The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology. The small heat shock protein 20  is a 17 kDa molecular chaperone with a central role in cardiac function ,4,5,6. TImportantly, the cardioprotective effects of Hsp20 are linked to Ser16 phosphorylation by cAMP-dependent protein kinase (PKA) ,11. ThisIn addition to evidence from experimental models, the identification of Hsp20 mutations in dilated cardiomyopathy (DCM) patients has further supported the functional significance of Hsp20 in cardiac muscle ,13. The Mechanistically, the protective properties of Hsp20 can be attributed to its association with various binding partners regulating cell survival or death, phosphorylation enzymes or subunits, as well as protein phosphatases or phosphodiesterases ,17,18,19Data presented herein underscore a significant role for the cardioprotective Hsp20-Ser16 phosphorylation in modulating the subcellular distribution and interaction of Hsp20 with 14-3-3, leading to dissociation of CFL2 from 14-3-3 and enhanced CFL2-mediated F-actin depolymerization. Conversely, the human Hsp20-P20L mutation which is causally linked to impaired cardiac function displays reduced phosphorylation and diminished physical interaction with 14-3-3 leading to aberrant regulation of CFL2-mediated F-actin depolymerization. Collectively, these findings link PKA-mediated Hsp20 phosphorylation to cytoskeletal changes pertinent to cardiac muscle physiology and pathophysiology.To determine whether 14-3-3 interacts with Hsp20 and CFL2 in the heart, we performed pull down assays using recombinant MBP-14-3-3 or MBP proteins and mouse cardiac protein extracts. Western blot analysis showed that in vitro expressed 14-3-3 binds Hsp20 and CFL2 in cardiac muscle a. To detAs Hsp20 function is affected by phosphorylation at Ser16 by PKA ,25, we eWe conclude that CFL2 and Hsp20 may bind directly and independently of each other to 14-3-3 in cardiac muscle. Moreover, binding of Hsp20 to 14-3-3 is modulated by PKA-mediated phosphorylation, indicating the presence of a regulatory protein complex with a putative functional significance during β-adrenergic stimulation.The observation that 14-3-3 binds directly to Hsp20 and CFL2 in cardiac muscle prompted us to investigate the minimal regions of CFL2 and Hsp20 involved in 14-3-3 binding. For this purpose, we first generated recombinant proteins from various deletion constructs of CFL2 a,b. BlotWe next mapped the minimal region of 14-3-3 that is required for association with CFL2 and Hsp20 by generating N- and C-terminal deletion constructs of 14-3-3 c. Blot op < 0.05, t test, two-tailed n = 3). This was associated with increased binding of phosphorylated GST-Hsp20 to MBP-14-3-3, in agreement to the findings from blot overlay and immunoprecipitation assays  treatment. ISO is an agonist of the β2-adrenergic receptor (β2AR) which elevates cAMP and thus, PKA activity. To this end, we first transfected HEK 293 cells with a myc-tagged Hsp20 expression vector and found by immunofluorescence that Hsp20 preferentially localizes to the cytosol a. This op < 0.05, t test, two-tailed n = 3). Moreover, the cytoskeletal fraction contained phosphorylated Hsp20 . We conclude that the naturally occurring Hsp20-P20L mutation displays diminished 14-3-3 interaction and mobilization to the cytoskeletal fraction under stress conditions, such as β-adrenergic stimulation.Subcellular analysis of Hsp20-P20L-transfected HEK 293 cells demonstrated no major alterations in the localization of the mutated versus WT protein b. This wAs the P20L mutation diminishes the association of Ser16 phosphorylated-Hsp20 with 14-3-3 , and givCollectively, these data suggest that the Hsp20-P20L mutation not only affects the direct interaction of this protein with 14-3-3, but it also impairs the regulation of the CFL2/14-3-3 interaction.p < 0.05 vs. CFL2 and ‡p < 0.05 vs. CFL2:14-3-3:Hsp20, t-test, two-tailed, n = 3). Importantly, compared to Hsp20-WT, the phosphorylated Hsp20-P20L protein displayed a significantly reduced effect on CFL2 activity . However, phosphorylated Hsp20-P20L exhibited significantly enhanced CFL2-mediated actin depolymerization when compared to non-phosphorylated Hsp20 . Preliminary findings on Hsp20-S16A and Hsp20-S16D indicate that PKA-treatment of Hsp20-S16A had no effect on CFL2 activity, while PKA-treated Hsp20-S16D exhibited enhanced CFL2-mediated F-actin depolymerization, similar to phosphorylated GST-Hsp20 (data not shown). Collectively, these findings reveal an important role of Hsp20 phosphorylation in the regulation of actin dynamics.CFL2 is a major regulator of actin dynamics and a key player in actin depolymerization . We ther2+ cycling and contractility. Among them, Hsp20 and its phosphorylation have emerged as important determinants of cardiac muscle function [β-Adrenergic stimulation of cardiomyocytes, such as under stress conditions, leads to activation of PKA and phosphorylation of key proteins governing Cafunction ,11. HoweIn cardiomyocytes, Hsp20 has been described to exhibit cytosolic localization ,28, howeTo date, 14-3-3 proteins have been estimated to interact with over 200 proteins ,34 that The identification of the mechanism mediating Hsp20 translocation to the cytoskeleton and modulation of F-actin depolymerization may have important implications in deciphering the involvement of Hsp20 in cardiac disease. Hsp20 has been shown to translocate from the cytosol to myofibrils following cardiac and skeletal muscle ischemia ,39. IschThe relevance of this regulatory mechanism to heart disease pathogenesis is further underscored by the molecular and cellular implications of the phosphorylation impairing Hsp20-P20L mutation that has been observed in dilated cardiomyopathy patients ,53. At tOur findings demonstrate that the impaired ability of mutant Hsp20 to become phosphorylated at Ser16, leads to significantly reduced binding to 14-3-3, and aberrant regulation of CFL2-mediated F-actin depolymerization , possiblIn conclusion, we herein describe a mechanism through which β-adrenergic stimulation modulates Hsp20 subcellular localization and function in cardiomyocytes. We show that the PKA-mediated phosphorylation of Hsp20 promotes its translocation to the cytoskeleton where it modulates the interaction of 14-3-3 with CFL2 and consequently impacts F-actin depolymerization. Through this mechanism, the stress-induced Hsp20 phosphorylation could safeguard cytoskeletal integrity and protection from injury during stress conditions. Conversely, the human phosphorylation impairing Hsp20-P20L mutation disrupts formation of the 14-3-3/Hsp20 protein complex, fails to displace CFL2 from 14-3-3 and leads to aberrant control of actin dynamics. Under stress conditions, this leads to cytoskeletal disruption and myofibrillar damage that is anticipated to affect cardiac contractility and contribute to cardiac pathology. Therapeutic approaches modulating Hsp20 phosphorylation may thus hold promise in combating heart disease.The generation of the full length 14-3-3 gamma (amino acids 1–247) expression construct was performed by RT-PCR on human muscle cDNA using primer-1 (5′ GCGAAGATGGTGGACC 3′) and primer-2 (5′ CTTAATTGTTGCCTTCGCCG 3′). Deletion expression constructs of 14-3-3 were generated using primer-1 and primer-3 (5′ CTTTGCTCTCGTACTGGGTC 3′) (amino acids 1–120) or primer-4 (5′ ACCCAGTACGAGAGCAAAGTG 3′) and primer-2 (amino acids 115–247). PCR products were cloned in the EcoRI/SalI sites of the pMALc2x vector . The authenticity of all constructs was confirmed by sequence analysis . The expression constructs of GST-Hsp20 (amino acids 1–160) containing the wild-type or the Hsp20-P20L variant, as well as the different GST-CFL2 constructs containing full length and deletion fragments of CFL2, have been previously reported ,57.Protein expression of all of the above constructs was performed as previously described . Recombi4 (pH 7.2), 2 mM EDTA, 10 mM NaN3, 120 mM NaCl and 1% NP-40, supplemented with protease inhibitors . Equivalent amounts of recombinant MBP and MBP-14-3-3 recombinant proteins bound to amylose resin (New England Biolabs) were mixed with 0.5 mg of cardiac homogenates at 4 °C for 16 h. The beads were washed with 10 mM NaPO4 (pH 7.2), 10 mM NaN3, 120 mM NaCl, 0.1% (v/v) Tween-20 and were subsequently analyzed by Western blot using Hsp20, phospho-Hsp20 (Ser16) , CFL2 and phospho-CFL2  primary antibodies and peroxidase-conjugated goat anti-rabbit  secondary antibody. Immunoreactive bands were detected using Pierce ECL Plus reagents .Pull down assays were performed as previously described ,59. Brie2), supplemented with 200 μM ATP (Sigma-Aldrich) and 1250 units of the PKA catalytic subunit. Samples were incubated at 30 °C for 1 h and subsequently used for pull down or blot overlay assays.Protein phosphorylation of cardiac protein homogenates or GST-Hsp20 recombinant protein was performed in vitro using the cAMP-dependent protein kinase (PKA), catalytic subunit (New England Biolabs), as previously described . Briefly3VO4, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween-20). GST-CFL2 phosphorylation was confirmed by Western blotting using an anti-phospho-CFL2 antibody .GST-CFL2 recombinant protein phosphorylation was performed as previously described . In brieImmunoprecipitation experiments were performed in mouse cardiac extracts, as previously described ,61. BrieProtein interactions were assessed in vitro by blot overlay assays, as previously described ,57,60,62In a different set of experiments, mapping of the minimal binding domain of 14-3-3 to CFL2 was performed using equal amounts of GST, GST-CFL2 (amino acids 