Some children develop allergic symptoms, including anaphylaxis, after the first intake of cow protein. Therefore, the tolerability of PHF in infants with cow's milk allergy (CMA) is important information. Partially hydrolyzed whey formula (PHWF) is well characterized, but those containing both whey and casein are also available. We evaluated the characteristics of two whey and casein PHFs, PHF1 and PHF2, Residual antigenicity of β-lactoglobulin (β-LG) and casein in the formulas was measured using ELISA. The molecular weight profile was determined using high-pressure liquid chromatography. IgE reactivity and allergenic activity of the formulas were evaluated by ImmunoCAP inhibition assay and by basophil activation test using blood from patients with CMA, respectively.n = 10) had casein-specific IgE. The antigenicity of β-LG in PHF1 was similar to that in PHWF1, but it was slightly higher than that in PHWF1 for casein. PHF1 had a higher IgE reactivity than PHWF1. However, PHF1 and PHWF1 had a similar ability to activate basophils. PHF2 had lower antigenicity of casein and β-LG, IgE reactivity and basophil activation than PHWF1.All the participants (These results suggest that the tolerability of PHF1 and PHF2 in patients with CMA is similar to and higher than that of PHWF1, respectively, and that the degree of IgE binding to PHFs does not necessarily correspond to basophil activation. Cow's mCow's milk-based infant formula is classified into conventional cow's milk formula (CMF), partially hydrolyzed formula (PHF), and extensively hydrolyzed formula (EHF) according to the degree of hydrolysis of milk proteins. PHF was designed and developed to reduce the sensitizing capacity of cow milk proteins while maintaining the palatability of the formula at a reduced processing cost compared to EHF . Most ofMultiple clinical trials have shown the reducing effect of PHF on atopic dermatitis compared to that of CMF , 6, althAlthough not as well as EHF, the reactivity of PHF in children with CMA is substantially reduced. It is reported that approximately 40%–75% of children with CMA can consume PHF –12, and For the above reasons, the tolerability of starter formulas in infants with CMA is important information. Milk proteins are broadly divided into casein and whey proteins. Although both contain some milk allergens, most children with CMA have higher specific IgE values for casein than for whey proteins , 19. Parex vivo testing method that is highly correlated with the clinical symptoms of food allergy . The exclusion criteria were as follows: (I) taking antihistamines within 3 days before the scheduled blood sampling date and (II) taking drugs, such as steroids, that act on the immune system within 1 week before the date. Since the aim of this study was to evaluate the allergenicity of formulas using the blood of children with CMA, the age range was set to 2–15 years old. This study was conducted between January and March 2022 and was registered in the UMIN Clinical Trials Registry (study ID: UMIN000046543).This was a cross-sectional study of patients with CMA evaluating basophil activation and IgE reactivity to milk proteins in infant formulas using peripheral blood samples. Participants were recruited from outpatients at Kanagawa Children's Medical Center. The inclusion criteria were as follows: (I) aged 2–15 years; (II) history of immediate symptoms to milk components; and (III) milk-specific antibodies .2.3.Four powdered milk formulas were used: PHF1 , PHF2 , PHWF1 , and CMF1 .2.4.The residual antigenicity of casein (CN) and β-lactoglobulin (β-LG) in each formula was determined using commercial ELISA kits  according to the manufacturer's instructions. The ELISA kits are based on polyclonal antibodies.2.5.g, 25°C, 15 min) followed by collecting the middle layer as defatted fractions. Subsequently, they were filtrated with a 0.22 µm filter  and applied to HPLC  with a poly-hydroxyethyl aspartamide column . The molecular weight pattern of the formulae was determined as previously described (Formulas were diluted with 0.1 M phosphate buffer (pH 7.4) and centrifuged  of blood samples were also collected for this study. For collection, 3 and 4 ml of the samples were collected for heparinized blood samples to be used in the BAT and for serum samples to be used in the ImmunoCAP inhibition assay, respectively.2.7.Serum levels of IgE antibodies to milk (f2), casein (f78), β-LG (f77), and α-lactalbumin (f76) were measured using ImmunoCAP , according to the manufacturer's instructions.2.8.A/ml. The diluted formulas and serum samples were mixed in a 3:2 ratio and incubated at room temperature for 2 h, as previously reported (A/ml)] × 100/anti-milk IgE of serum sample incubated without inhibitor (UA/ml). The half-maximal inhibitory concentration (IC50) values were calculated using statistical software .Formulas used as inhibitors were diluted with 0.1 M phosphate buffer (pH 7.4). Serum samples were diluted with IgE Sample Diluent  to make cow's milk-specific IgE 4–10 Ureported . Incubat2.9.−CRTH2+ cells with FSC/SSC characteristics of lymphocytes) was analyzed using flow cytometry. Basophil activation was expressed as the net percentage of CD203c positive basophils above the threshold defined by the negative control. Basophils were defined as non-responders when the percentage of basophils activated by anti-IgE was less than 10%  using an allergenicity kit  according to the manufacturer's instructions. Briefly, after incubating heparinized whole blood with the antigen at 37°C for 15 min, basophil activation was assessed using antibodies against CD3, chemoattractant receptor-homologous molecule on Th2 cells (CRTH2), and CD203c. PBS and anti-IgE antibodies were used as the negative and positive controls, respectively. Serially diluted formula samples were used to assess basophil activation for formulas. CD203c expression in at least 300 basophils . Statistical significance was set at P < 0.05.Data are expressed as mean ± standard deviation (SD). The difference compared to PHWF1 was analyzed by paired 3.3.1.The residual antigenicity of β-LG and casein was determined using commercially available ELISA kits . The antThe molecular weight profiles of the formulae were determined using HPLC . In CMF13.2.A/ml), casein (84.3 ± 179 UA/ml), β-LG (11.3 ± 16.4 UA/ml), and α-LA (5.66 ± 8.55 UA/ml) , wheat (20%), buckwheat (10%), and sesame (10%). They had specific IgE to milk (40.0 ± 28.3 U5 UA/ml) . Cases w3.3.4 and 105 µg/ml and in PHF2 at 103 and 104 µg/ml compared to PHWF1  and low β-LG (β-LG-specific IgE <3.5 UA/ml). The log IC50 values of PHF1 in Patients 1 and 4 of high β-LG and Patients 6 and 8 of low β-LG were almost the same as those of PHWF1. No difference was observed in the pattern of log IC50 values of three formulas between the two subgroups  at low concentrations. Less inhibition was observed in PHF1 at 10to PHWF1 . The IC5of PHWF1 . These rubgroups .3.4.2 and 104 µg/ml. PHF1 and PHWF1 showed no differences at any concentration tested (To evaluate the ability of the formulas to induce activation of basophils from patients with CMA, BAT was performed using their blood. None of the participants had non-responder basophils. All formulas activated basophils in a concentration-dependent manner. CMF1 activated basophils at low concentrations. PHF2 was less effective than PHWF1 at activating basophils at concentrations of 10n tested . The AUCn tested . There w4.in vitro and ex vivo methods. To the best of our knowledge, this is the first report evaluating the allergenicity of PHF using both the ImmunoCAP inhibition assay and basophil activation test using blood from patients with CMA.Because a starter formula is used when the presence of absence of CMA in an infant is unknown, tolerability of the formula in infants with CMA is important information. Regarding the allergenicity of PHF, there are some reports on PHWF, but there are few reports on the use of casein and whey protein hydrolysates. In this study, we compared the allergenicity of two PHFs containing casein and whey proteins, PHF1 and PHF2, with PHWF1, a typical PHWF product, using PHF2 had lower antigenicity of casein and β-LG by sandwich ELISA and lower allergenicity evaluated by IgE binding and BATs than PHWF1. This suggests that PHF2 may be less reactive than PHWF1 when ingested by children with CMA. Clinical studies evaluating the tolerability of PHF by an oral challenge test in patients with CMA have been reported, and a PHWF from the same manufacturer as PHWF1 used in this study and PHF2 can be ingested by 40%–60% , 11, 20 The participants in this study showed many individual differences in specific IgE to milk components. When they were divided into two subgroups based on β-LG-specific IgE values, the trends on basophil activation by the PHFs but on IgE binding to them differed between the groups. This result suggests that different IgE profiles on milk components may have different tendencies toward basophil activation. Further studies with more number of patients to confirm this possibility and elucidate the mechanism are required.PHF1 was similar in the antigenicity of β-LG determined by sandwich ELISA compared to that of PHWF1, but slightly higher than that of casein. In addition, all participants in this study had casein-specific IgE, and PHF1 inhibited the binding of IgE to milk antigens more than PHWF1 did. These results indicate that many IgE epitopes of casein remain in PHF1. In contrast, PHF1 and PHWF1 had similar abilities to activate basophils, although the participants were more likely to develop basophil activation to casein. Although casein is not used as a raw material for PHWF, casein with an intact size was reportedly detected in PHWF . In addiin vitro and ex vivo experiments. Although BAT shows a high correlation with the oral challenge test (in vivo evaluation, such as an oral challenge, is required. Second, some patients with CMA are reported to be sensitized only to whey proteins (This study compared the allergenicity of PHF1 and PHF2 with that of PHWF1 by ImmunoCAP inhibition assay and BAT using blood from children with CMA. This study has two strengths. First, we performed BAT, which is highly correlated with clinical symptoms , 22, usinge test , 28, resproteins , althougproteins , 29. To In this study, although PHF1 had more IgE epitopes of cow's milk proteins than PHWF1, the ability of PHF1 to activate basophils from patients with CMA was similar to that of PHWF1. This suggests that the risks of developing allergies when ingesting PHF1 or PHWF1 are similar. PHF2, which was previously reported to be effective in oral immunotherapy , was shoFood allergies are partially attributed to the loss or lack of tolerance to food allergens . Hydroly


We exhibited that Ova-IgE ICs induced the NLRC4-inflammasome components, including NLRC4, caspase-1, intracellular IL-1β, and secretion of IL-1β, as well as the granule contents MMP9, TIMP1, and TIMP2 proteins via TLR2 signaling; these responses were suppressed, when NLRC4 inflammasome got actived in the presence of ICs. Furthermore, Ova-IgE ICs induced mRNA expressions of MMP9, TIMP2, and ECP and protein expressions of MMP9 and TIMP2 in EoL-1 through FcɛRII. Interestingly, TLR2 ligand and Ova-IgE ICs costimulation elevated the number of CD63+ cells, a degranulation marker, as compared to the native IgE. Collectively, our findings provide a mechanism for the impacts of Ova-IgE ICs on eosinophilic responses via NLRC4-inflammasome and may help understand eosinophil-associated diseases, including chronic eosinophilic pneumonia, eosinophilic esophagitis, eosinophilic granulomatosis, parasitic infections, allergy, and asthma.Immune complexes (ICs) skew immune responses toward either a pro- or anti-inflammatory direction based on the type of stimulation. Immunoglobulin E (IgE) is associated with Th2 immune responses and known to activate innate immune cells. However, roles of antigen (Ag)-specific-IgE ICs in regulating human eosinophil responses remain elusive; therefore, this study builts upon the mechanism of which ovalbumin (Ova)-IgE ICs affects eosinophilic responses utilizing human EoL-1 cell line as a model. Eosinophils are granulocytes functioning through pattern recognition receptors (PRRs) and destructive granule contents in allergic inflammation and parasitic infections. One of the PRRs that eosinophils express is NLRC4, a member of the CARD domain containing nucleotide-binding oligomerization (NOD)-like receptor (NLR) family. Upon recognition of its specific ligand flagellin, NLRC4 inflammasome is formed and leads to the release of interleukin-1 FcεRII is also critical in internalization of IgE-allergen complexes and presenting Ags on major histocompatibility complex-II (MHC-II) molecules to the T-helper type 2 (Th2) cells [εRI has no enzymatic activity, its engagement with IgE and IgE ICs induces degranulation and release of histamine and prostaglandin from mast cells and also activation of Th2 responses by mediating interleukin-4 (IL-4), IL-13, and granulocyte macrophage colony-stimulating factor (GM-CSF) secretion by basophils [Allergic inflammation is an important pathophysiological condition associated with parasitic infections and certain diseases, including asthma and allergic rhinitis. These pathologic conditions are mainly mediated by immunoglobulin E (IgE)-dependent mechanisms, which influence the functioning of several immune cells, including eosinophils through the interactions with specific receptors , 2. IgE asophils , as wellasophils .ε heavy chains. Structural studies of IgE-Fc alone, and when it is bound to Fc receptors FcεRI and FcεRII/CD23, showed a bent Fc conformation, as well as allosteric communications between the two distant receptor-binding sites [ε-chain. Although, IgE-Fc portion has a symmetrical chemical sequence, its three-dimensional structure is asymmetrical [εRI and FcεRII/CD23 expressing cells such as mast cells, basophils, and eosinophils and can be exploited to tailor the immune responses for the benefit of the host.IgE differs from IgM, IgD, IgG, and IgA by its ng sites . The strmetrical , 9. Thesmetrical . Flexibimetrical , 12; howβ (IL-1β), IL-18 [Salmonella typhimurium, Pseudomonas aeruginosa, and Legionella pneumophila [εRII [Eosinophils are the innate immune cells involved in the initiation and the maintenance of Th2 immune responses . Even th), IL-18 . The inf), IL-18 . NAIP isumophila , 22. In la [εRII .