodetection recovery of PcAb sELISA for the same glycosylated TM was significantly higher than that of mAb sELISA. This may be due to the fact that mAb only targets specific epitope regions on the protein molecule, while PcAb can undergo specific immune recognition with multiple epitope regions on the target protein molecule. As a result, the mAb sELISA system is more susceptible to structural changes in the target analyte, leading to a significant decrease in its detection accuracy ,13. It iepitopes . Accordiepitopes ,19. ThesThe SDS-PAGE analysis of the in vitro digestion stability of TM influenced by different glycosylation treatments is shown in Exopalaemon modestus TM [Scophthalmus maximus parvalbumin [During the digestion process of untreated and differently glycosylated TMs in simulated intestinal fluid (SIF), the band intensity of both untreated and differently glycosylated TM proteins gradually decreased with increasing SIF digestion time b,d. It iestus TM , Scophthvalbumin , soy provalbumin , and milvalbumin . This isvalbumin ,21. It ivalbumin ,26, therExopalaemon modestus, and CD spectrum analysis also revealed a significant decrease in α-helical structure content accompanied by a marked increase in irregular coil structure. Fu et al. [Penaeus chinensis using ribose, oligogalactose, and chitooligosaccharides, and CD spectrum analysis showed a significant decrease in α-helical structure content accompanied by a significant increase in β−fold content. They also found that the maintenance of α−helical structure content plays an important role in the sensitivity of TM. In addition, untreated TMs (Tre and Chi) also showed a significant decrease in α−helical structure content, especially Chi, which may be due to the active groups of non-reducing sugars Tre and Chi being able to interact with the protein through non-covalent interactions such as hydrogen bonding and hydrophobic interactions [The impact of different glycosylation treatments on the secondary structure of TM can be understood through the analysis of CD spectra. The CD spectral analysis results of TM with different glycosylation treatments are shown in u et al.  synthesiractions ,26, leadThe results of the UV absorption spectral analysis of different glycosylated TMs are shown in max) corresponding to the maximum absorption peak of the intrinsic fluorescence spectra of untreated TM is around 305.2 nm. With the application of glycosylation treatments, the maximum fluorescence intensity at λmax of glycosylated TMs show varying degrees of increase, but there is no significant shift in λmax. Among them, the fluorescence intensity corresponding to λmax of Rib−TM decreases most significantly, followed by Chi−TM, Tre−TM, Glu−TM, and Lac−TM samples  (NCBI ID: ACB38288), it is known that TM contains multiple Lys(K) (29 aa/284 aa), which are susceptible to glycosylation reactions with reducing sugars [To understand the mechanism of the effect of glycosylation treatment on the IgG/IgE binding capacity, immunodetection, and in vitro digestibility of TM, the TM glycosylation sites with different glycosylation reactions were further identified using Q Exactive mass spectrometry. As shown in g sugars ,20. In aScylla paramamosain. Mass spectrometry analysis revealed three glycosylation sites  within TM IgE binding epitopes, which may be the main factors leading to a decrease in IgE binding ability. Studies have reported that glycosylation modification of Exopalaemon modestus TM with reducing sugars of different molecular weights disrupts or masks multiple key amino acid residues (Lys) within these TM epitopes, resulting in a significant decrease in IgE binding ability and ultimately reducing the allergenicity of different glycosylated TMs. Further analysis of the impact of different glycosylation sites on the binding epitopes of TM IgE reveals that Glu-TM contains two IgE binding epitopes that are glycosylated, namely E5b: 142–162 and E5c: 157–183. Additionally, three IgE binding epitopes  in Rib-TM were found to be modified. Furthermore, one glycosylated modification site (E5c: 157–183) was identified in Glu–TM. These results suggest that glycosylation modification leads to varying degrees of glycosylation of TM epitopes, causing their dilution or masking, ultimately resulting in a significant decrease in its IgG/IgE binding capacity and immunodetection recovery [Scylla paramamosain). Mass spectrometry analysis revealed that three glycosylation sites  were discovered in TM IgE epitopes, which may be the main factors contributing to decreased IgE binding ability. Another study reported that glycosylation modifications by different molecular weight reducing sugars disrupted or masked multiple key lysine residues in the TM epitopes of Exopalaemon modestus, leading to a significant decrease in its IgE binding capacity and ultimately weakening the allergenicity of different glycosylated TMs [Chlamys nobilis). Through indirect ELISA, basophil activation assay, and shotgun proteomics analysis, it was observed that six IgE binding epitopes of TM were modified by glycosylation, resulting in a significant decrease in its allergenicity.Further analysis of the effects of different glycosylation sites on the IgE binding epitopes of TM reveals that Glu-TM has two IgE binding epitopes that are glycosylated, namely E5b: 142–162 and E5c: 157–183. Rib–TM has three glycosylated IgE binding epitopes . One glycosylated modification site (E5c: 157~183) was identified in Glu-TM. Based on these results, it can be concluded that glycosylation leads to differential glycosylation of TM epitopes, resulting in the destruction or masking of these epitopes. As a result, the IgG/IgE ability of TM, immunodetection, and in vitro digestibility are significantly reduced ,35. Han recovery ,35. In aated TMs . Additioated TMs  performeIn conclusion, the IgG/IgE binding ability, immunodetection, in vitro digestibility, and structural changes in TM glycosylated with different types of sugars  and various glycosylation temperatures were investigated. The IgG/IgE binding ability of glycosylated TM, immunodetection recovery, and in vitro digestibility all show a significant decrease after glycosylation treatment. The extent of reduction depends on the degree of glycosylation, with Rib (pentose sugar) glycosylated TM exhibiting the greatest reduction, followed by Glu, Lac, Tre, and Chi. Additionally, as the glycosylation temperature increases, a decrease in binding ability to IgG/IgE, immunodetection recovery rate, and in vitro digestibility becomes more pronounced. Based on CD, UV, and intrinsic/extrinsic fluorescence spectroscopy analyses, significant structural changes are observed in glycosylated TM. The secondary structure undergoes rearrangement, with the typical linear α-helical structure of TM being disrupted and gradually transformed into β-turns and irregular coils during heat treatment. The three-dimensional structure becomes twisted and folded, and the surface hydrophobicity increases significantly, even leading to hydrophobic aggregation. Glycosylation sites are identified using HPLC-MS/MS, with three epitopes  of TM found to be susceptible to glycosylation modification. It is worth noting that Tre-TM and Chi-TM, which did not undergo glycosylation modification, also exhibit a significant decrease in the immunodetection recovery rate. This suggests that different sugar molecules may not only lead to a loss of IgG binding sites on TM through structural disruption and epitope modification but also produce non-covalent interactions between sugar molecules and the target protein, resulting in the masking or shielding of antigenic epitopes on the target protein, thereby reducing the recovery rate of the target protein in immunodetection.