1–166), GST-CFL2 (amino acids 1–55), GST-CFL2 (amino acids 19–154) and GST-CFL2 (amino acids 105–166) proteins that were separated by SDS-PAGE and allowed to interact with MBP-14-3-3 (amino acids 1–247), as described above. Western blot analysis with an MBP antibody (New England Biolabs) determined the minimal region of CFL2 required for 14-3-3 binding.In parallel, GST, GST-CFL2 and phosphorylated GST-Hsp20 recombinant proteins were allowed to interact with MBP-14-3-3 (amino acids 1–120) or MBP-14-3-3 (amino acids 115–247), as described above.The Actin Polymerization Biochem Kit  was used to investigate actin dynamics in vitro on a PerkinElmer LS 55 Fluorescence spectrometer , as previously described ,61. The HEK 293 cells  were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (ThermoFisher Scientific), as previously described ,59. The 3) for 1 h at 25 °C. Hsp20 (AbCam) or actin (Merck Millipore) primary antibodies were diluted in blocking buffer and applied to the cells for 1 h at 25 °C. Samples were washed again with PBS and counterstained for 1 h at 25 °C with the appropriate secondary antibody  (Invitrogen) diluted in blocking buffer. Following further washes with PBS, samples were mounted with Vectashield medium containing DAPI  and analyzed on a Leica confocal laser scanning microscope . Localization analysis for Hsp20 and actin was performed using Colocalization Threshold plugin of ImageJ.For immunofluorescence analysis, the myc-Hsp20 construct was transiently transfected in HEK 293 cells with Lipofectamine™ 2000 (ThermoFisher Scientific), according to the manufacturer’s instructions. Twenty-four hours after transfection, cells were fixed for 20 min at 25 °C with ice cold methanol, washed three times with phosphate-buffered saline (1× PBS) and permeabilized for 30 min at 25 °C in PBS containing 0.1% Triton X-100. The cells were then washed in PBS prior to incubation with blocking buffer  at 37 °C (Sigma-Aldrich) and then harvested for fractionation experiments, as described above.Cell fractionation experiments were performed as previously described . In brieThe competition of Hsp20 and CFL2 for 14-3-3 binding was examined by pull down assays. In particular, HEK 293 cells were transfected with the full length myc-CFL2 construct and after 24 h the cells were lysed in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl and 1% NP-40, supplemented with protease inhibitors (Sigma-Aldrich). Pull down assays were performed using myc-CFL2 HEK 293 cell lysates that were incubated with MBP-14-3-3 recombinant protein bound to amylose resin, in the presence of phosphorylated or non-phosphorylated GST-Hsp20 eluted protein. Protein binding to MBP-14-3-3 was determined by Western blot analysis using myc (Sigma-Aldrich) or phospho-Hsp20 (AbCam) antibodies.


MoS2 is a 2-D nanosheet which has strong high fluorescence-quenching ability when applied to a dye-labeled antibody (Ab). In this paper, we developed an Ab-functionalized MoS2-based fluorescent immunosensor, which utilized the fluorescence resonance energy transfer (FRET) between the MoS2 and fluorescence dye during the Ab–antigen interaction. The assay was performed on a low-cost cotton thread-based microfluidic platform due to the good wicking property and flexibility. Upon the optimization of assay conditions, the immunosensor demonstrated remarkable sensitivity of 50 per mL and specificity with a dynamic linear response range of 102–106 EID50 per mL for IBV standard solutions. The developed immunoassay successfully detected the IBV spiked chicken serum with satisfactory results. The foregoing presents its potential application for on-farm detection.Infectious bronchitis virus (IBV), an avian coronavirus, significantly affects the performance of both the egg-laying and meat-type birds causing the foremost of economic loss in poultry industry. This paper aims to develop a rapid, low-cost, and sensitive biosensor for IBV detection by using molybdenum disulfide (MoS I.Coronaviruses are enveloped, positive-strand RNA viruses of birds and mammals including humans. As a representative of the Gamma-coronavirus genus, the avian coronavirus (AvCoV) infectious bronchitis virus (IBV) may cause highly contagious respiratory disease in chickens and other galliforme birds 2) is an emerging material and one of such TMDs that can be synthesized in large scale and directly dispersed in aqueous solution and no treatment of surfactants or oxidation is required 2 become promising material in biosensing platforms and finding various applications 2 surface is a key enabling feature, because it enables strong affinity of protein-surface adsorption et al.2 biosensor by using the high fluorescence-quenching ability between MoS2 and dye-labeled single-stranded DNA probe for prostate specific antigen detection. High sensitivity and high selectivity with a detection limit for the PSA of 0.2 ng/mL were achieved. Tuteja et al.2-based electrochemical immunosensor for detecting 2-modified electrodes and et al.2 aptasensor for the detection of the malarial biomarker Plasmodium lactate dehydrogenase (pLDH). Zhang et al.2 for alpha fetal protein detection. MoS2 nanosheets surface was modified using polyethylenimine (PEI) polymer and gold nanopaticles. The immunosensor was able to analyze AFP in real human serum samples with limit of detection of 1.0 2 in biosensor applications.Nanomaterials have become powerful element in constructing new biosensors due to their unique optical, electronic and catalytic properties The thread-based microfluidics is promising alternative to conventional microfluidic systems due to it many special characters. The thread-based biomedical devices are low cost and broadly available, flexible, easily to handle, lightweight, easy to manipulate and facilitate to transported or stored in any forms. In addition, the wicking properties of thread enable the low volumes of sample solution through it efficiently. All above mentioned makes thread-based microfluidics an attractive matrix for the fabrication of low-cost and low-volume microfluidic diagnostic devices for handheld on-site diagnosis applications 2 for the detection of IBV. The principle is based on a homogeneous FRET immunoassay, the sensing mechanism is shown in 2 on the fluorophore during the antigen-antibody reaction was adopted. The antibody (Ab) probes are modified with fluorescent dye labelling (dyed-IBV-Ab) and MoS2(MoS2-IBV-Ab bioconjugates), respectively. In the presence of IBV, both the dyed-IBV-Ab and the MoS2-IBV-Ab bioconjugates will specifically bind with the target IBV due to the antigen-antibody reaction. After binding, the fluorescence of the dyed-IBV-Ab probe is largely quenched owing to the transfer of electrons or energy between the closely connected dye molecules and the MoS2. In the absence of IBV, the fluorescence of the dyed-IBV-Ab probe will not change. Therefore, the concentration of the IBV in the chicken blood sample can be quantitatively determined by analysis the fluorescence intensity in an assay.Here, we aimed at developing a biosensor for rapid detection of IBV. We developed a single-step immunosensor on cotton thread based on antibody-functionalized MoSII.A.2 nano-sheet dispersion, ovine serum albumin , hydrogen peroxide (H2O2), poly(L-lysine) (PLL), chicken serum and all other mentioned chemicals and solvents were purchased from Sigma-Aldrich . Anti-infectious bronchitis virus (Massachusetts) (IBV) was purchased MyBioSource, Inc. . Infectious bronchitis virus, low pathogenic avian influenza virus A H4N6 and H9N2 were kindly provided by our collaborator  and the detailed culture procedure can be found elsewhere 50 per mL (egg infectious dose 50%). Avian influenza virus A H4N6 (Avian influenza virus A/Duck/Czech/56 (H4N6)) was propagated in 11-day-oldembryonated chicken eggs by inoculation into the allantoic cavity MoSB.The labelling of IBV Ab with fluorescent dye is conducted by using the Alexa Fluor 488 Antibody Labeling Kit. Briefly, 1.0 mg/mL IBV Ab was mixed with 1 M sodium bicarbonate solution. Then C.2 acted as a nanoplatform to adsorb IBV Ab and was further employed as a biological probe. The MoS2 (0.5 mg/mL) dispersion in aqueous was first concentrated by centrifugation at 8000 rpm for 30 min to remove the surfactant. The MoS2 was resuspended in Milli-Q water and sonicated for 1 h to provide a homogeneous solution. Aqueous poly(L-lysine) solution was added to the resulting MoS2 at a concentration of 1 mg/mL and stirred thoroughly for 1 h followed by overnight incubation at 4 °C. The poly(L-lysine) backbone is electrostatically adsorbed to MoS2 surface. The PLL-MoS2 mixture was purified and concentrated by centrifugation three times at 8000 rpm for 30 min to remove the solvent and resuspended in PBS. IBV Ab was then added at a desired concentration into the obtained mixture followed by overnight incubation at 4 °C to produce the MoS2-IBV-Ab bioconjugates. With the chemical modification by poly(L-lysine), the antibodies can be bind to MoS2 via electrostatic and covalent interactions The MoSD.2O2 and 0.01 M HCl for 5 min respectively, followed by washing with a large amount of ultrapure water to remove the residual acid. Finally, cotton threads were dried at 37 °C for at least 2 h and stored for further use. Cotton threads were rendered hydrophilic with an air plasma . The design of the thread network is shown in 2-IBV-Ab bioconjugates. The cotton thread was blocked with 1% BSA in PBS for 30 min, rinsed with PBS (0.1% Tween 20) and Milli-Q water, and quickly dried under a stream of N2. Paraffin wax was applied on the end of the each thread channels to create isolated