β, MMP9, TIMP1, and TIMP2 responses. Taken together, these data suggest a new role for NLRC4, an alternative pathway for human eosinophilic functions via Ag-specific IgE ICs.We herein show that ovalbumin (Ova)-specific IgE ICs increase the eosinophilic granule contents, including MMP9, TIMP2, and ECP, in TLR2-primed EoL-1 eosinophil cell line after induction of Fc receptors. Also, unlike native IgE, in the absense of a specific Ag in the vicinity, TLR signaling along with Ova-IgE ICs gave rise to EoL-1 cells degranulation. Interestingly, Ova-IgE ICs suppressed the NLRC4 protein and the activation of NLRC4 inflammasome in EoL-1 human eosinophils, which, in turn, resulted in diminished production and secretion of IL-1μg/ml streptomycin, and 10 mM HEPES and incubated at 37°C and 5% CO2.The EoL-1 cell line was used as model for human eosinophils . The celμg Ova: μg IgE) ratio and incubated for 30 min at room temperature. Native human IgE was used in the experiments. To activate NLRC4 inflammasome formation, EoL-1 cells were seeded in antibiotic-free medium and stimulated with TLR2 agonist PAM3CSK4 as a first signal  and 4 hr poststimulation, TLR2 primed cells were transfected with FLA  using lipofectamine 2000 (Invitrogen) both with and without IgE-Ova immune complexes. Cells were lysed 24 hr after PAM3CSK4 stimulation. Supernatants were collected and assayed for IL-1β and IL-10 secretion and gelatinolytic activity.To form Ova-IgE ICs, chicken egg Ova (Grade V)  and mouse anti-Ova IgE monclonal antibody  were mixed at a 1 : 10 (β (CST), anti-MMP2 (CST), anti-MMP9 (CST), anti-TIMP1 (CST), and anti-TIMP2 (CST). Anti-GAPDH (CST) and antivinculin (CST) antibodies were used as housekeeping proteins. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated antirabbit and antimouse secondary antibodies (CST). Subsequently, the protein bands were visualized by electrochemiluminescence (ECL)  using the ChemiDoc XRS + System (Bio-Rad Laboratories). Band intensities were quantified using Image Lab Software (Bio-Rad Laboratories).Thirty microgram of proteins per lane were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated on 10%–12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane . For protein detection, the membranes were first probed with primary antibodies against anti-NLRC4 , anticaspase-1 (Abcam), anti-IL-1εR1α (BioLegend). Cell staining was assessed by flow cytometry on an Accuri C6 Flow Cytometer (BD Biosciences). FACS analysis was performed with FlowJo software .After stimulation, EoL-1 cells were washed in FACS buffer  with 2% fetal bovine serum (FBS)) and incubated for 45 min at 4°C in the dark with the following antibodies: APC antihuman CD63 (BioLegend), APC antihuman CD69 (BioLegend), APC antihuman CD23 (BioLegend), and APC antihuman Fcβ and IL-10 (BioLegend) antibodies diluted in PBS (1 : 250) and incubated overnight at 4°C. The wells were washed with PBST and blocked with blocking solution (10% FBS containing PBS) for 1 hr at room temperature. Biotin-conjugated antihuman IL-1β and IL-10 (BioLegend) antibodies diluted in blocking solution (1 : 250) were added and incubated for 1 hr at room temperature. HRP Avidin D diluted in blocking solution  was added and incubated for 30 min at room temperature. The TMB Peroxidase Substrate and TMB Peroxidase Substrate Solution B were mixed in 1 : 1 ratio and added to each well. After the color change was observed, the reaction was stopped by adding 1 N HCl. The absorbance value was measured at 450 nm with the spectrophotometer.The Sandwich ELISA method was used to measure the levels of cytokines secreted from EoL-1 cells. Nunc MaxiSorp 96-well plates were coated with purified antihuman IL1-Total RNA was isolated from samples  using RNAqueous©  according to manufacturer's instructions. All RNA samples were DNase treated with DNA-Free (Ambion) according to manufacturer's instructions before quantitative PCR.MMP9 primers:  , MMP2 prCt) before and after stimulation using the following formula:Each RT-PCR reaction was performed as previously described , 31. TheCt(stimulated)−Ct(control). Experiments were performed at least twice, and one representative experiment is depicted. Results were expressed as fold change in expression of stimulated cells relative to nonstimulated cells.The fold change in mRNA was determined by: Fold change 22, 1 μM ZnCl2) for two to four nights at 37°C. After incubation, gels were stained with Coomassie Brilliant Blue solution for 2 hr and visualized using the ChemiDoc XRS + System (Bio-Rad Laboratories). Band intensities were quantified using Image Lab Software (Bio-Rad Laboratories). Human recombinant MMP9 (hrMMP9)  was used as a standard control.MMP2 and MMP9 activities secreted from EoL-1 cells were investigated by gelatin zymography technique. Cell supernatants were electrophoresed into 7.5% (w/v) polyacrylamide gel copolymerized with gelatin (3 mg/ml) under nonreducing conditions. After electrophoresis, gels were incubated in renaturing buffer (2.5% Triton X-100) two times for 30 min at room temperature. After renaturation, gels were developed in zymogram activation buffer .Statistical analyses were performed using an unpaired two-tailed γR engagement during TLR priming steps inhibits the assembly and activation of the NLRP3 inflammasome complexes [S. typhimurium as the activation signal for NLRC4 inflammasome, we tested eosinophil responses in the presence of Ova-IgE ICs and IgE alone. First, we examined the influence of Ova-IgE ICs on the mRNA and protein expression of NLRC4 in EoL-1 cells after the priming step through TLR2 signaling. Because the human NLRC4 gene has four transcript variants, two different primer sets were used to detect all NLRC4 transcripts and showed that Ova-IgE ICs upregulated the expression of NLRC4 as compared to nonstimulated cells and TLR2-primed cells  their rapid (within hours) responsiveness to broad range of stimuli, (2) their expression profile of PRRs whose roles in regulating eosinophilic responses are largely unknown, (3) their ability to express human eosinophilic pan markers, including Siglec-8 and IL5R , 24, andomplexes , 33. Froed cells . Moreove Figures . CollectεR2, aka CD23) [εRI and low affinity IgE receptor FcεRII in EoL-1 cells. We demonstrated that TLR2 activation along with Ova-IgE ICs treatment significantly upregulated the expression of FcεR2 in EoL-1 cells while this costimulation did not change the number of FcεR1-expressing cells  degradation , 37. Numy change . Consisty change . Next, wy change . Additioy change . Last, winophils . Interes stimuli .β; however, the presence of Ova-IgE ICs significantly inhibited NLRC4 inflammasome proteins  are the most studied and well-characterized MMPs whose expressions and activations have been addressed in neoplastic pathologies and inflammatory disorders, including asthma . SeveralAdditionally, IgE ICs can either enhance or suppress the eosinophilic immune responses depending on the timing and the type of infection in the host. IgE-allergen ICs are known to induce a rapid activation of basophils and mast cells. Moreover, APCs can present innocuous antigens to T cells by the help of IgE. These findings underlined the importance of proximity of IgE epitopes on the antigen, which greatly affects the subsequent shape of antigen and antibody ICs, as well as the potency of antigen, which then determines the severity of allergic reactions . TherefoIgE immunobiology in the context of treatment strategies yet to be discovered. Applications of IgE-mediated ICs or IgE per se in clinical settings are not only limited to the allergy or asthma diseases but rapidly extending to the other diseases, including the treatment of cancer. In this regard, AllergoOncology is emerging as a new field, which suggests the exploitation of IgE antibodies to treat malignant diseases due to the presence of modulatory mechanisms for IgE antibodies . In thisIn conclusion, our study suggested a novel role for NLRC4 inflammasome in the suppression of eosinophil responses by antigen-specific IgE ICs. It is noteworthy that the clinical trials and drug development studies on EAD are limited and nonspecific ; this st