The aim of this study was to assess the impact of abutments with angled screw access channel on the retention of zirconia crowns.n = 7). Control group (Group STA) included straight screw access channel abutments. Study group (Group ASC) included angled screw access channel abutments. Following aging , the pull-off forces (N) were recorded by using retention test (1 mm/min). Types of failure were defined as ; Type 2; Cohesive failure when luting agent remained on both Ti-base and crown surfaces; and Type 3; Adhesive failure when luting agent predominantly remained on the crown (> 90%). Statistical analysis was conducted by using (IBM SPSS version 28). Normality was checked by using Shapiro Wilk test and Q-Q plots. Independent t-test was then used to analogize the groups.Seven implant replicas were inserted in epoxy resin blocks. Fourteen zirconia crowns for central incisor tooth were digitally fabricated and cemented to titanium bases (Ti-bases) with resin cement. Titanium bases were categorized into 2 groups (P < .05). Failure modes were Type 2 for group STA and Type 3 for group ASC.Mean ± standard deviation of retention force records ranged from 1731.57 (63.68) N (group STA) to 1032.29 (89.82) N (group ASC), and there was a statistically significant variation between the 2 groups (The retention of zirconia crowns to abutments with a straight screw access channel is significantly higher than abutments with angled screw access channel. The management of dental situations with screw-retained restorations has been applicable with the spread of abutment with Angled Screw access Channel (ASC) . In the The screw access channel of the ASC might be located anywhere between 0 and 25° from the implant long axis , 4 Ball The ASC abutment has larger screw access opening and fewer axial walls than the straight abutment as a reason of the greater angulation. Therefore, the surface area between the abutment and the crown is reduced.Recently, titanium bases (Ti-bases) have become a popular part in the digital workflow of implant supported restorations. One advantage is that the crown and the Ti-base can be extra-orally bonded together and then that one piece restoration can be screw retained to the implant . TherefoMoreover, restorations with angled screw channel can be digitally designed and fabricated through the advancements of the software programs and implant systems .Retention force is directly related to the abutment’s surface area. The geometry of the abutment may be a priority to chemical bonding as it provides more retention . The retTherefore, this in vitro study aimed to assess the impact of abutments with angled screw access channel on the retention force applied on zirconia crowns. The null hypothesis was that the angled screw channel Ti-bases have no effect on the retention of adhesively bonded zirconia crowns.Seven implant analogues were embedded into epoxy resin blocks. Implant replicas were maintained in position with a guide pin and connected to a laboratory surveyor. The surveyor was used to permit the fixation of the implant replicas into the resin blocks . Implantn = 14) samples that were grouped into 2 groups based on the angulation of the screw-access channel. Group STA (control group) included (n = 7) straight screw access channel Ti bases  angled screw access channel Ti bases [The sample size was calculated presuming 5% alpha error and 80% study power. Sample size was based on Rosner’s method  calculatTi bases  Fig. 2)n = 14) sSingle zirconia crown for central incisor teeth was digitized for each sample. For Group STA, straight screw access channel Ti-base  of size 4.5 × 6 × 2.5 mm was selected from the library on  CAD software. The screw access hole of the straight Ti-base is located at the incisal edge. The screw access hole of the restoration was designed to be located near the incisal edge Fig. .Fig. 3ScFor Group ASC, angled screw access channel Ti-base  of the same dimensions was selected from the library on  CAD software. The screw access channel was situated on the palatal surface with angulation 24 degrees  for the milling process of zirconia crowns .The crowns of both groups were designed precisely for easier pull off the retention test.Polytetrafluoroethylene tape was used to close the screw access hole. Aluminium oxide particles (120 µ) were used to sandblast the inner surface of the crown. The restoration was cleaned in an ultrasonic unit for approximately 1 min. The restoration was then thoroughly washed with water spray and dried with water/air followed by cleaning with Ivoclean. A single coat of primer was applied with a brush to the pre-treated surfaces. The primer was left for 180 s then was dissipated with a strong stream of air. Self-adhesive resin sealant  was used as a bonding agent to bind zirconia crowns to their corresponding Ti-bases . Spot cuThe samples were exposed to artificial aging through thermal cycling  according to ISO TR11405 (1994) and mechanical loading  in a mastication simulator, which is correspondent to relatively 1 year of masticatory cycles , 21. An Universal testing machine  was used to implement the retention testing with a customized device for axially separating crowns from abutment surface at 1 mm/min crosshead speed , 22. TheA software program  was used to register the records for the maximum pull-off force in newtons (N), for each sample. After the retention test, the abutment surfaces and the inner surfaces of the restorations were visually inspected with a Stereomicroscope  under × 20 magnification for remaining cement, and classification of failure modes  was used for the statistical analysis to estimate the differences between the groups. Normality was checked by using Shapiro Wilk test and Q-Q plots. Values were normally distributed; therefore, groups were compared by using independent t-test. The tests were two-tailed, and the level of significance was set at P < 0.05) as illustrated in Table Mean value of retention forces in group STA 1731.57 N) was higher than that of group ASC (1032.29 N). Therefore, there is statistically significant difference between pull-off retention forces of straight and angled screw access channel abutments  for the straight abutment group, indicating cohesive failure. While, in the angled screw access channel abutment, most cement remained on the intaglio of the crowns (Type 3), which identified an adhesive failure between the Ti-base surface and the luting agent. These observations assist the benefit of increasing means of retention to improve the adhesion of angled screw channel abutments to titanium surfaces.In this study, the methodology was used to mimic the standard clinical situations. Thermocycling and mechanical loading were performed to replicate the oral environment . ThermocThe retention of zirconia crowns to abutments with a straight screw access channels is significantly higher than abutments with angled screw access channel. Angled screw access channel abutment causes reduction in the pull-off retention force of zirconia crown. It is recommended to perform further invivo studies to prove the invitro findings of this study.


Infectious bronchitis virus (IBV) causes an acute and highly infectious viral disease of chickens, which brings huge economic losses to the poultry industry. Forsythoside A (FTA) is a natural ingredient with wide pharmacological and biological activities, which has been demonstrated to have anti-IBV activities. In the present study, we found that IBV replication reached the highest level of 7.44 ± 0.11 copies/μL at 48 h post infection (hpi) and caused cytopathic effect and apoptosis in baby hamster kidney (BHK) cells. FTA pretreatment significantly inhibited IBV replication in a dose-dependent manner with an inhibition rate of up to 51.1%. In the presence of interferon deficiency, FTA pretreatment also inhibited IBV replication, indicating that the inhibition of FTA on IBV replication did not rely on the innate immune response of host cells. Additionally, we found that FTA pretreatment attenuated inflammation, apoptosis, and autophagy induced by IBV infection. UV-irradiation-inactivated IBV did not affect the viability of BHK cells, but it upregulated the expressions of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), and nuclear factor kappa-B (NF-κB) proteins, suggesting that the apoptosis and autophagy induced by IBV infection were through activating the PI3K/Akt/NF-κB pathway. FTA pretreatment downregulated the PI3K/Akt/NF-κB pathway, and the PI3K activator and inhibitor were proven to show that FTA pretreatment inhibited IBV-induced apoptosis and autophagy by attenuating the PI3K/Akt/NF-κB pathway.Infectious bronchitis virus (IBV) is an acute and highly infectious viral disease that seriously endangered the development of the chicken industry. However, due to the limited effectiveness of commercial vaccines, there is an urgent need to develop safe and effective anti-IBV drugs. Forsythoside A (FTA) is a natural ingredient with wide pharmacological and biological activities, and it has been shown to have antiviral effects against IBV. However, the antiviral mechanism of FTA is still unclear. In this study, we demonstrated that FTA can inhibit cell apoptosis and autophagy induced by IBV infection by regulating the PI3K/AKT/NF-κB signaling pathway. This finding is important for exploring the role and mechanism of FTA in anti-IBV infection, indicating that FTA can be further studied as an anti-IBV drug. Avian infectious bronchitis (IB), an acute and highly infectious viral disease, has caused huge economic losses to the worldwide poultry industry since the 1930s . Avian iTo survive