zones to prevent the fluid flowing. The cotton thread was stretched on the glass slide with the paper support using double adhesive tape. The cotton threads with MoS2-IBV-Ab bioconjugates were stored at 4 °C and used within one week.Cotton threads (100%) were boiled by 2 M NaCl for 30 min, and then soaked in 0.01% HE.2 was characterized by the FEI-Tecani G2 transmission electron microscope (TEM) operating at 200 kV. The hydrodynamic diameters, size distribution and Zeta  system . The UV-Vis absorption spectra were analyzed on DR6000TM UV-Vis spectrophotometer  with a resolution of 1 nm. The fluorescence spectra were recorded by the Cytation 5 Multi-mode Reader . The fluorescent imaging (Ex/Em=488 nm/519 nm) was taken on a fluorescent microscopy  under the same settings, namely exposure time, magnification, etc. The fluorescence intensity was then analyzed by analyzed by ImageJ.The morphology of MoSF.In a typical assay, a small volume  was spiked in the chicken whole blood and loaded into the sensor. The average fluorescence intensity is then calculated to determine the sample concentration. To evaluate the specificity of the presented immunosensor, avian influenza A H4N6 and H9N2 were detected. The fluorescence signals with these non-specific analytes were recorded.The performance of the presented immunosensor for detecting real blood sample was validated. IBV of various concentration  system . As shown in 2, PLL-MoS2 dispersion and MoS2-IBV-Ab bioconjugates are 113.5±0.32 nm, 162.1±1.07 nm and 983±84.5 nm, respectively. The Zeta potential  was conducted, the result of which is shown in When preparing the thread, the MoSC.6 EID50 per mL was analyzed by the presented thread immunosensor to obtain the standard curve and calculate the limit of detection. A linear fit of the obtained relative fluorescence intensity difference with respect to varying concentration of IBV standards is plotted in 2 EID50 per mL with a correlation coefficient (R2) of 0.9828. The limit of detection calculated based on 50 per mL. The different fluorescence responses of the test zones on the cotton thread upon the detection of various concentrations of IBV standard solution are shown in 2-IBV-Ab are formed, resulting in largely quenched owing to the transfer of electrons or energy between the closely connected dye molecules and the MoS2.With the optimization settings as demonstrated in previous section, quantitative analysis of IBV was carried out on the cotton thread. The transportation of fluidic was investigated firstly by using food color and it was observed that the fluid could reach the test zones within 1 min due to the capillary wicking. In addition, a time duration of 6 min was found to be sufficient for a complete reaction, as shown in D.The specificity of the developed cotton thread biosensor was evaluated against non-specific virus, avian influenza A H4N6  was coated with the IBV Ab at 10 IV.2-based immunosensor on a thread-based microfluidic network for rapid IBV detection. The distinct quenching property of MoS2 on the fluorophore during the antigen-antibody reaction was adopted. The thread-based network interconnected by knots to achieve the fluid mixing and separation. IBV standards and spiked chicken blood sample were successfully detection with high specificity and a detection of limit of 50 per mL. The present immunosensor demonstrated a good linearity and validated with ELISA method. In comparison with conventional immunological tests, the presented method have many advantages, such as ease of local manufacture, small consumption of reagents and samples, high sensitive and short time of analysis. All these characteristics allow for the use of this technology for rapid, prompt on-site IBV detection.In this study, we reported a proof-of-concept MoS


The disruption of the blood–brain barrier (BBB) plays a critical event in the pathogenesis of ischemia stroke. TGR5 is recognized as a potential target for the treatment for neurologic disorders.This study investigated the roles of TGR5 activation in attenuating BBB damage and underlying mechanisms after middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were subjected to model of MCAO and TGR5 agonist, INT777, was administered intranasally. Small interfering RNA (siRNA) for TGR5 and BRCA1 were administered through intracerebroventricular injection 48 h before MCAO. Infarct volumes, brain water content, BBB permeability, neurological scores, Western blot, immunofluorescence staining and co- immunoprecipitation were evaluated.Endogenous TGR5 and BRCA1 were upregulated in the injured hemisphere after MCAO and TGR5 expressed in endothelial cells. Treatment with INT777 alleviated brain water content and BBB permeability, reduced infarction volume and improved neurological scores at 24 h and 72 h after ischemia. INT777 administration increased BRCA1 and Sirt1 expression, as well as upregulated expressions of tight junction proteins. Ischemic damage induced interaction of TGR5 with BRCA1. TGR5 siRNA and BRCA1 siRNA significantly inhibited expressions of BRCA1 and Sirt1, aggravated BBB permeability and exacerbated stroke outcomes after MCAO. The protective effects of INT777 at 24 h after MCAO were also abolished by TGR5 siRNA or BRCA1 siRNA.Our findings demonstrate that activating TGR5 could reduce BBB breakdown and improve neurological functions through BRCA1/Sirt1 signaling pathway after MCAO. TGR5 may serve as a potential new candidate to relieve brain injury after MCAO. Stroke is one of the most common causes of death and the main cause of long-term disability worldwide . It has central nervous system (CNS), studies have found that activating TGR5 alleviates brain damage and improves outcomes in a model of experimental autoimmune encephalomyelitis (EAE) and hepatic encephalopathy  Χ100% .BBB permeability was evaluated by EB extravasation using spectrophotometry as previously described . At 4 h The in vitro experiment was prepared as previously reported , with soThe method of double and triple immunofluorescence staining was performed as previously described , 29. RatWestern blot analysis was performed as previously reported . ProteinCo-IP was performed as previously described , 31.500 P < 0.05 was considered as significant.All data analyses were performed using SigmaPlot 11.0 and GraphPad Prism 6 . Parametric data was expressed as mean ± SEM. Data from different groups were compared using one-way ANOVA followed by post hoc Tukey tests. Non-parametric data  were analyzed with the Kruskal–Wallis test followed by Dunn’s post-hoc. In all statistical analysis, A total of 494 rats were used and 410 rats underwent MCAO induction. There were no deaths in sham group. For groups of MCAO, the mortality rate was 11.5% (47 of 410) (Supplementary Table P < 0.05 versus Sham). Double immuno-fluorescence staining demonstrated that TGR5 was expressed in endothelial cells at 24 h after MCAO . The administration of 0.48 mg/kg INT777 decreased cerebral infarction, restored neurological function and ameliorated brain water content at 72 h after injury . Based on the dose study, we chose to middle dosage of INT777 for all subsequent studies.Treatment with 0.48 mg/kg and 1.44 mg/kg of INT777 significantly reduced infarct volume, improved neurological scores and reduced brain water content of right ischemic hemisphere at 24 h post-MCAO compared to MCAO+vehicle group Fig. a-d , INT777 treatment significantly reduced EB dye leakage(P < 0.05 versus MCAO+vehicle)  , and INT777 further increased TGR5 and BRCA1 expression(P < 0.05 versus MCAO)  Fig. f. CO-IP AO) Fig. e.To further assess the role of TGR5 in stroke, TGR5 siRNA was administered by ICV injection to knockdown endogenous TGR5. Double immunofluorescence staining showed that both TGR5 and BRCA1 expressions increased in penumbra following MCAO while siRNA significantly reduced TGR5 or BRCA1 expression   Fig. a and b. 05) Fig. a and b.P < 0.05)  Fig. c-f, whenP < 0.05). However, INT777 increased expressions of Sirt1, ZO-1 and occludin, compared with MCAO group (P < 0.05)  protein expressions or variations are associated with alterations in BBB permeability . As showP < 0.05)   , play an important role of neuroprotection for brain pathologies which are mediated by TGR5 . McMilliAlthough the exact mechanisms of TGR5–mediated BBB protection are not well clarified, BRCA1 may play an important role in the TGR5-mediated signaling pathway. BRCA1, a well-known tumor suppressor implicated in familial breast and ovarian cancers, provides a protective role in atherosclerosis and neurological diseases –14. NoriSeveral evidence have confirmed that BRCA1 is a key regulator of Sirt1 in cancer research. BRCA1 inactivation events  are accompanied by decreased Sirt1 levels while overexpression of BRCA1 results in increased Sirt1 levels  by direcIn the current study, we found that INT777 increased the expression of Sirt1 after MCAO while TGR5 siRNA and BRCA1 siRNA inhibited the Sirt1 expression, reversed the effect of INT777 on Sirt1, which means TGR5 and BRCA1 can act as upstream regulators of Sirt1. Furthermore, our data demonstrated that TGR5 or BRCA1 knockdown significantly reverses the neuro-protection of INT777 on stroke outcomes, as well as decreasing ZO-1 and occludin expression. This finding supports the notion that the BRCA1/ Sirt1 signaling pathway plays a role in BBB protection induced by activation of TGR5 after MCAO.There are some limitations in the present study. First, TGR5 produces pleiotropic effects via different signaling pathways, such as alleviating inflammation and attenuating apoptosis . In thisAs summarized in Fig. Additional file 1.