Mantle cell lymphoma (MCL) is a rare B-cell malignancy with a predominantly aggressive clinical course and poor prognosis. Abnormal expression of Ambra1 is closely related to the occurrence and development of various tumors. However, the role of Ambra1 in MCL remains unknown. Here, we performed both in vitro and in vivo experiments to investigate how Ambra1 regulates MCL progression and whether Ambra1 modulates the sensitivity of MCL cells to the CDK4/6 inhibitor palbociclib. We discovered that MCL cells had decreased levels of Ambra1 expression relative to normal B cells. Overexpression of Ambra1 in MCL cells inhibited autophagy, reduced cell proliferation, migration, and invasion, and decreased cyclin D1 level. While knockdown of Ambra1 reduced MCL cell sensitivity to CDK4/6 inhibitor palbociclib. Furthermore, overexpression of cyclin D1 lowered the sensitivity of MCL cells to palbociclib, enhanced cell proliferation, migration, invasion, and autophagy, and inhibited cell apoptosis. When Ambra1 expression was inhibited, the in vivo antitumor effects of palbociclib on MCL were reversed. Ambra1 expression was downregulated but cyclin D1 expression was upregulated in MCL samples, demonstrating a negative correlation between Ambra1 and cyclin D1. Our findings suggest a unique tumor suppressor function for Ambra1 in the development of MCL. MCL accounts for approximately 3% to 10% of cases of non-Hodgkin lymphoma, and it is more common in male patients2. MCL is characterized by a characteristic chromosomal translocation, t, which leads to overexpression of cyclin D1. Lymph node disease is common, especially in the gastrointestinal tract, spleen, and bone marrow3. The clinical course is largely aggressive, and the prognosis is poor, with high recurrence and mortality rates4. Nearly all patients eventually experience refractory disease or relapse, with a lethal sequence of drug resistance and continuous relapse, making treatment challenging5. Therefore, deepening the research and understanding of MCL pathogenesis can bring hope for the development of new targeted drugs.Mantle cell lymphoma (MCL) is a subtype of B-cell non-Hodgkin lymphoma that is characterized by a heterogeneous clinical presentation6. After stimulation, CDK4/6 can bind to cyclin D to form a complex, and this cyclin D-CDK4/6 complex further phosphorylates the retinoblastoma (Rb) protein, making it unable to bind to the E2 transcription factor (E2F). As a result, E2F is activated and regulates cell cycle progression from G1 phase to S phase8. CDK4/6 is a key molecule involved in the development of various malignant tumors, and compared with pancycle inhibitor drugs, CDK4/6 inhibitors have stronger anticancer effects and less toxicity9. In addition, studies have shown that CDK4/6 is a very promising therapeutic target in treating MCL10.Cyclin-dependent kinases (CDKs) are key molecules that regulate the cell cycle in mammals, and CDK4 and CDK6 are important members of the family11. The autophagy function of normal cells is essential for disease resistance, but impaired autophagy leads to the occurrence of various diseases, such as neurodegenerative diseases, inflammatory diseases and cancers13. Autophagy is regulated by autophagy-related genes. Activating molecule in Beclin-1-regulated autophagy (Ambra1) is an activator of the autophagy-related gene Beclin-1. It binds to Beclin-1 and positively regulates the lipid kinase activity of the Beclin-1/VPS34 complex, thereby promoting autophagosome formation14. Studies have shown that Ambra1 deletion leads to increased cyclin D levels in cells and mice, promotes cell proliferation and reduces sensitivity to CDK4/6 inhibitors. Ambra1 deletion not only promotes lung adenocarcinoma growth, but a low level of Ambra1 is associated with poorer prognosis in lung adenocarcinoma patients15. In addition, abnormal Ambra1 expression is closely related to the occurrence and development of various tumors. However, the role of Ambra1 in MCL has not yet been reported.Autophagy is the process by which damaged organelles or unnecessary proteins are encapsulated in a double-membrane structure and directed to lysosomes for degradation, a process that is particularly important for maintaining neuronal activity and normal cell functionBased on the above background, we explored the role of Ambra1 in the development of MCL and its internal regulatory mechanism through in vitro and in vivo experiments. Our research may provide new targets and ideas for the drug-resistant treatment of MCL.This research was carried out after all subjects provided informed consent and was approved by the Clinical Research Ethics Committee of Xiangya Hospital of Central South University (K202203149). Fifteen MCL patients and 15 healthy participants were included in our study. All MCL patients were confirmed by examination in the Department of Hematology, Xiangya Hospital of Central South University. Healthy control subjects were free of MCL at the time of physical examination. Blood samples were centrifuged at 3000 rpm for 10 min and stored at -80 °C until further processing.16 or autophagy inhibitor chloroquine (CQ) was further added for 24 h. The cyclin D1 overexpression vector was constructed and transfected into Jeko-1 and Z138 cells. The ov-Ambra1, ov-cyclin D1, si-Ambra1 and corresponding negative control (NC) vectors were provided by Genepharma . The above sequences were transfected into cells using Lipofectamine 3000  reagent.IM-9 human peripheral blood B lymphocytes and MCL cell lines  were purchased from Shanghai Zeye Biotechnology Co., Ltd. IM-9 cells were grown in RPMI-1640 medium supplemented with 10% FBS and 1% double antibiotic. Jeko-1, Z138, JVM2, and Rec-1 cells  were cultured in RPMI-1640 medium containing 10% FBS and 1% double antibiotic. The Ambra1 interference and overexpression vectors were constructed for transfection of JVM2 and Rec-1 cells or of Jeko-1 and Z138 cells, respectively. The groups were as follows: NC (oe-NC), ov-Ambra1; NC (si-NC), si-Ambra1#1, si-Ambra1#2, and si-Ambra1#3. A 2 μΜ concentration of the CDK4/6 inhibitor palbociclib-ΔΔct method were used to normalize gene expression. The primer sequences were as follows: Ambra1-F: GAGCAGGATCCAGAGAGCAC, Ambra1-R: CCTCTGGGCGTAGTATGCAG; cyclin D1-F: ACCTCTTCACCTTATTCATGGCT, cyclin D1-R: GCCTTTCCCGACCCTGCTAC; GAPDH-F: ACAGCCTCAAGATCATCAGC, GAPDH-R: GGTCATGAGTCCTTCCACGAT.Total RNA was extracted by TRIzol . cDNA was reverse transcribed using a cDNA reverse transcription kit  with total RNA as a template. Ultra SYBR Mixture  was used for qRT‒PCR experiments. GAPDH and the 2Total protein was extracted with RIPA lysis buffer , and the protein concentration was quantified by a BCA protein assay kit  and mixed with SDS‒PAGE loading buffer. Proteins were transferred to a PVDF membrane after separation by gel electrophoresis, and the membrane was blocked with 5% skim milk solution and incubated with antibodies specific for LC3-II/I , Beclin1 , ATG5 , p62 , cyclin D1 , and β-actin  at 4 ℃ overnight. Then, the membrane was incubated with secondary antibodies. The membrane