and replicate within a host, viruses have evolved various strategies to evade the host’s innate immunity responses –10. FoHost cellular kinases can regulate the replication and the relevant pathogenesis of viruses, which can be used to identify the novel therapeutic targets –25. ThForsythia suspense, exerts various pharmacological activities, including anti-inflammatory, antibacterial, antioxidant, antiviral, and neuroprotective properties through diverse mechanisms (in vivo and in vitro (Forsythoside A (FTA), the main active ingredient in the traditional Chinese medicine chanisms –43. Itchanisms , 45. Spein vitro , 45. Howin vitro , 47. Bas50 of IBV infected for 48 h, at this time, the activity of BHK cells decreased to 65.4%. In Firstly, we observed the IBV infection in BHK cells. As shown in To avoid the cytotoxicity of FTA, an MTT assay was used. P < 0.001), but it was notably reversed in FTA (25 and 50 µM) groups (P < 0.05). The mRNA expression of pro-inflammatory cytokines were assayed by a real-time polymerase chain reaction (RT-PCR) assay. In P < 0.001), but FTA pretreatment can downregulate them in a dose-dependent manner (P < 0.01).The EdU immunofluorescence stain assay was used to study the effect of FTA pretreatment on cell proliferation. As shown in P < 0.001), while FTA pretreatment downregulated it in a dose-dependent manner (P < 0.05). In P < 0.0.5), while FTA pretreatment can significantly block the trends (P < 0.05).Additionally, to study the effect of FTA on apoptosis induced by IBV infection, we analyzed the apoptosis rate using the FCM assay, and detected the level of pro-apoptotic proteins (caspase-3 and BAX) and anti-apoptotic protein (Bcl-2) using the western blot assay and caspase-3 activity assay kit. In P < 0.05), and p62 protein was significantly downregulated in the IBV group (P < 0.05). However, FTA pretreatment was ameliorated the trends (P < 0.05).To further investigate the effect of FTA pretreatment on autophagy induced by IBV, we tested the levels of Beclin-1, p62, and LC3B proteins using the western blot assay. As shown in In conclusion, these data showed that FTA pretreatment can attenuate the inflammation, apoptosis, and autophagy induced by IBV infection in BHK cells.P < 0.05) while downregulated in a dose-dependent manner in the FTA groups (P < 0.05).To investigate the mechanism of FTA protecting BHK cells from IBV infection, we focused on the expression of PI3K/AKT signaling pathway. In P < 0.01) (P < 0.05).To further determine the relationship between FTA’s inhibition and PI3K/Akt/NF-κB, UV-irradiation-inactivated IBV was used to infect BHK cells. We found that UV-irradiation-inactivated IBV did not affect the BHK cell viability , but it  < 0.01) . And FTAIn conclusion, all results indicate that the PI3K/Akt/NF-κB signal pathway was activated by IBV infection, and FTA can inhibit it.To determine the relationship between the mechanism of FTA inhibiting apoptosis and autophagy induced by IBV and the PI3K/AKT/NF-κB pathway, the PI3K activator 740Y-P (10 µM) and PI3K inhibitor LY294002 (10 µM) were used to over-express or inhibit expression of PI3K protein. In Additionally, the PI3K activator 740Y-P exacerbated the expressions of PI3K, Akt, and NF-κB-p65 proteins induced by IBV infection. It also enhanced the apoptosis and autophagy of BHK cells. However, these changes were attenuated by FTA pretreatment . FTA andIn summary, our results findings indicated that IBV infection induced autophagy and apoptosis partially through the PI3K/Akt/NF-κB signaling pathway, while FTA pretreatment prevented these effects.IBV brings huge economic losses to the poultry industry. However, the commercially available vaccines have shown limited efficacy. Although the antiviral activities against IBV of FTA have been demonstrated, the diverse mechanisms are still unclear. Our study aims to explore the effect and mechanism of FTA on IBV infection in the BHK cells. We found that IBV could infect BHK cells and induce apoptosis at 12 hpi. Additionally, FTA pretreatment for 2 and 12 h significantly inhibited IBV replication. Further research proved that this was not dependent on the innate immune response of host cells, such as IFN activation. Furthermore, our experiments demonstrated that FTA can significantly inhibit cellular processes induced by IBV infection such as inflammation, apoptosis, and autophagy. These effects may be achieved by regulating the PI3K/Akt/NF-κB signaling pathway. Overall, these results were consistent with previous studies , 45, indin vitro  and was obtained highly purified (≥98%) with an average molecular weight of 624.59. The PI3K agonist 740Y-P  was purcThe 3-−2, 5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich . The pMD18-T plasmids expressing IBV-N were purchased by Sanhon Biotech .2.A fibroblast cell line baby hamster kidney (BHK) cells (Cat No. FS-0185) and the African green monkey kidney (Vero) cells (Cat No. CCL-81) were purchased from American Type Culture Collection . The cells were cultured in Dulbecco’s modified Eagle medium , supplemented with 10% fetal calf serum  and 1% penicillin-streptomycin  in a constant temperature incubator at 37°C with a humidified atmosphere containing 5% CO50 of IBV and periodic rocking. After incubation for 1 h, the cells were washed thrice with phosphate buffer saline  to remove any unbound virus and replenished with fresh DMEM supplemented with 3% FBS. Subsequently, the cytopathic effect was observed under a light microscope .The IBV strain Beaudette US (Cat No. VR-22) was obtained from the ATCC , 24, amp50) for 0, 6, 12, 24, 36, 48, 60, and 72 h . To determine the non-cytotoxic concentration of FTA, BHK cells were treated with FTA  for 72 h, and Vero cells were treated with FTA  for 72 h .The design of this experimental as shown in 50 of IBV or UV-IBV for 48 h. In FTA groups, cells were treated with FTA  for 2 or 12 h prior IBV infection .In FTA anti-IBV research, the experiment divided into three groups: negative control group (NC), positive control group of IBV or UV irradiation inactivated IBV infection (IBV or UV-IBV), and FTA treatment for IBV infection groups (FTA). In the NC group, cells were not subjected to any treatment. In IBV or UV-IBV group, cells were infected with 10 TCID50 of IBV for 48 h. In FTA + IBV group, BHK cells were treated with FTA (50 µM) for 12 h before IBV infection. In Y-P or LY group, BHK cells were treated with PI3K activator 740Y-P (10 µM) for 2 h or PI3K inhibitor LY294002 (10 µM) for 1 h. In Y-P + IBV or LY + IBV group, BHK cells were treated with PI3K activator or PI3K inhibitor before IBV infection. In Y-P + IBV + FTA or LY + IBV + FTA group, BHK cells were treated with PI3K activator or PI3K inhibitor before FTA treatment and then infected with IBV .In FTA anti-IBV mechanism research, the experiment divided into six groups: negative control group (NC), positive control group of IBV infection (IBV), FTA treatment for IBV infection group (FTA + IBV), PI3K activator 740Y-P or PI3K inhibitor LY294002 treatment group (Y-P or LY), FTA PI3K activator or PI3K inhibitor treatment for IBV infection group (Y-P + IBV or LY + IBV), and combination of PI3K activator or PI3K inhibitor and FTA treatment of IBV infection group (Y-P + FTA + IBV or LY + FTA + IBV). In the NC group, BHK cells were not subjected to any treatment. In the IBV group, BHK cells were infected with 10 TCID50/mL.BHK cells and Vero cells were seeded in 96-well plates and infected with serial 10-fold dilutions of samples in 8 replicates. The plates were incubated for approximately 7 days at 37°C, and the titers of IBV were calculated using the Reed-Muench method and are shown as the TCID4 CFU/well and treated with IBV, UV-irradiation-inactivated IBV, or FTA for 0, 6, 12, 24, 36, 48, 72 h, respectively. Subsequently, the cells were incubated with colorless DMEM (80 µL/well) and 5 mg/mL MTT (20 µL/well) solution at 37°C for 4 h. After washing three times with PBS, 0.05% DMSO (100 µL/well) was added, and the plate was gently shaken for 10 min. The optical density (OD) value of wells at 490 nm was measured using an enzyme-linked immunosorbent assay reader   . The perThe replication of IBV in BHK and Vero cells was tested by the plaque assay. IBV-infected cells were seeded in 6-well plates and overlaid with DMEM containing 3% FBS and 3% methyl cellulose . The pla50 of IBV for 0, 12, 24, 36, and 48 h. Following the manufacturer’s instructions, the BHK cells were digested with trypsin, collected by centrifugation, and washed with PBS. Then stained with annexin V-FITC and PI, all cells were visualized by a fluorescence microscope.The IF assay was performed to determine the apoptosis and proliferation of BHK cells. Cell apoptosis was tested using commercial Annexin V-FITC/propidium iodide (AV/PI) dual staining commercial kits . BHK cells were seeded in 6-well plates and infected with 10 TCID50 of IBV for 48 h. Following the user manual, all cells were labeled by EdU and Hoechst 33342 and observed under fluorescence microscope.Cell proliferation was assayed using the BeyoClick EdU-488 Cell Proliferation Assay Kit (Beyotime) . The BHKAll results were visualized by a fluorescence microscope  at a magnification of 400×, and the signals were counted in five random visional fields. The Image pro software (USA) was used to analyze these images.50), the 50 µL of cellular lysate supernatant, 40 µL of detection buffer, and 10 µL of caspase-3 substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (2 mM) were added into the plates and incubated for 60 min . Briefly, after the BHK cells in 96-well plates were treated with FTA  and IBV (10 TCIDn amount .50), the