The Antarctic sea ice area expanded significantly during 1979–2015. This is at odds with state-of-the-art climate models, which typically simulate a receding Antarctic sea ice cover in response to increasing greenhouse forcing. Here, we investigate the hypothesis that this discrepancy between models and observations occurs due to simulation biases in the sea ice drift velocity. As a control we use the Community Earth System Model (CESM) Large Ensemble, which has 40 realizations of past and future climate change that all undergo Antarctic sea ice retreat during recent decades. We modify CESM to replace the simulated sea ice velocity field with a satellite-derived estimate of the observed sea ice motion, and we simulate 3 realizations of recent climate change. We find that the Antarctic sea ice expands in all 3 of these realizations, with the simulated spatial structure of the expansion bearing resemblance to observations. The results suggest that the reason CESM has failed to capture the observed Antarctic sea ice expansion is due to simulation biases in the sea ice drift velocity, implying that an improved representation of sea ice motion is crucial for more accurate sea ice projections. Climate models typically fail to capture the observed Antarctic sea ice expansion during recent decades. Here, the authors show that the observed expansion is reproduced in a climate model after removing biases in the sea ice drift velocity. In this study, we focus on the expansion: during 1979–2015 the Antarctic sea ice area increased at a statistically significant rate that was approximately a third as fast as the Arctic sea ice retreat  Large Ensemble, which has 40 realizations that all use identical historical and future forcing but differ in their initial conditionseld Fig. . The obsAlthough the satellite-derived sea ice motion fields begin in 1979, here we focus on the period 1992–2015 due to issues with the sea ice motion data prior to a satellite sensor transition in December 1991 , we replaced the simulated surface wind in the sea ice momentum calculation with ERA-InterimWe investigated the relationship between sea ice drift velocity and sea ice area in the simulations, and we found no clear connection between the trends  The use of just three ObsVi ensemble members may be insufficient to resolve the influence of sea ice motion biases on the sea ice trend in CESM due to internal variability. (ii) Despite substantial improvement, there are still notable differences between the observations and the ObsVi runs in terms of the spatial structure of the changes Fig. . (iii) TIn conclusion, like most current climate models, CESM does not simulate the observed Antarctic sea ice expansion. These results show that this can be improved by manually correcting sea ice drift velocity biases. Some of this improvement can be captured by instead correcting biases in the surface winds in the sea ice momentum equation. The main candidates for explaining the remainder of the discrepancy between simulated and observed sea ice changes include model biases in the sea ice rheology, sea ice drag coefficients, and ocean surface currents, as well as ice velocity biases due to the coarse model resolution. Our results suggest that an improved representation of sea ice motion is crucial for more accurate sea ice projections.28, which is managed by the National Snow & Ice Data Center (NSIDC). This dataset includes sea ice velocity fields for both hemispheres, which are derived from satellite measurements and also draw on buoy measurements as well as free drift estimates calculated from NCEP-NCAR reanalysis geostrophic winds. It provides sea ice velocities that are interpolated onto a 25-km resolution Equal Area Scalable Earth (EASE) grid with daily temporal resolution from October 1978 to January 2016 at the time the data were downloaded. Here we use data during 1992–2015. As discussed above, we omit the earlier years due to data issues prior to a December 1991 satellite sensor transition -F8, -F11, and -F13 SSM/I, and the DMSP-F17 Special Sensor Microwave Imager/Sounder (SSMIS). The ice concentration is provided on a 25-km resolution polar stereographic grid. We use the NSIDC Sea Ice Index35 for the observed ice area time series, as well as for the observed ice extent time series plotted in Supplementary Figs. For the observed sea ice concentration, we use the monthly-mean Sea Ice Concentrations from Nimbus-7 SMMR and DMSP SSM/I-SSMIS Passive Microwave Data33, which has been suggested to provide a somewhat reliable estimate for the Southern Ocean surface fields37. The reanalysis goes back to 1979, and we use wind data during 1979–2015. The wind product is reported on a 0.75∘ grid resolution with 6-h frequency. We interpolate it to the model grid using bilinear interpolation.We use reanalysis surface winds from ERA-Interimτ = 1 h to constrain the sea ice drift velocity to resemble observations. The momentum equation is sub-cycled during each sea ice model time step (using the CESM parameter xndt_dyn) in order to avoid numerical instability. In locations where the satellite-retrieved sea ice velocity data is not available but there is simulated ice, we use the ice momentum equation with ERA-Interim surface winds as in the ERAWind runs. In the ERAWind runs, the default ice momentum equation is used but the surface wind used to generate the atmosphere-ice stress is replaced with ERA-Interim winds; note that the model wind field is altered only in the calculation of the atmosphere-ice stress in the sea ice momentum equation.In the ObsVi runs, the sea ice momentum equation is replaced with a relaxation to the satellite-derived ice velocity field,The three ObsVi runs are branched from the corresponding LENS runs on January 1, 1960. For each ObsVi run, we spin-up the model during simulation years 1960–1991 by relaxing the sea ice velocity to the observed mean annual cycle (averaged over 1992–2015), and then the simulation is continued during 1992–2015 using the full time evolution of the observed ice motion field. In other words, we computed the mean annual cycle in the daily observational field, and the ice motion is relaxed to this field every year during 1960–1991, although increases in greenhouse gas forcing and other forcing changes during this period are equivalent to the LENS runs.6 km2 in the first year (blue lines in Supplementary Fig. Due to the change in sea ice momentum forcing in 1960, the Antarctic sea ice area increases rapidly during the first few months such that the annual-mean ice area increases by around 1 × 10The three ERAWind runs are similarly branched from the corresponding LENS runs on January 1, 1960. Since the ERA-Interim winds are available for a longer time period, we spin-up the ERAWind runs with the 1979 forcing repeating every year during simulation years 1960–1978 and then use the full time evolution of the wind field during 1979–2015, thereby allowing further spin-up during 1979–1991 before the 1992–2015 analysis period.We find that the Antarctic sea ice area also initially increases in the ERAWind runs (blue lines in Supplementary Fig. Additional simulations to investigate the sensitivity to spin-up conditions are presented in the 38.Although the 1992–2015 ice area trends agree better with observations in the ObsVi runs than in the ERAWind runs, the ERAWind runs show better agreement with observed year-to-year changes in the ice area. This is listed in Supplementary Table 40. We find that the seasonal structure of the 1992–2015 ice area trend varies considerably between the three ObsVi runs (Supplementary Fig. Although this study focuses on annual-mean trends, some previous studies have examined seasonal variations in the observed trendsThe changes in the ice momentum equation in all of the runs in this study apply in both hemispheres. However, in the Arctic we do not find that any of these changes lead to substantially more accurate simulations of the sea ice area trend or the year-to-year variability (Supplementary Table Supplementary InformationPeer Review File


Hyperphosphataemia increases cardiovascular mortality in patients with kidney disease. Direct effects of high inorganic phosphate (Pi) concentrations have previously been demonstrated on endothelial cells (ECs), including generation of procoagulant endothelial microvesicles (MVs). However, no mechanism directly sensing elevated intracellular Pi has ever been described in mammalian cells. Here, we investigated the hypothesis that direct inhibition by Pi of the phosphoprotein phosphatase PP2A fulfils this sensing role in ECs, culminating in cytoskeleton disruption and MV generation. ECs were treated with control (1 mM [Pi]) vs. high (2.5 mM [Pi]), a condition that drives actin stress fibre depletion and MV generation demonstrated by confocal microscopy of F-actin and NanoSight Nanoparticle tracking, respectively. Immuno-blotting demonstrated that high Pi increased p-Src, p-PP2A-C and p-DAPK-1 and decreased p-TPM-3. Pi at 100 μM directly inhibited PP2A catalytic activity. Inhibition of PP2A enhanced inhibitory phosphorylation of DAPK-1, leading to hypophosphorylation of Tropomyosin-3 at S284 and MV generation. p-Src is known to perform inhibitory phosphorylation on DAPK-1 but also on PP2A-C. However, PP2A-C can itself dephosphorylate (and therefore inhibit) p-Src. The direct inhibition of PP2A-C by Pi is, therefore, amplified by the feedback loop between PP2A-C and p-Src, resulting in further PP2A-C inhibition. These data demonstrated that PP2A/Src acts as a potent sensor and amplifier of Pi signals which can further signal through DAPK-1/Tropomyosin-3 to generate cytoskeleton disruption and generation of potentially pathological MVs. Phosphate compounds play a pivotal role in all cells, in processes as disparate as energy homeostasis; nucleotide, nucleic acid and phospholipid metabolism and signaling through protein phosphorylation events. Recently renewed interest in the functional and pathological effect of orthophosphate (Pi) has been driven by evidence that elevated serum Pi concentrations are strongly associated with an increased risk of cardiovascular events and mortality, particularly in patients with hyperphosphataemia, a hallmark of