was incubated with ECL solution for 1 min, and images were acquired in the imaging system .Suspended cells were evenly distributed by pipetting, and a small amount of the suspension was then smeared on the slide and left to dry. Triton X-100 (0.3%) was used for 30 min at 37 °C for permeabilization. Then, 5% BSA was used for blocking at 37 °C for 60 min. A suitable dilution of the anti-LC3 primary antibody  was added dropwise onto the slide for overnight incubation at 4℃. Then, the slide was incubated with 50–100 μL goat anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor 488  at 37 °C for 90 min. DAPI working solution was added for nuclear staining at 37 °C for 10 min. The slides were sealed with buffered glycerin and observed under a fluorescence microscope .4 cells were centrifuged in a 1.5 mL sterile centrifuge tube to obtain a cell pellet, and the cells were resuspended in the corresponding drug-containing medium according to the grouping configuration and then seeded in a 24-well plate. After 6, 12, and 24 h of treatment according to the group designations, a reaction solution containing 20% CCK-8 reagent was prepared, and 300 μL of the CCK-8 reaction solution was added to each well. After incubation at 37 °C and 5% CO2 for 4 h, the reaction solution was transferred to a 96-well plate, and the absorbance at 450 nm was measured on a Bio-Tek microplate reader.A total of 2 × 106 cells/mL in serum-free medium; 100 μL of this suspension was added to each well. The plate was placed in a 37 °C incubator for 48 h. The upper insert was removed, and the cells in the lower compartment were aspirated and counted by flow cytometry. For invasion experiments, a sterile pipette tip, an EP tube, Matrigel and a Transwell chamber were incubated at 4 °C overnight for precooling one day before the experiment. Then, 100 μL of ice-cold, serum-free DMEM was added to each well to dilute the Matrigel to a final concentration of 200 μg Matrigel per well. Then, the cells were incubated at 37 °C for 30 min, and we aspirated the supernatant. The subsequent steps were the same as those used for the migration experiments.For migration experiments, the lower compartment of the Transwell chamber was filled with 500 μL of 10% FBS complete medium. The cells were digested with trypsin to form a single-cell suspension, which was then diluted to a density of 1 × 105 cells. Five hundred microliters of binding buffer, 5 μL of Annexin V-APC, and 5 μL of propidium iodide were used. The reaction was performed for 10 min at room temperature in the dark. Within 1 h, flow cytometry  was used for observation and detection.Cells treated as described above were directly collected by centrifugation, washed twice with PBS, and centrifuged at 2000 rpm for 5 min each time to collect approximately 3.2 × 107 treated Jeko-1 cells were injected through the tail vein in a volume of 50 μL. The corresponding cell lines were inoculated in different groups: si-control group and si-Ambra1 group, 20 mice in each group. After injection, detailed observations and recordings were performed twice a week18. Then, each large group was randomly divided into two groups: si-control group and si-control + palbociclib group; si-Ambra1 group and si-Ambra1 + palbociclib group. Each group contained 10 mice. Fifteen days after cell injection, the mice were treated with or without palbociclib (3 mg/100 g body weight in a volume of 1 mL) by daily gavage19. Four weeks’ post-injection, the experiment was terminated. Mice were sacrificed by intraperitoneal injection of 150 mg/kg sodium pentobarbital.All animal experiments were approved by the Ethics Committee for Animal Experimentation of Xiangya Hospital of Central South University (Prot. ID 202203003). All methods are reported in accordance with ARRIVE guidelines. Beginning on the day before transplantation, 40 male CB17 SCID mice were intraperitoneally injected weekly with 0.2 mg of an anti-mouse interleukin 2 receptor beta monoclonal antibody (TMβ1) to eliminate natural killer cells. Then, the mice were randomly divided into two groups, and 4 × 10P < 0.05 indicated that the difference was statistically significant.GraphPad Prism 8.0 software was used for statistical analysis. Student's t test or one-way ANOVA was used for comparisons between two or among multiple groups, respectively. Pearson correlation analysis was performed to assess the correlation between Ambra1 and cyclin D1 expression in MCL patients. The study followed the principles set forth in the World Medical Association Declaration of Helsinki and was approved by the Institutional Ethics Committee of Xiangya Hospital of Central South University (K202203149). All animal experiments have been approved by the Ethics Committee for Animal Experimentation of Xiangya Hospital of Central South University (Prot. ID 202203003). We confirm that informed consent was obtained from all from all subjects  included in the study.First, we measured Ambra1 expression in MCL cells. Compared with that in IM-9 human peripheral blood B lymphocytes, Ambra1 expression in MCL cells  was significantly decreased. Among the MCL cell lines, Ambra1 expression was lower in Jeko-1 and Z138 cells  is a rare but aggressive B-cell hematological malignancy characterized by the t translocation resulting in overexpression of cyclin D123. Ambra1 is considered to be an autophagy and transport protein that plays a role in neurogenesis and cancer cell invasion24. Ambra1 can coordinate cellular responses to stress conditions such as starvation, and it is actively involved in cell proliferation, cytoskeleton modification, apoptosis, mitochondrial removal, and cell cycle downregulation25. In melanoma, Ambra1 deficiency accelerated tumor growth and reduced overall survival in a Braf/Pten mutant mouse model of melanoma. Furthermore, that study demonstrated that Ambra1 deletion promotes melanoma invasiveness and metastasis by increasing cell motility/invasion26. In addition, Ambra1 has been shown to modulate paclitaxel-induced apoptosis in breast cancer cells through the Bim/mitochondrial pathway, thereby altering the sensitivity of cells to paclitaxel27. Importantly, Zhao B et al. revealed that Ambra1 acts as a tumor suppressor, and its expression is reduced in uveal melanoma28. These studies suggest that Ambra1 plays a dual role in diseases. Consistent with the study of Zhao B et al., our results suggest that Ambra1 is a potential tumor suppressor in MCL. We found that the Ambra1 gene exhibited lower expression in MCL cells. Moreover, in MCL cells, overexpression of Ambra1 inhibited autophagy, affected the cyclin D1 level, inhibited proliferation, migration and invasion, and promoted apoptosis.Autophagy is a recycling process highly conserved in eukaryotic cells, and dysfunction of this process leads to many disease pathologies29. Chaikovsky AC et al. reported that loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition15. Consistent with the study of Chaikovsky AC et al., we also found that Ambra1 deletion reduced MCL cell sensitivity to the CDK4/6 inhibitor palbociclib. In vivo, Ambra1 deficiency reversed the effect of palbociclib. In MCL cells, cyclin D1 binds to CDK4/6 to phosphorylate Rb, releasing the block of G1 to S phase transition. Palbociclib is a specific and potent oral CDK4/6 inhibitor that induces complete, prolonged G1 arrest (pG1) in Rb + MCL cells30. In addition, inhibition of the master regulator of