BHK cells were digested with trypsin, collected by centrifugation, and washed with PBS. Then stained with annexin V-FITC and PI, the cells were analyzed by FCM (Becton Dickson). The cells were divided into (annexin V/PI) early (annexin V positive and PI-negative) and late apoptotic (annexin V positive and PI-positive).The flow cytometry (FCM) assay was used to test the apoptosis of BHK cells. The Annexin V-FITC/propidium iodide (AV/PI) dual-staining commercial kits were purchased from Beyotime Biotechnology . Followi6 cells were seeded in 6-well plates for at least 12 h before treatment with IBV (10 TCID50) and FTA . Total RNA from cells was isolated using the RNApure Tissue & Cell Kit  and SteadyPure Virus DNA/RNA Extraction Kit . Additionally, total RNA was used for reverse transcription using HiFi-MMLV cDNA Kit  and oligo-dT priming as per the manufactures’ instructions. For qPCR, cDNA was amplified using UltraSYBR Mixture  in Bio-Rad IQ5 Thermal Cycler (Bio-Rad)  . The priCT−∆∆ method or y = −3.3049x + 36.652 .The β-actin was used as a reference gene. The expression fold changes were calculated using the 2The total protein lysate from BHK cells was extracted using an extraction kit  and quantified by using a BCA protein assay kit . The β-actin images were reused because they are part of the same internally controlled experiment. The protein samples (20 µg/lane) were separated on 8%–15% polyacrylamide gel electrophoresis  and transferred onto nitrocellulose filter membranes . After that, NC membranes were incubated with primary antibodies overnight at 4°C and a secondary antibody at room temperature for 40 min . All imaThe primary antibodies against the following proteins were used: rabbit anti-β-actin , rabbit anti-caspase-3 , rabbit anti-Bcl-2 , rabbit anti-BAX , rabbit anti-Beclin-1 , rabbit anti-p62 , rabbit anti-LC3 , rabbit anti-PI3K , rabbit anti-Akt antibod , and rabbit anti-NF-κB-p65 . The secondary antibodies were used: Alexa Fluor488 labeled goat anti-rabbit IgG (H + L) .t-test was used for comparisons of two sample means. One-way ANOVA analysis with Dunnett’s test was used for multiple comparison. P-values <0.05 was considered statistically significant. The samples were randomly divided into different groups by the random number method. Investigators were not blinded during the experiment and outcome assessment. Most experiments were repeated with similar results.All data are presented as the mean ± standard error of the mean (SEM) from three independent experiments performed. Statistical calculations were performed using GraphPad Prism version 8  . Student


Hepatitis B virus (HBV) infection is the most common cause of liver cirrhosis. Portal venous system thrombosis (PVST) is a major complication of liver cirrhosis. Recently, it has been shown that C-type lectin-like receptor 2 (CLEC-2) and galectin-1 participate in the activation and aggregation of platelets, thereby promoting the development of thrombosis. This cross-sectional study aims to evaluate the association of serum CLEC-2 and galectin-1 levels with PVST in patients with HBV-related liver cirrhosis.Overall, 65 patients with HBV-related liver cirrhosis were included, of whom 23 had PVST and 42 did not have. Serum CLEC-2 and galectin-1 levels were measured using enzyme-linked immunosorbent assay kits. PVST was assessed by contrast-enhanced computed tomography and/or magnetic resonance imaging scans. Subgroup analyses were conducted according to the degree and location of PVST.p = 0.006) and galectin-1 (p = 0.009) levels than those without. Patients with partial/complete PVST or fibrotic cord , but not those with mural PVST , had significantly higher serum CLEC-2 and galectin-1 levels than those without PVST. Patients with superior mesenteric vein thrombosis had significantly higher serum CLEC-2 (p = 0.013) and galectin-1 (p = 0.025) levels than those without PVST. Patients with main portal vein thrombosis had higher serum CLEC-2 (p = 0.020) and galectin-1 (p = 0.066) levels than those without PVST, but the difference in serum galectin-1 level was not significant between them.Patients with PVST had significantly higher serum CLEC-2 (Serum CLEC-2 and galectin-1 levels may be associated with the presence of PVST in HBV-related cirrhotic patients, but this association should be dependent upon the degree of PVST. Portal C-type lectin-like receptor 2 (CLEC-2) is a type II transmembrane receptor composed of an extracellular ligand-binding C-type lectin-like domain, stalk region, single transmembrane helix, and short cytoplasmic tail . It is eGalectin-1 is the firstly discovered member of the galectin family and belongs to the prototype galectin containing a carbohydrate-binding domain, which can exist as monomers or dimers . It is aSome animal studies have demonstrated a relationship of CLEC-2 and galectin-1 with venous thrombosis, but human studies are lacking , 15–17. 2.2.1.This study followed the Declaration of Helsinki and was approved by the Medical Ethical Committee of the General Hospital of Northern Theater Command . All patients who were admitted to the Department of Gastroenterology of the General Hospital of Northern Theater Command between January 2020 and August 2022 were selected. Inclusion criteria were as follows: (1) patients were diagnosed with HBV-related liver cirrhosis; (2) patients underwent contrast-enhanced computed tomography (CT) and/or magnetic resonance imaging (MRI); and (3) patients had already agreed to donate their blood samples and their blood samples remained. Exclusion criteria were as follows: (1) patients with a diagnosis of malignancy or history of splenectomy, splenic artery embolization, transjugular intrahepatic portosystemic shunt, or liver transplantation, which are well-known local risk factors of PVST; (2) patients with other thrombotic diseases; (3) patients with coronary heart disease  or acute2.2.Diagnosis of liver cirrhosis was based on medical history, clinical manifestations, cirrhosis-related complications, laboratory tests, imaging, and histology. HBV-related liver cirrhosis would be defined, if cirrhotic patients were diagnosed with HBV infection or had positive hepatitis B surface antigens. Based on the contrast-enhanced CT/MRI imaging, PVST was defined as thrombosis within portal venous system vessels, including left portal vein, right portal vein, main portal vein (MPV), superior mesenteric vein (SMV), splenic vein (SV), or the confluence of SMV and SV . Based o2.3.Demographics, laboratory tests , and major complications of liver cirrhosis  at admission were collected. Child-Pugh score and model for end-stage liver disease (MELD) score at admission were calculated . Data ac2.4.Blood samples used in the present study were obtained from the remaining samples of cirrhotic patients prospectively collected by our group. After fasting for 12 h, venous blood samples were collected by gel-procoagulant tubes from cirrhotic patients and centrifuged at 3000 rpm for 10 min at room temperature. The supernatant was carefully collected to obtain serum and stored at a −80°C refrigerator until further analyses. Serum CLEC-2  and galectin-1  levels were measured using enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions.2.5.p < 0.05 was considered statistically significant. SPSS version 20.0  and GraphPad Prism version 8.0.1  were used for all statistical analyses.Continuous variables were expressed as median  and mean ± standard deviation, and their differences between groups were compared with the Mann–Whitney U test  or T-test . Categorical variables were expressed as frequency (percentage), and their differences between groups were compared with the Chi-square test or Fisher’s exact test. Subgroup analyses were conducted according to the degree and location of PVST. A two-tailed 3.3.1.Overall, 65 patients were included . Their m3.2.p = 0.612) and MELD score  were statistically similar between patients with and without PVST  and MELD score  were statistically similar between patients with mural PVST and those without PVST  and MELD score  were statistically similar between patients with partial/complete PVST or fibrotic cord and those without PVST  and MELD score  were statistically similar between patients with MPV thrombosis and those without PVST  and MELD score  were statistically similar between patients with SMV thrombosis and those without PVST  serum CLEC-2/galectin-1 level was positively associated with the presence of PVST in HBV-related cirrhotic patients; (2) patients with partial/complete PVST or fibrotic cord, but not those with mural PVST, had significantly higher serum CLEC-2 and galectin-1 levels than those without PVST; (3) patients with MPV or SMV thrombosis had a significantly higher serum CLEC-2 level than those without PVST; and (4) patients with SMV thrombosis, but not those with MPV thrombosis, had a significantly higher serum galectin-1 level than those without PVST.Platelets are denucleated blood cells from megakaryocytes in the bone marrow . ThromboCLEC-2 is an important platelet-activating receptor . Its ligIIbβ3 on the platelets surface, changing from a low-affinity/resting state to a high-affinity/active state, exposing the high-affinity binding sites of fibrinogen .XQ: conception and design, administrative support. YZ, XX, SX, JC, and XQ: provision of study materials or patients. YZ, XX, SX, and JC: collection and assembly of data. YZ and XQ: data analysis and interpretation. YZ, XZ, XX, XG, SX, SM, JC, and XQ: manuscript writing. YZ, XZ, XX, XG, SX, SM, JC, and XQ: final approval of manuscript. All authors contributed to the article and approved the submitted version.