chronic kidney disease (CKD) , but alsEven though mechanisms for the sensing of [Pi] in bacteria, yeast ,12 and p+-linked PiT1 (SLC20A1) Pi transporters, allowing Pi to act on intracellular phosphatases and subsequently induces a global increase in both protein Tyr and Ser/Thr phosphorylation  acutely induced hyperphosphorylation of Src at Tyr419 within 1.5 h in cultures of EA.hy926 ECs D,E. To cTo show that these Pi-induced changes in PP2A and Src were leading to functionally important downstream signals culminating in increased MV output, the resulting effects on DAPK-1 were studied. From the scheme in ®. Western blotting of the GFP-Trapped proteins using pan-specific antibodies against phospho-Ser/Thr and phospho-Tyr demonstrated Serine and/or Threonine phosphorylation within the GFP-TPM-3 protein, and reproducible hypophosphorylation under high Pi conditions  has 7 Snditions C,D. No TAn earlier study on Tropomyosin-1 (TPM-1)  showed pFinally, to demonstrate a functional link between the Pi effects on DAPK-1 phosphorylation and MV output in In this study, we provide evidence for the first time that an elevated intracellular Pi concentration can be sensed directly in a mammalian cell line by the Pi-responsive protein phosphatase PP2A.An important feature of the proposed mechanism  is the cApart from a recent interesting study on plasma membrane Pi transporter proteins , no sensIt is important to emphasise that the evidence presented here is not intended to exclude the possibility that Pi is also sensed by proteins other than PP2A—in EA.hy926 cells or in other mammalian cell types. At least in vitro, physiologically attainable Pi concentrations have been shown to activate or inhibit the biological activity of a number of important regulatory proteins, including phosphoprotein tyrosine phosphatases (PTPs) ,18,19 anIt has long been suspected that Pi exerts important “Pi toxicity”  effects The data presented in In conclusion, this study shows how, in endothelial cells, DAPK-1 and TPM proteins downstream from PP2A/Src can give functionally important cytoskeletal rearrangements and output of MVs, which, as we have shown previously , are pro2 IU·mL−1) and streptomycin (100 mg·mL−1) at 37 °C in a humidified 5% CO2 atmosphere. Unless otherwise stated, cells were seeded for experiments in 35-mm six-well plates at 3 × 105 cells/cm2 and used at 70% confluence. Experimental incubations were performed in MEM  with 2 mM L-glutamine, penicillin (102 IU·mL−1) and streptomycin (100 mg·mL−1) at pH 7.4 with 1.8 mM [Ca2+] and 1 mM [Pi]. To model hyperphosphatemia, NaH2PO4 was added to raise the [Pi] to 2.5 Mm—a concentration that has been used extensively elsewhere  for 1.5 h and used subsequently for GFP-TrapThe inhibitory effect of Pi ions on PP2A catalytic activity was determined in pulled-down PP2A from EA.hy926 cell lysates using a PP2A Immunoprecipitation Phosphatase Assay Kit  as described in the manufacturer’s instructions. Activity was determined colorimetrically by measuring Pi release from an L-threonine phosphopeptide (K-R-pT-I-R-R).® Lab-Tek®). After treatment with 1 and 2.5 mM [Pi] for 1.5 h, they were fixed with 3.7% formaldehyde and permeablised with 0.1% saponin in PBS, pH 7.5. F-actin was detected using Alexa-Fluor®-594-phalloidin . Nuclei were counterstained by DAPI. The cells were visualised by confocal microscopy using an Olympus FV1000 CLSM. Cells expressing transcytoplasmic actin stress fibres were blind-scored  were plated in 8-well slide-chambers  or by Wilcoxon matched-pairs signed rank test (for nonparametric data). One-way ANOVA combined with Tukey’s or Dunnett’s posthoc test was applied for multiple comparison tests as appropriate. Actin stress fibres and their distribution frequency were blind-scored and analysed by nonparametric ANOVA combined with a Chi-square test. p values < 0.05 were considered statistically significant.Unless otherwise stated, data are presented as the mean


Antifreeze proteins (AFPs) are specific proteins, glycopeptides, and peptides made by different organisms to allow cells to survive in sub-zero conditions. AFPs function by reducing the water’s freezing point and avoiding ice crystals’ growth in the frozen stage. Their capability in modifying ice growth leads to the stabilization of ice crystals within a given temperature range and the inhibition of ice recrystallization that decreases the drip loss during thawing. This review presents the potential applications of AFPs from different sources and types. AFPs can be found in diverse sources such as fish, yeast, plants, bacteria, and insects. Various sources reveal different α-helices and β-sheets structures. Recently, analysis of AFPs has been conducted through bioinformatics tools to analyze their functions within proper time. AFPs can be used widely in various aspects of application and have significant industrial functions, encompassing the enhancement of foods’ freezing and liquefying properties, protection of frost plants, enhancement of ice cream’s texture, cryosurgery, and cryopreservation of cells and tissues. In conclusion, these applications and physical properties of AFPs can be further explored to meet other industrial players. Designing the peptide-based AFP can also be done to subsequently improve its function.  Numerous organisms advance exclusive adaptive mechanisms for surviving in exceptionally cold environments. Approximately two-thirds of the earth’s surface contains water, where the average surface temperature of seas and oceans varies within −2 to 30 °C, depending on the latitude ,2. In AnThese peptides and proteins contain ice-binding affinity and are able to reduce the freezing temperature of a solution non-colligatively with a negative influence on its melting temperature, a phenomenon known as the thermal hysteresis (TH). Furthermore, AFGPs can prevent recrystallization of ice where large ice crystals are developed at the expenditure of the smaller ones, and thus avoid the ice regrowth-caused mechanical cell damage. Presently, it is commonly agreed that the AFGPs function through adsorbing their flat ice-binding surfaces over the specific ice crystals’ planes, hence inhibiting or preventing the further growth of ice .Due to special features and functions of these proteins, their capabilities have been utilized in cell biology, food industry, and biotechnology. In this review, we describe the types, features, properties, and characterizations of antifreeze proteins. Furthermore, it also covers the biotechnological study of AFPs via different tools and web servers to easily analyze the features and properties of AFPs. Finally, we try to elevate the role of these proteins/peptides in diverse aspects of industrial, agricultural, and medical proposes.In general, AFPs can be considered in terms of two features: TH and ice recrystallization inhibition (IRI) features. They belong to a subgroup of ice-binding proteins (IBPs) depressing the hysteresis freezing point known as the ‘thermal hysteresis gap’. Two hysteresis intervals are involved in this TH gap, including a melting hysteresis (MH) interval within melting point (MP) and the equilibrium melting/freezing point, and a freezing hysteresis interval ,7. In thUnder this condition, the ice crystals burst. Flavobacterium frigoris PS1 (FfIBP) binds to the basal plane and a lemon shape is created, while hyperactive AFPs can bind to the ice crystals’ basal plane [AFPs can also bind to the particular ice crystals’ planes within the TH gap, resulting in exclusive ice morphology . For insal plane .Gadus macrocephalus Type IV AFP (GmAFP IV) and AFP derived from berry were purified within affinity chromatography [Recently, most research concerning AFPs has focused on exploring, recognizing, purifying, evaluating, and detecting methods, analyzing their mechanisms, and characterization as well as bioinformatics studies to support the potential utilizations of AFPs in different aspects. For instance, ice affinity purification is a classic method for AFPs purification and is used to extract AFPs from the solution . Similartography ,18.Experimental methods had been used to assess the mechanism of antifreeze proteins containing site-directed mutation as well as ice-etching ,20. As pHigh-resolution microscopy as well as immuno-fluorescence could be utilized to reveal the variations in distribution of AFPs . OrbitalRecently, screening and bioinformatics tools were effective and accurate to analyze the diverse types of AFPs. For instance, the hydration structure of AFPs could be analyzed by random tool modeling ,31, meanAFPs are one of the main proteins that remain functional in cold temperatures naturally encompass bony fish, insects, plantae, fungi, Nematoda, amphibia, bacteria, and diatoms  Figure .AFGPs are produced by various organisms, categorized into five different classes (AFGP and types I–IV) depending on their sources and structural features . Arctic The Type I AFP which has a straight α-helix chain with molecular mass of 3.3–4.5 kDa and length of almost 58 Å is abundantly derived from diverse fish species as well as yeasts. It comprises 37 amino acids, where most of the residues are alanine. Furthermore, it involves three repeats of Thr-Ala-Ala-X-Ala-X-X-Ala-Ala-X-X .Besides, the Type II AFP includes the greatest number of non-glycoproteins and thermal hysteresis proteins THPs, and exists in various species, including Atlantic herring, sea raven, and smelt. The structures of the protein are stabilized through disulfide bonds within cysteines, and the general tertiary structures are categorized as two α-helices and two β-sheets. The molecular mass is in the range of 11–24 kDa ,45.The Type III AFP exists in eelpout and both Northern and Antarctic zoarcid fish. The molecular mass is in the range of 6.5–14 kDa. These AFPs are classified into two groups in terms of their affinity towards quaternary (QAEs) and sulfopropyl (SP) amines. Type III AFP contains no cysteine residues and no amino acid bias. The two anti-parallel triple-stranded β-sheets make their secondary structures more compact. The three-dimensional (3D) structure of this type of AFPs forms the shape of a spherical or rather a triangular prism with two moderately flat faces. The