cellular metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation31. After further addition of the autophagy inhibitor CQ, we found that Ambra1 deletion partially abrogated the inhibitory effect of palbociclib on autophagy and increased the intracellular cyclin D1 level.A study by Liu J et al. in prostate cancer showed that Ambra1 knockdown increased cisplatin-induced apoptosis. Furthermore, cisplatin-induced autophagy was upregulated by Ambra1 overexpression or downregulated by Ambra1 knockdown in prostate cancer cells32. Cyclin D1, a major cell cycle regulator, is one of three unlinked D-type cyclin gene family members that also regulates transcription34. Cyclin D1 regulates the migration and invasion of MCL cells and can be viewed as a true operator in the pathogenesis of MCL35. By overexpressing cyclin D1, we found that overexpression of cyclin D1 also lowered the sensitivity of MCL cells to palbociclib; enhanced cell proliferation, migration, invasion, and autophagy; and inhibited cell death. In addition, Ambra1 expression was negatively correlated with cyclin D1 expression. This is also the first time that we report that Ambra1 and cyclin D1 play an important role in the mechanism of MCL sensitivity to palbociclib. Our observation also raises another possibility that the administration of CDK4/6 inhibitors , alone or in combination with cyclin D1 inhibitors, might be a possible therapeutic strategy for MCL. This possibility needs more exploration and confirmation in our future studies.Targeting the cell cycle is a rational approach for MCL therapy, as aberrant expression of cyclin D1 and dysregulation of CDK4 underlie cell cycle progression and MCL cell proliferation36. Recent research showed that RNF2 ubiquitylates and degrades Ambra137. Moreover, noncoding RNAs (including circular RNAs and microRNAs) have been regarded as key regulators of Ambra138. Through direct binding to the 3'-untranslated region of Ambra1 Mrna (Supplemet), circRNA-BCL2 may subsequently modify the production of miRNA-198 to control Ambra1 expression38. In addition, miR-200b inhibits autophagy and induces apoptosis by directly targeting the autophagy-associated gene Ambra139. However, the exact genetic and epigenetic mechanisms that govern Ambra1 expression remain unclear, and future research is required to define how Ambra1 downregulation occurs in MCL. In addition, how Ambra1 affects cyclin D1 expression, migration, invasion, and apoptosis in tumor cells through autophagy and the molecular mechanism by which Ambra1 regulates cyclin D1 expression are not clear. Due to time and funding constraints, we are not currently able to solve these questions well. In the future, with sufficient time and funding, we will continue to explore how Ambra1 affects cyclin D1 expression, migration, invasion, and apoptosis in tumor cells through autophagy and the molecular mechanism by which Ambra1 regulates cyclin D1.Importantly, Ambra1 expression is controlled by caspase-mediated cleavage and calpain-mediated degradationIn conclusion, our study demonstrated that abnormal expression of the autophagy-related regulator Ambra1 affected the cyclin D1 level and interfered with the sensitivity of MCL cells to the CDK4/6 inhibitor palbociclib. Reversing the abnormal expression of Ambra1 could inhibit the occurrence and development of MCL. Our study provides a reference and basis for the clinical treatment of MCL and provides a new strategy for overcoming drug resistance in MCL.Supplementary Information.


Background: SGLT2i directly inhibit the cardiac sodium-hydrogen exchanger-1 (NHE1) in isolated ventricular cardiomyocytes (CMs). However, other studies with SGLT2i have yielded conflicting results. This may be explained by methodological factors including cell isolation techniques, cell types and ambient pH. In this study, we tested whether the use of protease XIV (PXIV) may abrogate inhibition of SGLT2i on cardiac NHE1 activity in isolated rabbit CMs or rat cardiomyoblast cells (H9c2), in a pH dependent manner.Methods: Rabbit ventricular CMs were enzymatically isolated from Langendorff-perfused hearts during a 30-min perfusion period followed by a 25-min after-dissociation period, using a collagenase mixture without or with a low dose PXIV (0.009 mg/mL) present for different periods. Empagliflozin (EMPA) inhibition on NHE activity was then assessed at pH of 7.0, 7.2 and 7.4. In addition, effects of 10 min PXIV treatment were also evaluated in H9c2 cells for EMPA and cariporide NHE inhibition.Results: EMPA reduced NHE activity in rabbit CMs that were not exposed to PXIV treatment or undergoing a 35-min PXIV treatment, independent of pH levels. However, when exposure time to PXIV was extended to 55 min, NHE inhibition by Empa was completely abolished at all three pH levels. In H9c2 cells, NHE inhibition by EMPA was evident in non-treated cells but lost after 10-min incubation with PXIV. NHE inhibition by cariporide was unaffected by PXIV.Conclusion: The use of protease XIV in cardiac cell isolation procedures obliterates the inhibitory effects of SGLT2i on NHE1 activity in isolated cardiac cells, independent of pH. While the inhibition of NHE1 by SGLT2i in isolated cardiomyocytes has been replicated in several studies involving different cell types (Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have revolutionized the medical treatment of diabetic and heart failure patients . Large cll types , other sll types . In a prIt has been reported that enzymes used in cell isolation procedures can harm channel proteins embedded in the plasma membrane. For instance, protease XIV and XXIV, which are specific serine proteases, were found to degrade hERG1 potassium current and channel in HEK cells and cardiomyocytes . ConsequWe also questioned whether the PXIV effects on NHE1 by Empa was specific for freshly isolated rabbit cardiac cells or more a general phenomenon applicable to other cardiac cells as well. We therefore also used another cardiac cell type. Therefore, in this study, we aimed to investigate whether the exposure time of serine protease XIV disrupts inhibition of NHE1 by SGLT2i in freshly isolated rabbit cardiac cells. We next tested whether 2) this effect was dependent on pH, 3) was also present in cardiac precursor cells, and 4) NHE inhibition by the classic NHE inhibitor cariporide was also affected by PXIV. We conclude that inhibition of the NHE by EMPA is strongly dependent on duration of protease incubation both in rabbit cardiomyocytes and rat cardiac precursor cells, and that it is independent on pH.ad libitum. Male New Zealand White rabbits (2.5–3.5 kg) were anaesthetized with 35 mg/kg ketamine and 10 mg/kg xylazine subcutaneously and heparinized with a bolus of 1000 IU heparin (intravenously). Subsequently, the animals were euthanized by pentobarbital 50 mg/kg intravenously, the thorax was opened and the heart was quickly removed and submerged in ice-cold perfusion solution (composition see below). After cannulation of the aorta, the heart was mounted on a Langendorff perfusion setup, and perfused for 15 min at constant pressure (50 mmHg) at 37°C with a modified Tyrode’s solution containing (in mM) 128 NaCl, 4.7 KCl, 1.45 CaCl2, 0.6 MgCl2, 27 NaHCO3, 0.4 NaH2PO4 and 11 glucose (pH maintained at 7.4 by equilibration with a mixture of 95% O2 and 5% CO2) to remove remaining blood from the heart. Next, perfusion was changed to a nominally calcium-free