Ice cream made from Saemimyeon (high amylose content rice variety) exhibited the highest hardness value (49.55 N cm−2) among the varieties tested, but had relatively low viscosity (4030 cP), overrun (17.95%), and drip-through (0.75 g/min) values. These findings suggest that rice varieties with different amylose contents are suitable for making ice cream and have the potential to expand the rice processing market and increase its value.Ice cream consumption has increased over the years. In this study, we investigated the potential of using rice varieties with varying amylose contents for ice cream production. We analyzed the physical and chemical properties and sensory quality characteristics  of rice-based ice cream made from five varieties with low and high amylose levels. To make the ice cream, we ground rice into a fine powder and combined it with skim milk powder, butter, sugar, glycerin esters of fatty acids, locust bean gum, and water to form a gelatinized mixture. This mixture was then aged, frozen, and hardened. The ice cream’s key quality characteristics, such as viscosity , hardness (4.27–49.55 N cm In the context of global warming, mainly caused by the persistent emission of greenhouse gases (GHGs) that exacerbate climate change, agriculture is regarded as both a source and sink of atmospheric GHGs. Agriculture  accounts for about 18% of global GHG emissions, with livestock and rice cultivation contributing about 6% and 1%, respectively .Orzya sativa L.) is a major source of energy, consumed as a staple food by more than half of the world’s population, especially in Asia [Rice  varieties used in the experiments were Baegokchal [The rice , the volume was increased to 100 mL with distilled water, and the mixture was incubated for 20 min at 30 °C. Absorbance was measured at 620 nm using an ultraviolet-visible spectrometer . The absorbance at 620 nm and the amylose concentrations were adjusted with a standard curve based on potato (Solanum tuberosum L.) starch [Rice amylose content was determined using the methods of Juliano , accordi) starch . 2 = 0.982) [Rice crude protein content was determined using near-infrared (NIR) spectroscopy . Rice flours (0.6 g) were added to the micro insert ring bound to the mini sample cup, the air gap in the sample was removed using the sample cup disposable back, and NIR spectroscopy was performed using visible and near-infrared wavelengths (400–2500 nm) under ambient temperature conditions (25 °C). The software ISI scan  was used for the calculation of crude protein (R= 0.982) .Rice moisture content was measured using a moisture meter , and pasting properties were analyzed using a rapid viscosity analyzer . Rice flour  was added to an aluminum container for rapid viscosity analysis and 25 mL of distilled water was added. The samples were incubated at 50 °C for 1 min, then heated to 95 °C for 3.45 min, maintained at 95 °C for 2.7 min, cooled to 50 °C for 3.91 min, and maintained at 50 °C for 1.24 min. The parameters recorded were initial pasting temperature, peak viscosity (P), trough viscosity (T), final viscosity (F), breakdown viscosity (P − T), and setback viscosity (F − T) .Total solids, total sugars, pH, viscosity, and hardness of the rice ice creams were measured. The total solids were determined by measuring the moisture content using the air oven method. Rice ice cream (3 g) was transferred to a pre-weighed flat bottom aluminum dish, which was moved to a hot air oven set to 105 °C for 5 h. Dried rice ice cream was then placed in desiccators containing silica gel as a desiccant. After 30 min, the dish was weighed. Total solids were calculated by employing the following formula.1: Weight of empty dishW2: Weight of empty dish and rice ice creamW3: Weight of empty dish and dried rice ice creamWTotal sugars were measured using a hand refractometer , and pH was measured using a pH meter .The viscosity of rice ice cream was measured using a viscometer  with a spindle (LV-04/64) after aging at 4 °C . All mea−1, the distance was 3.0 mm, and the cell load was 19.6133 N (Newton). The hardness was the peak value of the first compression.Hardness was measured using a rheometer  after hardening the rice ice cream in a cube frame (24 mm × 24 mm × 24 mm) and aging it for 5 min at ambient temperature (25 °C) . The harOverrun is the increase in the volume of ice cream that occurs due to the incorporation of air during batch freezing. The method of measuring overrun involves separately weighing the ice cream mix and frozen ice cream in an affixed volume container (approximately 175 mL), then calculating the percentage increase in volume using the formula described by Akbari, Eskandari, Niakosari, and Bedeltavana . The ove1: Weight of ice cream mixW2: Weight of ice creamWThe rate at which ice cream melted through a mesh screen at room temperature was measured using the procedures specified by Bolliger et al. . At ambiColor was measured using a colorimeter . The parameters evaluated from the CIELAB system were lightness (L*), red/green (a*), and yellow/blue (b*). Rice ice cream was added to the measurement cell (50 × 15 mm), so that there was no empty space, and then hardened, and each sample was measured 10 times.The ice creams were stored at −18 °C for over 24 h after production and remained at ambient temperature (25 °C ± 1 °C) for 5 min before sensory analysis . A 25 g n = 3), and color measurement carried out 10 times. The results were evaluated by the analysis of variance and Duncan’s multiple range test (p < 0.05) using SAS . Correlation analysis was used to determine relationships among variables. The correlation between rice  and rice ice cream , using physicochemical properties, was determined using the Pearson correlation coefficient.Values are expressed as the mean ± standard deviation (p < 0.05).The amylose content, crude protein, and pasting properties of rice are presented in Rice grains contain a substantial amount of storage protein that can be classified as either prolamin, glutelin, albumin, or globulin . Additiop < 0.05). Saeilmi showed the highest breakdown viscosity, followed by Saemimyeon, Miho, Baegokchal, and Dodamssal (p < 0.05). These results clearly distinguish the rice characteristics and act as an important variable in the quality characteristics of rice ice cream manufactured using rice with different amylose contents.The initial pasting temperature of rice ranged from 67.15 °C to 84.14 °C, the peak viscosity from 147.03 to 403.11 cP, the trough viscosity from 64.08 to 204.89 cP, the final viscosity from 82.50 to 386.41 cP, the breakdown viscosity from 99.33 to 205.33 cP, and the setback viscosity from 18.41 to 181.53 cP. As the amylose content increased, the initial pasting temperature, peak viscosity, trough viscosity, final viscosity, and setback viscosity of Baegokchal, Miho, Saeilmi, and Saemimyeon  showed a positive relationship. According to research, such as that of Hasjim, et al. , it has −2, overrun ranging from 17.95% to 46.99%, and drip-through rate ranging from 0.75 to 0.98 g/min.The physicochemical properties of rice ice cream, including total solids, total sugars, pH, viscosity, hardness, overrun, and drip-through rate, are shown in Erkaya, et al.  found thp < 0.05). This correlation can be attributed to the varying total sugars content produced during the gelatinization of each type of rice at 85 for 30 min. Varavinit et al. [The total sugar content decreased as the amylose content increased . The hardness of the rice ice cream was highest when prepared using Saemimyeon (49.55 Ncm−2), but the hardness of the rice ice cream prepared using Baegokchal was 4.27 Ncm−2, a difference of 11-fold, depending on the type of rice used. However, the Baegokchal overrun value was 46.99%, which was more than double that of Saemimyeon (17.95%). As such, hardness is affected by overrun, as well as rheological properties of the mixture [p < 0.05). Baegokchal did not pass through the mesh until the 120 min mark, when all other samples were melted and were clustered on the top of the mesh . According to Lamberts et al. [The color values of rice ice cream are shown in s et al. , the bris et al. . For thip < 0.05). On the other hand, the b* value, which ranged from yellow to blue, was yellow, with a positive value ranging from 16.40 to 20.29 for all of the rice ice creams. Saemimyeon had the highest b* value of 20.29, whereas Baegokchal had the lowest at 16.40 (p < 0.05). Based on these results, the L* and a* values decreased, while the b* value increased as the amylose content of the processed rice used for the production of rice ice cream increased.The degree of redness , representing red to green, was weakly green, with a negative value ranging from −4.01 to −3.33 for all of the rice ice creams. The rice ice cream prepared using Baegokchal had the highest a* value of −3.33, while the lowest was the ice cream made with Saemimyeon at −4.01 (p < 0.01). The total sugars content, pH, and viscosity of the rice ice cream showed the highest negative correlation with initial pasting temperature at −0.956, −0.947, and −0.963, respectively, while the amylose content also showed a high negative correlation with these variables at −0.897, −0.877, and −0.932, respectively. Rice ice cream hardness showed the highest correlation with setback viscosity (r = 0.894), whereas overrun showed a negative correlation with trough viscosity (r = −0.895). The setback value is an indicator of the retrogradation of rice starch, which occurs in the third stage of the series of RVA tests. Retrogradation refers to the process in which starch molecules break the initial gelation and reform a gel network by interacting with water molecules [The relationship between the processed rice  and the physicochemical properties of rice ice cream  are shown in olecules . This prolecules , the setp < 0.001), and also showed a high negative correlation with setback viscosity (r = −0.827) and trough viscosity (r = −0.824). The redness value (a*) showed the highest negative correlation with setback viscosity , and also showed a high negative correlation with final viscosity (r = −0.823). The yellowness value (b*) showed the highest positive correlation with final viscosity , while trough viscosity and setback viscosity showed a very high positive correlation . Setback and final viscosity are closely related to the amylose content [When analyzing the correlation between the color value of rice ice cream and rice physicochemical properties, the lightness value (L*) of rice ice cream showed the highest negative correlation with final viscosity . Among the samples, Miho and Saeilmi scored the highest in terms of texture preference , while Dodamssal received the lowest score (3.9). The chewy texture was also evaluated, and the results indicated that Baegokchal scored the highest (6.2), followed by Miho, Saeilmi, Saemimyeon, and Dodamssal, with significant differences between the samples . These findings emphasize the importance of texture in determining the overall acceptance of rice ice cream.However, the samples exhibited notable differences in terms of texture, chewy texture, and rice flavor , indicating that the choice of rice did not significantly affect the color of the ice cream. A higher F-value indicates a higher proportion of the variance explained by the model, which suggests that the corresponding factor has a significant overall effect. Thus, the F-values for texture and chewy texture were high, indicating that these attributes had a more important impact on the overall evaluation. In fact, previous research has shown that texture is the most important factor influencing consumer preferences when choosing ice cream, followed by taste and aroma . TherefoIn conclusion, based on the sensory evaluation results, it is suggested that texture may be considered a more important attribute for determining the overall acceptance of rice ice cream. Therefore, the processing quality characteristics of rice ice cream should be carefully considered and adjusted based on the rice variety to meet consumer needs and preferences.In this study, rice ice cream was produced by incorporating processed rice with different amylose contents and comparing the resulting quality characteristics. The physicochemical properties of ice cream, including overrun and drip-through rate, were measured and compared among the different rice varieties. Significant differences were observed in the pH, total sugars content, and total solids of the ice cream with different amylose contents. The correlation between the physicochemical properties of rice and ice cream was shown, based on the different properties of rice ice cream. The correlation between the quality properties of rice and rice ice cream showed that the setback and final viscosity were highly correlated with hardness and color value. By utilizing the relationship between rice varieties and processing quality characteristics, this data can be used for the production of rice ice cream that meets consumer needs.This preliminary investigation provides some useful information regarding the suitability of employing rice varieties with different amylose contents as alternative raw materials in ice cream making with regard to the nutraceutical, nutritional, and physiochemical properties. This may help promote rice processing and create a new market for rice production.