prism’s side has a key role in ice-binding interaction ,47.Longhorn sculpin fish and is wholly different from the other types of AFPs. It is a protein enriched in glutamine and glutamate amino acids. It has the molecular weight of nearly 12 kDa and consists of 108 amino acids, which is much greater than other identified sculpin AFPs. Interestingly, its structure is completely made up of helices with a considerable number of α-helical folds, where the hydrophobic sides are in front of the interior and four amphipathic helices are organized anti-parallel, with the polar sides fronting the solvent [Lately, Type IV AFP has been revealed in  solvent .n, where the disaccharide β-D-galactosyl---N-acetyl-α-D-galactosamine is linked as a glycoside to the hydroxyl oxygen of the Thr residues. The residues are repeated in the structure of protein for 4–30 amino acids, and proline is rarely involved in the isoforms. The molecular weight is within 2.7–32 kDa. Overall, AFGPs having molecular weights between 20 and 33 kDa are referred to as AFGP 1–4, and that less than 20 kDa are denoted as AFGP 5–8 [The AFGP is available in the blood sera of Antarctic notothenioid and indicated that this type of AFP contains some repeating residues of (Ala-Ala-Thr)AFGP 5–8 ,49.Coleoptera) Tenebrio molitor, Microdera punctipennis, Dicentra canadensis, Rhagium inquisitor, and Anatolica polita [Even though AFPs are produced by a large group of insects, only a few of the structures are recognized, including the beetles . Later, scientists detected antifreeze protein from wheat (Triticum aestivum). Various investigations were conducted on plants’ antifreeze proteins in carrots (Daucus carota), ryegrass (Lolium perenne), Solanum tuberosum, Solanum dulcamara, Forsythia suspense, Picea abis, and Picea pungens, among many others [Initially, researchers demonstrated the presence of AFP in winter rye leaves , including AFPs and their recombinants, are principally employed in various areas such as medicine and food industries. Fish recombinant AFPs are applied to improve food conservation over freezing . Ice nucEncouraging findings were reported in various food technology investigations on the use of bacterial INPs to increase the ice nucleation temperature, with subsequent decrease in freezing times and the size of ice crystals. Thus, these further enhance the quality of frozen solid food. As a result of creating ice at higher temperatures, a lower energy cost is needed for food freezing. It was also shown that freezing food with less cooling was possible when utilizing biological ice nuclei created by bacteria ,51. In tIce recrystallization is among the difficulties in the ice cream industry. It is initiated by temperature fluctuation over storage, where small-size ice crystals are changed into the larger ice crystals through double freezing. The organoleptic and texture features of ice cream are destroyed by the large size of ice crystals. Hence, AFPs are recommended as an inhibitor for the natural growth of ice in freezing dairy yields. Ice cream products with optimal ice crystal structure play a crucial role in maintaining a creamy and smooth texture. AFPs offer continuity of the ice cream’s smooth structure via inhibition of the crystallization ,55,56. IAFPs’ ice recrystallization prevention feature is used in frozen fish and meat. An initial study had used some pieces of bovine meat saturated in a concentrated drain of AFP solution to make it dry . They foIn other investigations, the impacts of pre-slaughtering AFGPs injection to lambs were evaluated in terms of the quality of lamb meat after thawing ,57. The w/v each) are commercially utilized as the cryoprotectants. Though, these sugars lead to sweetness, which is non-desirable for some consumers and for certain food products [Based on these findings, by developing economical and consumer-acceptable techniques, frozen storage damages can be reduced by adding AFGP into the meat before freezing. To extend the products’ shelf lives, frozen storage is needed. Nevertheless, another problem would be initiated regarding freeze-induced denaturizing of the muscle proteins. Using cryoprotectants somewhat prevents this denaturization, where sucrose and sorbitol  still offers better protection compared to the conservational cryoprotectants. It results in no sweetness, which is a commercial advantage. Through discovering this innovative AFP capability, an exploration into other food uses is encouraged ,58.Since 1960, the frozen dough method has attracted a huge deal of interest to cope with the problems of the short shelf-life of conventional doughs. However, this method can result in the reduced retention capacity and the weakened dough structure. Although utilizing freeze-tolerant yeasts or strong wheat flour could prevent such drawbacks, the prolonged fermentation time has led to the deterioration in the bread texture ,60.2) retention capacity was increased, the yeast mortality was decreased, and the delayed crystal creation resulted in a TH phenomenon over the freezing and thawing cycle. The stronger fermentation capacity, and greater CO2 retention capacity, were found via the CaAFP-added group compared to SPI- and BSA-added groups [To overcome this issue, Zhang et al.  studied d groups ,61.Myoxocephalus aenaeus), in vitro, and industrial strain of baker’s yeast (Saccharomyces cerevisiae) [Nevertheless, there is no proper yeast candidate for the industrial bakery with optimum gassing capacity in frozen dough. Moreover, it is unlikely that considerable enhancements of this attribute can be provided by classical breeding programs. A study was conducted to express a recombinant gene of antifreeze peptide GS-5 from the polar fish grubby sculpin (evisiae) . In bothevisiae) .Recent studies showed that antifreeze proteins utilized in frozen dough could enhance and improve the total gas and gassing rate. Moreover, using high concentrations of AFPs from carrot can increase the quality and texture of frozen dough through keeping the volume of loaf within freezing process . EffectsA gel structure is included in numerous water-containing substances, in which water molecules are held by a polymer network to make an inter-tangled texture possessing good viscosity. Jelly, noodles, boiled eggs, dough, tofu, and cakes are all damaged by the freezing–thawing procedure through forming ice crystals which alter the internal texture .w/v) agarose gel. AFPs for maintaining the gel structures can be used for different purposes. The “gelatin freezing technique” is expanded to create porous materials, where AFPs made a considerable contribution. Creating such an ice-motivated structure within a gel or other water-containing substance is one of the encouraging applications of AFPs [The accumulation of AFP on embryonic ice crystals inhibits the ice blocks’ creation, a preservative for the polymer network and its water-holding feature over the freeze–thaw procedure. Only 0.05–0.1 mg/mL of AFP is needed for the protection of 0.5%  surface coating with antifreeze proteins was completed quickly through a one step-dipping technique lacking complex surface alteration. ABP-fused AFPs can maintain the proper orientation of the AFP on a surface, allowing the use of their full anti-icing features. Most prominently, AFPs impede ice creation on the surface of Al in comparison with the bare Al and the usual hydrophilic Al coatings. This impact is caused by the TH activity of Cn-AFP and capability in reducing the freezing point ,71,72,73Bacterial biofilms are a major concern in clinical medicine, with more than 400,000 estimated annual catheter-related bloodstream infections in the U.S. alone. Current treatments are unable to clear most of these catheter-related infections. Thus, treatment is restricted to device removal and replacement, which further increase the risk of re-infection and treatment costs. Described herein is a new anti-infective class that is effective against infectious agents such as bacteria, viruses, fungi, and protozoa .Ixodes scapularis tick can also contribute to the anti-infective activity [Ixodes scapularis tick antifreeze glycoprotein (IAFGP) binds to bacteria while altering their microbial biofilm creation in vitro (Serratia marcescens (SM), Escherichia coli (EC), Pseudomonas entomophila (PE), Staphylococcus aureus (SA), and Listeria monocytogenes (LM) [The anti-infective agents include antifreeze proteins and peptides from a variety of organisms in addition to the synthetic proteins and peptides whose amino acid compositions are derived from antifreeze proteins, such as an arthropod antifreeze protein. Nevertheless, antifreeze glycoprotein and glycopeptides from activity . They arin vitro . IAFGP cnes (LM) ,77.Mainly, cryomedicine is related to cryosurgery and various cryopreservations. Cryopreservation is a necessary stage in different biomedicine regions such as gene therapy, tissue engineering, and tissue preservation screening of drugs. To achieve these aspects, new platforms and reagents with antifreeze as well as compatible osmoprotective properties have been expanded recently for cryopreservation .Generally, destructive ice creation and recrystallization take place both outside and inside of cells over freezing procedures. A two-step freezing technique can solve these problems, where the cells are initially chilled at 0 °C in a preservation fluid. During the first phase, only the material surrounding the cell is frozen, where dehydration is performed. At the second stage, the cells are frozen completely with liquid nitrogen (−196 °C). Previous dehydration reduces the formation of ice crystals between the cells in this phase .In general, a programmable freezer is used in this technique, where the whole process will take about 2–3 h. Vitrification is another procedure, where the cells are frozen quickly to reach −196 °C with greatly concentrated (1–3 M) cryoprotectants , acetamide, propylene glycol) which penetrate the cells . This teThe detergents applied in the two-step freezing technique are also toxic, however, they are much more diluted compared to those utilized in vitrification. Within cold-tolerant organisms, the function of AFPs combined with different organic and inorganic materials, including