solution containing (in mM) 16.8 HEPES, 146.4 NaCl, 3.3 KHCO3, 1.4 KH2PO4, 1.0 NaHCO3, 2.0 MgCl2, 0.01 CaCl2, 11.0 glucose, pH 7.3 (NaOH). After 15 min, collagenase type B , collagenase type P , trypsine inhibitor , 0.2 mg/mL hyaluronidase (Sigma H-3506), and creatine (10 mM) were added, and the heart was perfused for another 30 min at a constant flow in a recirculating manner. During the final 30-min period, protease XIV (Sigma H-3506) was either absent, present only during the last 10 min of perfusion, or present throughout the entire 30-min perfusion. Subsequently, the left ventricular wall was further dissociated using the following after-dissociation protocol: the tissue was cut into small pieces, incubated in the enzyme-containing nominally calcium-free solution with or without PXIV  and cultured in high glucose  Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS) and 100 U/ml of Penicillin/Streptomycin. In brief, H9c2 cells were digested with trypsin (0.25%), recovered by centrifugation  and re-suspended in plating medium. Cells were grown in an atmosphere of 95% O3 1.0, KHCO3 3.3, KH2PO4 1.4, CaCl2 1.3, MgCl2 2.0, NaCl 144, pH adjusted with NaOH to 7.0 or 7.2 or 7.4. In a separate set of experiments, isolated rabbit cardiomyocytes which had been exposed to 0.009 mg/mL Protease XIV for 35 min during enzymatic dissociation protocol  with normal solution, which resulted in an almost instantaneously intracellular acidosis, followed by the recovery from acidosis, which was monitored for the next 5 min. The effect of 1 µM EMPA  or 0.02% DMSO  was tested on recovery of acidosis and these compounds were continuously present during the NH4Cl pulse and the washout period of NH4Cl. Additionally, H9c2 cells, were subjected or not to 0.009 mg/mL Protease XIV during the 10 min NH4Cl pulse and the washout period of NH4Cl during which either DMSO, EMPA or cariporide (10 μM) were also present, to address the questions whether NHE inhibition by EMPA or the classic NHE inhibitor cariporide was dependent on the presence of PXIV in this different cell type.  for 30 min in a 37 °C water bath  in HEPESprotocol , were re+ pulse . Shortlyll type. . The iniα = 0.05). For normally distributed data, the Paired t-test (unpaired 2-tailed) was used for 2-group analysis. For non-normally distributed data, the Mann-Whitney test was used for 2-group analysis, and the Kruskal-Wallis test with Dunn’s multiple comparisons test was used for multiple-group analysis. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Statistical analysis was performed using GraphPad Prism v9. Results are presented as mean ± SEM for normally distributed data or median ± IQ for non-normally distributed data. Data normality was examined using the Shapiro-Wilk test  in the presence of EMPA, whereas the slope of the linear fir is −1.44  in the presence of DMSO, indicative of inhibition of [H+] recovery by EMPA at pH 7.4. The individual and average slope values for 35 min PXIV at the different extracellular pH’s, are depicted in We first established the presence of NHE1 inhibition by EMPA with our standard cell isolation procedure without PXIV treatment. Typical examples from rabbit ventricular CMs clearly demonstrate the failure to recover from intracellular acidosis in the presence of EMPA compared to DMSO . NHE1 acum value . At an e by EMPA . We nexten by +] , the slo+] tracing for EMPA shows a significant negative slope, similar to the DMSO condition, indicative of pH recovery (We then investigated whether prolonged exposure to PXIV (55 min) during the enzymatic dissociation protocolaffects NHE1 inhibition by EMPA or influences pH effects on inhibition . The mearecovery . The absrecovery .+] changes in response to an NH4+ pulse and subsequent NH4+ washout in cells treated solely for 35 min with PXIV (extracellular pH of 7.2), showing a clear inhibitory effect of EMPA on NHE1 activity , and the accompanying averaged and individual slope values  Protease XIV can completely abrogate the inhibition of NHE1 by empagliflozin in primary cardiomyocytes and cultured cardiomyoblast cells, and 2) Empagliflozin’s inhibition of NHE1 is independent of extracellular pH levels ranging from 7.0 to 7.4. These results highlight the significance of experimental conditions in generating reproducible data and emphasize the careful selection of enzymes used in cell isolation procedures tuned to the specific mechanism under investigation.+ current  of the effect of proteases are unknown, this study emphasizes that study methods are key to the interpretation of the results. We speculate that our observations potentially are applicable to the function of other extracellular sarcolemma-bound proteins as well.In 2017 we were the first to report the inhibition of NHE1 activity by empagliflozin in isolated cardiac cells from rabbit hearts . In that current . DockingIn the first reports demonstrating NHE1 inhibition by SGLT2 inhibitors, the extracellular pH during measurements at 37° was set at 7.2 , whereasIn conclusion, robust inhibition of the NHE1 by EMPA was observed in non-PXIV treated rabbit cardiomyocytes and rat H9c2 cardiomyoblast cells. However, prolonged exposure to PXIV in cardiac cells abolished NHE1 inhibition by EMPA, highlighting the need for caution when using of PXIV in cardiac cell isolation procedures to examine the effects of SGLT2 inhibitor on cardiac NHE1 activity.+]I decay curve over a full range of pHi’s, whereby the obtained decay rate constant λ then reflects NHE activity independent of the pH. However, with EMPA or Cariporide there often was no [H+]I decay at all (resulting in an undefined decay constant), making exponential fitting strategies inappropriate. We therefore chose to use linear fitting of [H+]I changes following the first 50 s after withdrawal of NH4+, with the slope of the linear fit as an index of NHE activity. Although there were sporadically differences in the surge of the [H+]I between experiments resulting in a slightly different pH trajectories over which NHE activity was determined, most linear fits were similar for their pH trajectories over which the slope or NHE activity was determined. This justifies the use of slopes of linear fits as index of NHE activity as a measure of NHE activity.Ideally, NHE activity is determined by exponential fitting of the [H