The voltage-dependent potassium channel Kv1.3 is a potential target for cancer therapies. This channel exhibits a complex repertoire of physiological functions such as proliferation, activation, insulin sensitivity, nerve action potential and apoptosis among others. Furthermore, the expression and activity of Kv1.3 remodels in various types of tumors. Kv1.3 forms heteroligomeric complexes by association with a number of ancillary proteins that fine-tune the function of the complex. In addition, protein kinase signaling networks are essential for comprehending tumorigenesis. Kv1.3, but also its partners, undergoes phosphorylation-dephosphorylation cycles shaping and remodeling the biology of the channelosome. Therefore, this review compiles protein kinase mechanisms on Kv1.3 to understand regulatory mechanisms during tumor development.The voltage-gated potassium channel Kv1.3 plays a pivotal role in a myriad of biological processes, including cell proliferation, differentiation, and apoptosis. Kv1.3 undergoes fine-tuned regulation, and its altered expression or function correlates with tumorigenesis and cancer progression. Moreover, posttranslational modifications (PTMs), such as phosphorylation, have evolved as rapid switch-like moieties that tightly modulate channel activity. In addition, kinases are promising targets in anticancer therapies. The diverse serine/threonine and tyrosine kinases function on Kv1.3 and the effects of its phosphorylation vary depending on multiple factors. For instance, Kv1.3 regulatory subunits (KCNE4 and Kvβ) can be phosphorylated, increasing the complexity of channel modulation. Scaffold proteins allow the Kv1.3 channelosome and kinase to form protein complexes, thereby favoring the attachment of phosphate groups. This review compiles the network triggers and signaling pathways that culminate in Kv1.3 phosphorylation. Alterations to Kv1.3 expression and its phosphorylation are detailed, emphasizing the importance of this channel as an anticancer target. Overall, further research on Kv1.3 kinase-dependent effects should be addressed to develop effective antineoplastic drugs while minimizing side effects. This promising field encourages basic cancer research while inspiring new therapy development. Ion channels are crucial components of the cellular machinery that regulates the flow of ions across cell membranes. These proteins participate in the generation and maintenance of the electrical potential, which is vital to many biological processes, including nerve conduction, muscle contraction, and hormone secretion. The wide heterogeneity of ion channels is classified on the basis of channel ion selectivity and the opening mechanism . For exaTo date, phosphorylation is the most thoroughly studied PTM of Kv channels. Because all Kv channels are susceptible to phosphorylation, the literature contains various examples. Overall, the phosphorylation of Kv channels principally affects protein trafficking, clustering, and gating mechanisms . In addiRegarding the Kv1 or Shaker family, tyrosine phosphorylation downregulates Kv1.1, Kv1.2, and Kv1.5 channel activity at the plasma membrane ,5. This The phosphorylation of the other Kv families shares similar effects. For instance, whereas PKA-mediated phosphorylation increases the currents in Kv7.1, Kv7.2, and Kv7.3—members of the KCNQ family—tyrosine phosphorylation of these channels downregulates their expression in neurons . MoreoveOverall, the regulation of ion channels via phosphorylation is exceptionally complex and variable. Some Kv channels participate in the proliferation and migration of normal and tumor cells, contributing to metastasis. Thus, some channels, such as voltage-dependent Kv1.3, are highly related to tumorigenesis. Because the altered expression of Kv1.3 correlates with several types of tumors and cancer cells, this protein is widely considered to be an important anticancer target ,19,20. KPhosphorylation constitutes the most common, basic, and important PTM for regulating protein activity and function. In fact, approximately one-third of eukaryotic cell proteins carry one or more phosphoryl groups that are involved in a myriad of regulatory processes. Many of these pathways represent key points in several cell signaling pathways . InteresPhosphorylation involves the covalent binding of a phosphoryl group transferred from a nucleoside triphosphate  to a serine (Ser), threonine (Thr), or tyrosine (Tyr) residue in a protein. This reaction is catalyzed by protein kinases, which are encoded by approximately 2% of the genes in the total human genome. The attachment of this bulky negatively charged group creates an increased hydrophilic and polar state that might induce considerable changes to the three-dimensional structure of the modified protein. Moreover, the phosphorylated amino acid can react with side chains within the same or adjacent proteins and thus establish protein complexes. Phosphorylation is reversible, with a phosphoryl group removed by a protein phosphatase.Phosphorylatable Ser, Thr, and Tyr residues are not randomly distributed; in contrast, they are embedded in clear motifs known as consensus sequences within a protein. Hence, the pattern of each consensus sequence confers specific affinity for a unique protein kinase ,30. MoreIn addition, regulation via phosphorylation increases the regulatory complexity when a protein carries different consensus sequences recognized by numerous kinases, each of which can differentially modulate target protein activity. In some cases, phosphorylation is hierarchical. Thus, a specific target for kinases may only appear after only a phosphoryl is previously added to a specific residue, which enables the phosphorylation of nearby residues ,31. In oTogether, these processes exert an extremely refined regulatory effect on protein function. Notably, the removal of a phosphate group is a much less specific process, as reflected by the number of phosphatases (approximately 150) versus the number of kinases (more than 500) encoded in the human genome .In general, reversible covalent modifications are effective regulatory mechanisms, and phosphorylation in particular precisely modulates the most subtle processes . For thiAs mentioned above, the enzymes critical for adding and removing a phosphate group are kinases and phosphatases, respectively. While kinases show extremely specific and tightly regulated activity, the activity of phosphatases is less specific. Regarding targets, kinases are broadly classified into protein kinases, lipid kinases and carbohydrate kinases. Among these enzymes, protein kinases clearly constitute the largest and most diverse group .The first observation of protein kinase activity dates back to the mid-1950s, almost two decades after the discovery of phosphorylation . RegardlA second method of classification involves the phosphorylation target residue. Notably, serine is the most phosphorylated amino acid , while threonine and tyrosine phosphorylation are less common modifications  . Hence, Other useful classifications relate to their localization (cytosolic and membrane-spanning protein kinases) or subcellular compartment targeting. Indeed, the compartmentalization of kinases is vital for their proper function and is often controlled by anchoring proteins. The binding of protein kinases to these scaffolding proteins modulates the catalytic activity of the scaffolding proteins, recruiting them to be in close proximity to upstream and downstream effectors. Moreover, one anchoring protein can bind several kinases and phosphatases and thus accurately modulate signal transduction .In contrast, protein phosphatases exhibit functions opposite that of protein kinases. Phosphatases exhibit faster kinetics and less specificity than kinases. Phosphatases have been studied less extensively than kinases because their structures have been more challenging to identify. To date, more than 200 protein phosphatases have been classified into three families: (i) phosphoprotein phosphatases (PPPs), (ii) metallo-dependent protein phosphatase (PPMs), and (iii) protein-tyrosine phosphatases (PTPs). The PPP and PMP families dephosphorylate most pSer and pThr residues and even some pTyr residues in different domain sequences. In general, the PTPs, including the aspartate-based phosphatases, carry the same catalytic domain, with each showing different selectivity. Notably, although less than 2% of all phosphorylation events involve tyrosine, approximately one-half of all protein phosphatases exert their effects on this phosphorylated amino acid ,37.The voltage-gated potassium channel Kv1.3 was first identified in 1985 , and shoKv1.3 is a delayed rectifier channel, driving a K+ efflux after plasma membrane depolarization. The threshold for activation is approximately −35 mV ,45. The Kv1.3 is modulated either by accessory subunits or by PTMs. Early reports suggested that the molecular weight of Kv1.3 immunoprecipitated from Jurkat T cells was 65 kDa, in contrast with the protein found in vitro, which is translated into a 58-kDa product. The authors of this study claimed that PTMs caused the discrepancy and pointed to phosphorylation as the main PTM candidate .Although initial studies have described dual PKA- and PKC-dependent regulation of Kv1.3 in Xenopus oocytes  and in tAlthough Kv1.3 harbors different putative PKA phosphorylation sites , the effThe Kv1.3 amino acid sequence carries several putative PKC-dependent phosphorylation domains. Interestingly, all domains except for one domain have been conserved as mouse, rat, and human orthologs. Rat and human Kv1.3 carries four PKC phosphorylation domains: one in the cytoplasmic loop between the S2 and S3 segments, two between the S4 and S5 cytoplasmic loop, and the one in the C-terminus . One of Nevertheless, the general effect of PKC phosphorylation on Kv1.3 might differ depending on the predominant kinase isoform that is modified; its maturation stage, which is related to its expression; and the regulating cofactors involved in the cell type. For instance, untransformed human T lymphocytes express higher levels of