glycerol, glucose, ions, lipids, mineral salts, peptides, and carbohydrates. Therefore, utilizing optimal combinations of these substances can result in the maximized AFP performance differing between the usages ,81.Pseudopleuronectes americanus). Comparatively high concentrations of these proteins were furthermore operative in preventing the extracellular ice recrystallization for RBCs [AFPs are particularly effective in frozen solutions for inhibiting ice recrystallization. Initially, in 1969, a study mentioned and highlighted the crucial functions of the proteins in freeze-tolerant organisms. In vitro examination of red blood cells (RBCs) had illustrated the effects of AFP on the recovery of cryopreserved cells , which cfor RBCs . Previoufor RBCs .Studies showed that the injury gained at various phases over the cryopreservation process may result in the follicular depletion. Therefore, preservation within sub-zero conditions would help keep ovarian function. The results indicated the cryoprotective impacts of antifreeze proteins, particularly a high concentration of LeIBP (AFP), on ovarian tissue over the warming/vitrification procedure. The results offered a basis for further investigation on the mechanisms and impacts of AFPs on human ovarian tissues ,85.Moreover, other studies showed that AFGP8 also had a positive effect on bovine oocytes against cold condition injuries during the vitrification process . Type IIIn the early twentieth century, marine AFPs were initially utilized to protect the cell membranes of oolemma at hypothermic temperatures via AFGP from Antarctic and Arctic fish. Although most cryopreservation trials using AFPs had confirmed that adding AFPs could enhance the freezing and thawing sperm viability, numerous reports indicated no beneficial effect despite the freezing technique, storage temperature, and biological specimen . AFPs haCyprinus carpio sperm [Fish AFPs were investigated previously, for their effectiveness when supplemented in the sperm preservation media for various organisms at freezing and chilling steps. In ram, bovine, chimpanzee, buffalo, and fish, sperm quality was enhanced using cryopreservation and chilled storing through AFPs . Anotherio sperm .It has been demonstrated that Type I and III AFPs had cryoprotectant effects on the embryonic tissues in zebrafish  as well Cryoprotective agents (CPAs) are chemical substances which prevent ice crystal formation in zero to sub-zero conditions in organ transplantation. Current CPAs consist of small molecules (frequently known as penetrating CPAs), AFPs, and synthetic polymers . PresentIn organ maintenance, proper solutions and materials are required for organ stabilization and blood elimination. Currently, various components are used as CPAs, encompassing ethylene glycol, 1,2-propanediol, DMSO, formamide, glycerol, lactose, sucrose, and D-mannitol. These components cause intense cell death due to their high toxicities to organs. Therefore, the massive removal of CPAs upon warming is needed before the transplantation process .AFPs and some synthetic polymers are special CPAs that prevent ice crystal formation at low temperatures. Despite the fact that AFPs that are derived naturally from fish, insects, and plants play an important role in prevention of ice crystal formation, the low purity and high cost of these proteins are the major drawbacks. Moreover, AFPs usage in organ tissues’ preservation may possess immunogenicity to induce immune responses .Arabidopsis thaliana and several crop plants) which expressed different AFPs [The AFPs’ capability in thermal hysteresis prevents the damage caused by common minor frost measures in early autumn and late spring in the frost-sensitive crop plants. Therefore, the expression of the high-activity AFPs permits them to continue the freezing at nearly −5 °C. Over two decades, the effectiveness of this concept was examined in various investigations producing transgenic plants (ent AFPs .Originally, AFPs from fish were used in these assessments but the insect AFPs were found to exhibit higher activity. Then, they were used for plant transformation investigations. Transgenic plants produced in some studies showed the enhanced cold-tolerance of 1–3 °C in comparison with their wild-types. However, no studies with transgenic plants have found an adequate level of protection as yet. Progress up to this point shows the possibility of obtaining more significant results ,104.Research shows that AFPs can be applied for agriculture and aquaculture products; however, those methods have not been used at present. Previously, the Type I AFP expressed in the winter flounder was evident to prevent the growth of ice lattices in cold conditions, where such observation had led to the improved economic situation of dead fish . SimilarThe ability of organisms to tolerate cold conditions is a natural phenomenon in nature. The development of AFPs reveals a quintessence to comprehend this self-protection circumstance. Over the last decade, our understanding on the utilization of AFP have confirmed the promising usages of this protein with its specific function in different medical and industrial fields. Developments in both structural and physicochemical biological and genetic approaches significantly contribute and allow us to obtain an unprecedented perception towards the mechanisms of membrane protection and ice-binding of AFP. The useful applications of AFPs have been proven in numerous investigations. However, the time-consuming purification process of AFPs has limited the huge usage and functionality of these beneficial biomolecules in various applications.This review focused on potential properties of AFPs with different sources, structures, and characteristics. Further amounts of AFP obtained via large-scale methods will permit its functional developments in medical and industrial fields. Support from government agencies is needed for the medical application of AFPs regarding its mutagenicity, carcinogenicity, and toxicity. Hence, the specimen should be made in a facility satisfying the regulation of good fabricating practice. The necessary data will be provided by more assessments on abundant aspects of AFP to solve these problems.


The neuroanatomical and molecular substrates for cognitive impairment in Parkinson Disease (PD) are far from clear. Evidence suggests a non-dopaminergic basis, and a crucial role for cerebellum in cognitive control in PD. We investigated whether a PD cognitive marker (response inhibition) was differently controlled by g-amino butyric acid (GABA) and/or by glutamate-glutamine (Glx) levels in the cerebellum of idiopathic PD patients, and healthy comparators (HC). Magnetic resonance spectroscopy of GABA/Glx (MEGA-PRESS acquisition sequence) was performed at 3 Tesla, and response inhibition assessed by the Stroop Word-Color Test (SWCT) and the Wisconsin Card Sorting Test (WCST). Linear correlations between cerebellar GABA/Glx levels, SWCT time/error interference effects and WCST perseverative errors were performed to test differences between correlation coefficients in PD and HC. Results showed that higher levels of mean cerebellar GABA were associated to SWCT increased time and error interference effects in PD, and the contrary in HC. Such effect dissociated by hemisphere, while correlation coefficients differences were significant in both right and left cerebellum. We conclude that MRS measured levels of cerebellar GABA are related in PD patients with decreased efficiency in filtering task-irrelevant information. This is crucial for developing pharmacological treatments for PD to potentially preserve cognitive functioning. Converging evidence indicates that alterations in neurotransmitters beyond the dopamine system are present in Parkinson’s Disease (PD), and may disturb fronto-striatal related cognition . CognitiBased on the strong cerebellar-cortical interactions during information processing , we hypoIn the present case-control correlational study, 25 consecutive subjects diagnosed with PD according to the UK Parkinson’s Disease Society Brain Bank diagnostic criteria  in the eq = 1) in order to detect only effects that would have clinical significance. Based on the aforementioned assumptions, the minimum number of necessary samples to meet the desired statistical constraints is 16 per group.Since there are no published data on the relationship between cerebellar GABA and Glx (Glutamate/Glutamine complex) levels and response inhibition abilities, either in PD or in HC, to determine a sufficient sample size for a two tailed z test on the difference between two independent correlations, a power analysis was conducted using an alpha of 0.05, a power of 0.80, and a very large effect size  age between 18 and 65 years, (2) at least eight years of education, and (3) suitability for MRI scanning. Exclusion criteria were: (1) known or suspected history of alcoholism, drug dependence or abuse, other neurological disorders, (2) personality disorder, any present mental disorder (unipolar depressive and anxiety disorders of mild to moderate severity were suitable for recruitment) and past major mental disorders , according to DSM-5 criteria, (3) major medical illnesses, i.e., diabetes not stabilized, obstructive pulmonary disease or asthma, hematological/oncological disorders, B12 or folate deficiency as evidenced by blood concentrations below the lower normal limit, pernicious anemia, clinically significant and unstable active gastrointestinal, renal, hepatic, endocrine or cardiovascular system disease, newly treated hypothyroidism, (4) IQ below the normal range according to TIB  , (5) diaSociodemographic characteristics, clinical features, and dopamine replacement therapy dosages for the PD group are summarized in The study was approved (by a written statement containing a waiver) and undertaken in accordance with the guidelines of the Santa Lucia Foundation Ethics Committee. All participants gave their written informed consent for research after they had received a complete explanation of the study procedures. Information about the potential publication of research results was included in the form, and a signed consent to the processing of personal data obtained from all participants.https://github.com/richardedden/Gannet3.0/archive/master.zip. Due to a lack of consent of the participants, structural and chemical MRI data cannot be shared publicly, and can only be made available upon reasonable request if data privacy can be guaranteed according to the rules of the European General Data Protection Regulation (EU GDPR). The respective research group has to sign a data use agreement to follow these rules. This statement is in line with our institute’s policies and requirements by our funding bodies.The batch-processing tool for the quantitative analysis of GABA-edited MR spectroscopy spectra used in this study is available for immediate download at Demographic and neurological features were collected at enrolment by a trained neurologist (CP or FEP) with expertise on parkinsonism. Disease stage was measured by the modified Hoehn and Yahr scale , and theAfter having been screened for global cognitive impairment using the Mini-Mental State Examination (MMSE), all study subjects underwent the Mental Deterioration Battery (MDB) . The latDetails on methodology for neuropsychological and psychopathologic evaluations have been published elsewhere .Two traditional set-shifting tests, i.e., the Modified Wisconsin Card Sorting Test short form (M-WCST-sf) , and theMagnetic resonance scanning was conducted on a Philips 3.0 T Achieva system with a 32 channel receiving only head coil . Head position was fixed with foam padding to minimize movements.3), an efficient and reliable sequence for detecting brain level of endogenous GABA [T1-weighted structural magnetic resonance images were acquired for spectroscopic voxel placement . T2 and FLAIR sequences were acquired to clinically screen for possible brain pathology. GABA and Glx measurements in the left and right cerebellar hemispheres were obtained using the MEGA-PRESS acquisition sequence , a Matlab-based quantitative batch analysis tool specifically developed for GABA MEGA-PRESS spectra . Gannet PD and HC were first compared in terms of sociodemographic characteristics , and cerebellar GABA, Glx  levels, and Glx/GABA ratios  cerebellar Glx levels divided by GABA levels to assess the excitation/inhibition balance in the voxel) using chi-square and unpaired t-tests. Paired t-tests were used to compare cerebellar GABA, Glx levels and Glx/GABA ratios between hemispheres within diagnostic groups. In PD, linear correlations (significance was tested by Fisher’s r to z transformation) between left, right and mean (left+right/2) GABA, Glx levels and Glx/GABA ratios and dopamine replacement therapy dosages  were computed to verify potential medication effects on GABA and Glx signals measured by MRS . Equally1 and z2 are the Fisher’s transformed values of the two correlations and N1, N2 the respective sample size. Significance of the z test was set at p < 0.01 (two tailed) after correction for multiple comparisons (0.05/3 response inhibition scores).Linear correlations between cerebellar GABA, Glx levels, Glx/GABA ratios and response inhibition performance, separately for PD and HC, were performed for the M-WCST-sf P, the SWC-sv IE-T and the SWC-sv IE-E. To provide a direct test of a model assuming a different relationship in PD and HC between cerebellar GABA, Glx levels, Glx/GABA ratios and response inhibition performance, the significance of a potential difference between correlation coefficients in the two groups was tested. This was calculated as follows:n = 6) was diagnosed with a depressive disorder not otherwise specified. Severity of anxiety and depressive symptoms is reported in The two diagnostic groups did not differ for age, gender, and educational attainment. Within the PD patients’ cohort, one patient (5%) met diagnostic criteria for apathy . As for A significant negative correlation in the patients’ group was observed between HARS total score and daily levodopa equivalents see . No otheThe cerebellar metabolite levels and their excitation/inhibition balance did not differ between PD and HC sees . ConcentIn PD, mean cerebellar Glx concentration was correlated with daily levodopa dosages, with a tendency toward significance for the correlation between the latter and Glx level in the right cerebellar hemisphere see . No otheIn HC, mean cerebellar GABA level positively correlated with the SWCT-sv IE-T score see . In PD, p = 0.08). A positive significant correlation was observed in HC between the SWCT-sv IE-T score and the excitation/inhibition balance in the right cerebellar hemisphere. No other significant correlation was present in both groups between the measured cerebellar metabolites, the excitation/inhibition balance within the cerebellar voxel and scores indexing response inhibition abilities.No significant correlations were observed between mean, left and right Glx levels and neuropsychological measures indexing response inhibition abilities. However, given the significant correlation between daily levodopa dosage and mean cerebellar Glx level, and in an attempt to correct for this effect, partial correlations were calculated between the latter and WSCT-sf P, SWCT-sv IE-T and SWCT-sv IE-E scores in PD. Again, no significant correlation emerged when the effect of dopamine replacement treatment was removed, apart from a tendency toward significance for the relationship between the WSCT-sf P score and mean cerebellar Glx levels  and between the SWCT-sv IE-E score and left cerebellar GABA levels (negative in HC and positive in PD). A significant difference in correlation coefficients was also observed for the relationship between right GABA levels and the SWCT-sv IE-T score (negative in HC and positive in PD) see .Here the role of cerebellar tonic inhibition in cognitive functioning was investigated under a pathological clinical condition and compared to what observed under normal physiological condition. Specifically, the correlation between GABA and Glx cerebellar levels (as measured by MRS)  and propSuch results crucially demonstrate, in the first place, that the GABAergic neurochemical profile in the cerebellum is linked to response inhibition in both HC and patients diagnosed with PD. Since response inhibition is one of the most sensitive measures for characterizing the cognitive phenotype in PD , our resOur results also substantiate that the cerebellum is a critical node in the distributed neural circuits subserving cognition  in PD alA receptors by the tonically released GABA, tonic inhibition exerts a powerful action in cognitive functions by controlling neuronal excitability. Since it enforces a dynamic control of motor coordination [Although the role of persistent cerebellar GABAergic inhibition in shaping brain function has been intensively studied, evidence is limited to animal studies and the motor and learning domains. Mediated through an activation of extrasynaptic GABAdination  regulatiIndeed, the cerebellum is thought to mediate cortical information processing via closed cortico-cerebellar loops . The uniHowever, we found that the relationship between cerebellar GABA-dependent tonic inhibition and response inhibition was reversed in PD patients. Actually, potential changes in cerebellar output in PD are still largely unknown. Indirect evidence suggests some functional changes in the cerebellar-cerebral circuitry, which may support compensatory mechanisms to the basal ganglia dysfunction ,37. IndeNevertheless, the reported inverted association (respect to what observed in HC) in PD patients may also be a disease-related change. It is, indeed, possible that GABA concentrations in the cerebellum are increased in the early stages of the disorder. This change would be a consequence of dopamine depletion in the basal ganglia, and related to the executive dysfunctions observed in early PD . The eviActually, a first limitation of the present study is that the investigation of the relationship between GABA/Glx levels in the cerebellum and cognition in a sample of medicated PD patients may be influenced by the treatment itself. However, while dopamine replacement therapy was negatively correlated to mean cerebellar Glx levels, Glx did not affect response inhibition performance. Thus, the intervening effect of treatment on our main result is unlikely, also considering the null difference in cerebellar metabolite levels and their excitation/inhibition balance between medicated patients and the small sub-sample of unmedicated PD patients. Second, it might be argued that MRS cannot distinguish between synaptic and intracellular stores of GABA, thus impeding a detailed and definitive interpretation of the neurophysiological significance of our findings. Nevertheless, according to recent consensus, MRS is most sensitive to extracellular unbound GABA, which is involved in tonic inhibition . Since sA further potential limitation of the present study is the relatively small sample size, which might have increased the risk for type I error. However, a very large effect (Cohen’s q > 0.90) was observed for the difference in HC and PD correlation coefficients between mean cerebellar GABA levels and response inhibition performance, thus suggesting that the analyses were not underpowered (post-hoc computed beta = 0.80). Nevertheless, a further study including a larger sample is warranted also considering the significant  test of difference between HC and PD correlation coefficients between the excitation/inhibition balance in the cerebellum and measures of response inhibition.Here we demonstrate for the first time, that cognitive efficiency in counteracting interference is related to GABA-dependent tonic inhibition in the cerebellum both under physiological and pathological conditions. We also underline that increased tonic inhibition in the cerebellum, relative to response inhibition, is associated to normal levels of performance in PD patients. Given the cross-sectional nature of the present study we could not establish the potential longitudinal “cost” of the observed change in the relationship between GABAergic neurotransmission and cognition in PD. However, our finding provides strong evidence that the cerebellum should be considered as a primary site for systems-level compensation in the disorder . Conside