PKCβ, while in Jurkat T cells derived from a patient with T-cell leukemia, PKCα is the predominant kinase . Hence, The insulin-like growth factor-1 (IGF-1) signaling pathway involves the activation of several Ser/Thr protein kinases, including the phosphatidylinositol-3 (PI3) kinase , phosphoAdult neurogenesis is thoroughly controlled and restricted to two confined areas of the forebrain: the dentate gyrus of the hippocampal formation  and the Although serine-dependent Kv1.3 phosphorylation has been described , the chaCo-expression of Kv1.3 with v-Src  or EGF receptor (EGFR) tyrosine kinases increases channel phosphorylation by eight- and fourfold, respectively, in HEK293 cells ,71. A siKv1.3 participates in the proliferation of several cell types by controlling the cell cycle . Kv1.3 fInterestingly, certain elements of the MAPK signaling pathway can be activated downstream of Kv1.3 activation. For instance, Kv1.3 abundance controls the phosphorylation of ERK1/2, a member of the MAPK family that is indirectly activated by either growth factor receptors or tyrosine kinase receptors and thus transduces signals from the plasma membrane to the nucleus. Hence, it is unsurprising that ERK1/2 is intricately involved in cell activation, migration, and proliferation. For this reason, the implications of ERK1/2 dysregulation in cancer have been well documented . In macrFas-induced apoptosis, triggered by the Fas receptor activation of the lymphocyte-specific protein tyrosine kinase (lck)—a member of the sarcoma (Src) family—phosphorylates Kv1.3. Interestingly, although the lack of lck completely abolishes tyrosine phosphorylation, channel inhibition is mediated only through an additional mechanism . Shortly2+ influx during leukocyte activation and proliferation [Although lck and Kv1.3 are not directly associated, both bind to Dlgh1 (SAP97), a scaffold protein in the MAGUK family, forming a protein complex in T cells . The proferation . Thus, hferation .Another context in which both Src-family kinases and ROS play independent and dual roles in Kv1.3 is microglial activation. During acute brain insults, such as oxygen/glucose deprivation (OGD), PTKs and K+ channels contribute to microglia activation. In particular, Kv1.3 maintains membrane potential hyperpolarization to promote ROS production and thus combat the threat. However, ROS levels must be tightly regulated, which is realized through a negative feedback mechanism. Thus, ROS promote the phosphorylation of the channel and its subsequent inhibition, thereby limiting the respiratory burst. On the other hand, PTKs in the Src family phosphorylate the channel, leading to the same suppressive effect .Kv1.3-mediated ROS generation is mediated during mitochondrial respiration. An increase in cell respiration relies on both Kv1.3 Tyr447 phosphorylation mediated by ERK1/2 and on a voltage sensor in HEK293 cells. As a result, ROS production increases, generating a proliferative phenotype .As noted above, EGF efficiently downregulates Kv1.3 channel opening. By activating the EGFR pathway, current suppression relies on the phosphorylation of Tyr479. Similarly, v-Src phosphorylation and EGFR cotransfection change the kinetics of channel inactivation but accelerate C-type inactivation. Both the maximal current decrease and channel inactivation are mediated by separate signaling pathways. Hence, in the case of channel inactivation, in addition to Tyr phosphorylation, Ser/Thr phosphorylation and other mechanisms may be involved .The tyrosine kinase activity of the insulin receptor suppresses Kv1.3 current with no effect on channel inactivation ,85,86. TThis regulatory effect on the Kv1.3 current is particularly important in the olfactory bulb (OB), where both the insulin tyrosine kinase receptor and Kv1.3 are highly expressed ,89, withIn animals with at least one abolished insulin receptor kinase gene allele, the current through Kv1.3 was diminished due to the lower expression of the channel. Although heterozygous animals present with partial preservation of the kinase activity, the modulation of Kv1.3 is strongly susceptible to changes in expression. Interestingly, although Kv1.3 can reciprocally downregulate insulin receptor kinase activity, Kv1.3 knockout mice did not show altered channel expression .In addition to metabolic diseases, such as diabetes mellitus, insulin is an immunomodulator in the central nervous system (CNS) . For insThe tyrosine receptor kinase (Trk) family constitutes another tyrosine kinase-dependent regulator of Kv1.3 in the olfactory bulb. This group of kinases comprises three members: TrkA, TrkB, and TrkC. However, only TrkB carries an active kinase domain. The target neurotrophins are nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3) . Both KvThe TrkB receptor increases Kv1.3 expression ,98. ThisAlthough it is still not clear whether these outcomes are results of direct phosphorylation of the channel or crosstalk between signaling pathways, the causal relationship between phosphorylation and channel activity is evident . In addiProtein phosphorylation requires close proximity between an effector kinase and a target, and scaffolding proteins link several proteins together to optimize signal transduction. Kv1.3, with several molecular domains involved in protein interaction, binds different scaffold proteins. Thus, the channel functions as an adaptor for different proteins through a non-conducting function in addition to its current-conduction action . SometimFor ion channels, the membrane-associated guanylate kinase (MAGUK) family has been extensively described. The postsynaptic density 95 (PSD-95) protein carries three binding sites: a guanylate kinase (GK), a PDZ, and an SH3 domain. Kv1.3 carries proline-rich sequences and PDZ-binding motifs that can bind the GK-SH3 and PDZ, respectively. Whereas PSD-95-mediated clustering of Kv1.3 relies merely on the PDZ domain, the modulation of the current, kinetics, and inactivation are mediated through the GK-SH3 domain. PSD-95 also binds to the insulin receptor kinase, generating the Kv1.3/insulin receptor kinase/PSD-95 complex. In this context, the SH3 domain in PSD-95 interacts with proline-rich sequences in the insulin receptor kinase catalytic domain, preventing the phosphorylation of Kv1.3 tyrosine. Therefore, in the presence of PSD-95, insulin receptor kinase-induced suppression of the current through Kv1.3 is abolished . InsulinSAP97 is another MAGUK family protein that promotes Kv1.3 tyrosine phosphorylation mediated by lck action in T cells. Notably, although fyn can mask lck function in T cells, no interaction between these kinases and SAP97 has been reported . NonetheAs previously described, both Kv1.3 and insulin tyrosine kinases are highly expressed in the OB ,89, and When certain receptor tyrosine kinases (RTKs), such as insulin receptor and TrkB, are activated, they are phosphorylated, creating new and specific recognition sites for scaffolding proteins. Therefore, the pattern of tyrosine phosphorylation may generate another filter that, depending on the recruited adaptor, a specific kinase may phosphorylate Kv1.3 with a particular outcome .Finally, the abovementioned tyrosine kinase Src targets caveolin-1 (CAV1), which, in turn, binds Kv1.3 through a caveolin-binding domain in the N-terminus of the channel ,107. AltKv channels form heterooligomeric structures (Kvα subunits) with peripheral accessory subunits. These auxiliary proteins modulate several Kvα morphogenic, structural, and physiological properties, as well as channel subcellular localization. Overall, the main accessory proteins comprise Kvβ subunits and KCNEs as well as, ancillary BK channel subunits, namely K+ channel-interacting proteins (KChips) and dipeptidyl aminopeptidase-like proteins (DPPL), specifically for Kv4 channels . BecauseKvβ subunits were the first Kvα accessory proteins to be characterized. They are encoded by three different genes  that produce six different splicing variants ,112. KvβThe first observation of Kvβ phosphorylation was made serendipitously by scientists studying PKA-dependent Kv1.3 phosphorylation . SubsequKvβ1 accentuates the inactivation of Kvα with respect to the current, playing an essential role in cardiac cells. Kvβ1.3 can be phosphorylated by either PKA or PKC ,117,118.Kvβ2 lacks a ball-and-chain domain that would enable its regulation of Kvα inactivation. However, Kvβ2 is the most abundant and widely expressed Kvβ subunit, participating in the processing and targeting of Kvα subunits. In addition, Kvβ2 presents aldo-keto reductase activity by binding NADPH with high affinity . AlthougAgain, adaptor proteins physically link the Kvβ subunit and the kinase. Notably, ZIP1 and ZIP2 proteins, which bind both Kvβ2 and PKC-θ, facilitate phosphorylation .The KCNE family of ancillary subunits includes five members with one transmembrane domain. Each subunit is encoded by a single gene, whose mutations have been associated with several diseases. KCNEs participate in the trafficking and gating properties of Kv channels, although outcomes differ by the stoichiometry, specificity for the Kv subunit, and PTMs .All five KCNEs carry multiple putative phosphorylation sites for many kinases, but direct phosphorylation has seldom been reported to date. However, one example is the PKC-mediated Ser82 phosphorylation of KCNE3, which leads to changes in the electrophysiological pattern of the Kvα current ,121. On The mere expression of KCNEs is essential for controlling physiological phosphorylation levels under some conditions. In fact, the lack of KCNE2 or KCNE4 in mice induced fluctuations in the phosphorylation rate of certain components of signaling cascades in response to ischemia/reperfusion injury (IRI) ,129. On KCNE subunits also form complexes with adaptor proteins to reduce the distance between the kinase and Kvα subunit. For instance, Yotiao (AKAP9) is a scaffold protein that binds KCNEs, Kvα subunits, and PKA ,128.As mentioned above, the human genome encodes more than 500 kinases, and more than 90% of human proteins are phosphorylated. Hence, protein phosphorylation constitutes a widespread regulatory mechanism whose dysregulation might affect several types of cells to different degrees. Notably, approximately 50% of altered human kinases have been reported to correlate with a disease. In fact, many alterations in phosphorylation-dependent kinase functions result in cancer . For thi2+ flux required for T-cell activation is impaired [One well-documented disease characterized by the dysregulation in protein phosphorylation is acquired immunodeficiency syndrome (AIDS), which is caused by human immunodeficiency virus (HIV). In the case of Kv1.3, the HIV-induced outcomes vary depending on the cell type. In lymphocytes, the course of infection is characterized by an initial decrease in CD4 T-cell number and function . The virimpaired .However, HIV exerts an opposite effect on Kv1.3 phosphorylation in oligodendrocytes. The viral protein Tat interacts with Kv1.3, interrupting channel regulation mediated via phosphorylation. Therefore, the channel is more active, which results in increased oligodendrocyte injury .Interestingly, tyrosine kinases are most highly expressed in the nervous system. For this reason, many types of kinase dysfunction can trigger CNS diseases. As mentioned above, the insulin receptor is highly expressed in the OB, playing an important regulatory role mediated through Kv1.3 action . Thus, dAs illustrated in the previous sections of this review, both Kv1.3 and kinases are key regulators of the cell cycle, proliferation, and apoptosis ,22,69. TSeveral epidemiologic studies have shown that soy-based diets correlate with a lower cancer risk. The reason is related to the soy isoflavone genistein, which effectively blocks protein tyrosine kinases. Therefore, genistein affects Kv1.3 function via two separate pathways. First, it inhibits the tyrosine kinases that phosphorylate the channel. However, genistein also inhibits Kv1.3 in a rapid kinase-independent manner, which might involve a direct interaction. This compound is a potential therapeutic target for both cancer prevention and as an adjunct therapy. However, further basic and clinical studies are needed before the definitive approval of genistein for use in clinics ,139,140.Altogether, Kv1.3 phosphorylation is undoubtedly subject to many therapeutic approaches. Nonetheless, the entangled phosphorylation pathways and their heterogeneous outcomes have hindered the development of putative pharmacological drugs.Kv1.3, which is involved in cell proliferation and tumor progression, carries several consensus sequences that bind different serine/threonine and tyrosine kinases . In addiThus, an a priori basic PTM generates intricate regulatory mechanisms, complicating the study of these modifications. Paradoxically, the study of PTMs requires exhaustive research because many altered phosphorylation events correlate with disease. Moreover, the ubiquitous expression of Kv1.3 may lead to the extrapolation of these alterations to the whole body, especially the immune and nervous systems. Therefore, it is of vital importance to increase our understanding of Kv1.3 phosphorylation so that particular attention can be devoted to the design of specific anticancer therapeutic treatments.


Substance abuse is common among patients with schizophrenia, is related toworse course and outcome of illness. Unfortunately, little is known about how substanceabuse affects the cognitive function of schizophrenia patients, whose cognitive function isoften already comprised. Neurocognitive functioning includes inhibition control and decision-making, and both schizophrenia and substance use disorder are related to impairmentsof inhibition control. However, the influence of substance abuse on inhibition capacitiesamong schizophrenia patients is unclearThis study measured the influence of substance use disorder on inhibition capacities and risky decision-making in a group of 39 schizophrenia patients that were evaluated using a socio-demographic questionnaire and clinical assessment using the Positive and Negative Syndromes Scale for Schizophrenia. To assess inhibition control we utilized the Matching Familiar Figure Test (MFFT) and the Stroop task, and to evaluate decision-making we used the Iowa Gambling Task (IGT) and self-report questionnaire, the Barratt Impulsiveness ScaleUnivariate analysis found significant differences between the groups with regard to criminal history , smoking status , and total BIS score . Our model did not find a significant effect of substance abuse on the first response time and number of errors on the MFFT or in the total interference index of Stroop performance and net score on risky decision-making in the IGT.The two groups did not differ significantly either in first response time or in number of errors on the MFFT , nor did they differ in the total interference index of the Stroop task (F(1)¼0.49, p¼.49, d¼0.25, 95% CI ).The analyses did not detect any statistically significant effect of substance abuse on inhibition control or risky decision-making processes in outpatients diagnosed with schizophrenia, despite increased impulsivity, criminal history and smoking status. These results neither support nor disprove previous findings.None Declared


The golden age has passed for antibiotic discovery, and while some antibiotics are currently in various phases of clinical trials in the United States, many pharmaceutical companies have abandoned antibiotic research. With the need for antibiotics, we should expand our horizon for therapeutic mining and can look toward understudied sources such as ice cores. Ice cores contain microorganisms and genetic material that have been frozen in time for thousands of years. The antibiotics used by these organisms are encoded in their genomes, which can be unlocked, identified, and characterized with modern advances in molecular biology, genetic sequencing, various computational approaches, and established natural product discovery pipelines. While synthetic biology can be used in natural product discovery approaches, synthetic biology and bioengineering efforts can also be leveraged in the selection and biodesign of increased compound yields, potency, and stability. Here, we provide the perspective that ice cores can be a source of novel antibiotic compounds and that the tools of synthetic biology can be used to design better antimicrobials. Antimicrobial resistance (AMR) is a major global health threat and is estimated to contribute to 10 million human deaths by 2050 . MicroorAccording to the Baas Becking hypothesis, microbes are everywhere but nature selects . We haveThe potential antimicrobial-mediated ecological interactions locked within ice cores can be revealed through examination of genetic records and attempts to culture the microbes themselves. Antagonistic interactions between organisms in the form of predator–prey relationships can put selective pressure on prey and give rise to species with defensive molecules that may potentially have antimicrobial activity. Antimicrobials can also be derived from symbiotic relationships where one organism secretes antimicrobial compounds that protect a host from pathogens. The ecological as well as nonlethal, alternative, effects of sub-inhibitory levels of antimicrobials have been discussed in more detail by others ,12. HereIn the ice core metagenomic analysis (ICEMAN) pipeline, samples are examined from a sequencing, metagenomic approach as well as from a laboratory culturing approach. The sequence-based protocol, termed antibiotic discovery and analysis of metagenomes (ADAM), can be executed in a complementary fashion to the culturing-based environmental examination (EVE) protocol, thus maximizing the potential for recovering antibiotic compounds.ADAM relies on the assembly of DNA reads that can construct an accurate metagenomic representation. This is then subject to the examination by multiple computational programs that detect signatures of antibiotic genes such as MetaBGC  MetaBGC,While the ADAM approach relies on sequenced genetic information, EVE seeks to directly sample ice cores for extant microbial life that could be metabolically active provided an optimal growing environment. Organisms are known to enter cryptobiosis due to adverse environmental conditions. Recent research has shown that nematodes and viruses from permafrost can be revived and cultured ,19. ThroWhile synthetic biology approaches can be used to help identify ice core-derived antimicrobial compounds, synthetic biology can also be used to help optimize antibiotics against AMR through design and selection . The lisSynthetic biology approaches such as heterologous gene expression, pathway and metabolic engineering, and spatial programming can be used to not only produce and identify ice core-derived antimicrobial compounds but also up-regulate production in downstream purification processes . For exaMicroorganisms within ice cores are a possible repository for novel antimicrobial compounds that could be used in our ongoing struggle with AMR. Here, we outline the ICEMAN pipeline that incorporates both sequence-based and culture-based approaches to identify antibiotic compounds of interest in an orthogonal manner, maximizing the potential for antibiotic discovery from ice cores. Synthetic biology approaches can be applied for initial compound production as well as optimization steps.A point of consideration in the context of reviving ancient microbes is that of biosafety and biosecurity. Organisms and viruses originating from samples thousands of years old should be treated with care and following best practices in molecular biology to ensure the safety and security of not only the researchers handling the samples but also the public and current biosphere. Environmental contamination by an unsuspecting pathogen could be a threat to human, animal, and plant health. ICEMAN research should be conducted with proper oversight and handling protocols. ICEMAN taps into the ecological and genetic potential within ice cores, and a focused effort to mine and characterize antibiotic compounds within these samples could contribute to the health and wellness of society.