To this end, a target-based virtual screening was performed over a representative set of Peruvian flora-derived natural compounds. The best poses obtained from the ensemble molecular docking process were selected. These structures were subjected to extensive molecular dynamics steps for the computation of binding free energies along the trajectory and evaluation of the stability of the complexes. The compounds exhibiting the best free energy behaviors were selected for in vitro testing, confirming the inhibitory activity of Hyperoside against Mpro, with a iK value lower than 20 µM, presumably through allosteric modulation.Over 750 million cases of COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), have been reported since the onset of the global outbreak. The need for effective treatments has spurred intensive research for therapeutic agents based on pharmaceutical repositioning or natural products. In light of prior studies asserting the bioactivity of natural compounds of the autochthonous Peruvian flora, the present study focuses on the identification SARS-CoV-2 M Solving the associated quadratic equation allowed the calculation of the free concentration of inhibitor for given values of the total enzyme and inhibitor concentrations, [E]T and [I]T, and the inhibition curve was built by plotting ratio of the initial velocities, v([I])/v([I] = 0), as a function of total inhibitor concentration:The inhibition potency of compounds was assessed by determining the inhibition constant, Along these calculations, inhibitor depletion (due to binding to enzyme) was considered; thus, no approximation for the free inhibitor concentration  was made.kcat is the catalytic rate constant, Km is the Michaelis-Menten constant, [S] is the substrate concentration, and Ki is the intrinsic  inhibition constant. From this, the inhibition curve can be written as:In the case the inhibitor acts through a purely competitive mechanism, the previous quasi-simple equilibrium can be expanded as a simple competitive inhibition model as follows (Equation (6)):iappK in the previous equation is equivalent to the IC50. It is important to emphasize that IC50 is not an appropriate inhibition potency index, because it is an assay-dependent parameter .By approximating the free compound concentration by the total compound concentration and neglecting compound depletion, the K is the equilibrium constant for the conformational equilibrium between the inactive conformation E* stabilized by inhibitor binding and the active conformation E stabilized by substrate binding (K = [E]/[E*]), and, again, Ki is the intrinsic inhibition constant. From this, the inhibition curve can be written as:If the inhibitor acts through an allosteric non-competitive mechanism, the previous quasi-simple equilibrium (Equation (6)) can be expanded as a simple allosteric inhibition model as follows (Equation (9)):iappK is estimated from the inhibition curve by non-linear least-squares regression analysis, the true inhibition constant Ki can be calculated using Equation (8) or Equation (11).Once the apparent equilibrium constant


Freezing is an effective technology with which to maintain food quality. However, the formation of ice crystals during this process can cause damage to the cellular structure, leading to food deterioration. A good understanding of the relationship between food microstructure and ice morphology, as well as the ability to effectively measure and control ice crystals, is very useful to achieve high-quality frozen foods. Hence, a brief discussion is presented on the fundamentals/principles of optical microscopic techniques (light microscopy), electronic microscopic techniques (transmission electron microscopy (TEM) and scanning electron microscopy (SEM)), as well as other non-invasive techniques  and their application to measuring ice formation rates and characterizing ice crystals, providing insight into the freezing mechanisms as well as direct monitoring of the entire process. And, in addition, this review compares (the negative and positive aspects of) the use of simple and cheap but destructive technologies  with detailed microscopic technologies at the micro/nanometer scale but with pretreatments that alter the original sample (SEM and TEM), and non-destructive technologies that do not require sample preparation but which have high acquisition and operational costs. Also included are images and examples which demonstrate how useful an analysis using these techniques can be. The study of food microstructure has become increasingly important for researchers, since it can have a clear influence on different nutritional values, rheological behaviors, textural characteristics, and sensorial qualities, as well providing information about precise use conditions in the application of traditional processes or modern innovative technologies . In partSpecifically, the crystallization of water during freezing is the most important step of the freezing process, since ice nucleation occurs and initial crystals begin to grow in a dispersed dendritic form in the supercooled water, ending the freezing process, with the growth of concentric rings of solid ice at 0 °C and the final cooling of the solid ice . Thus, oIn practical terms, ice morphology  is a relevant parameter during the freezing process of solutions and/or liquid foods , since iIn this context, ice morphology plays a key role in the quality of frozen foods along the cold chain: (1) A rapid freezing rate is very important because it determines the ice morphology  within a food product. In tissue foods, a slow rate of freezing causes an ice morphology with large ice crystals in extracellular locations, resulting in poor frozen food quality. In contrast, a quick freezing rate produces an ice morphology with small crystals at locations inside and outside of the cells, and, as a consequence, high-quality frozen foods. (2) The freezing temperature should be kept constant, because any fluctuations in the storage freezing temperature tend to reduce the quality of frozen foods; for example, drip losses after thawing, which are mainly due to ice morphology changes as a result of recrystallization .Thus, techniques for the direct visualization of ice morphology have been developed through qualitative and quantitative measurements ,13, contBased on the key role of ice morphology for the quality of frozen food products, it is necessary to have updated and easily accessible scientific information for researchers related to the different techniques available for the observation and measurement of ice morphology in different experimental conditions, the temperature and freezing technique applied, as well as different food matrices. Therefore, the aim of this review is to discuss the use of microscopy in the study of the ice morphology and microstructure of frozen foods, highlighting the effects of the different low-temperature processes on the food quality properties. A brief discussion of the fundamentals and principles of the different microscopic techniques , as well as other non-invasive or destructive techniques  is carried out. Additionally, examples of their application to the study of the changes in frozen foods’ structure are shown, as well as their effectiveness in measuring the ice formation rate and characterizing the crystals’ morphology, providing information on the freezing mechanisms and allowing for the constant monitoring of the process.Our review was carried out using the keywords  in the Scopus and ScienceDirect databases from 2008 to 2023.Based on the >500 articles chosen from the Scopus and ScienceDirect databases, the paper selection was restricted to articles related to the different microscopic techniques used in ice crystal visualization such as optical microscopy  , electroAdditionally, among all the articles, only 109 were related with microscopy techniques to analyze ice crystals in food matrices.LM is commonly used to provide microscale evidence on the structure of different foods, since this direct observation technique has advantages that facilitate its use such as a modest start-up capital, low maintenance costs and convenience, full color information with high resolution in the images obtained , and the samples can be totally wet and these can be analyzed under ambient conditions, but the LM technique presents disadvantages such as a limited depth of focus, difficulty removing the presence of dirt or foreign bodies that prevent the visualization of samples, magnification lenses are fixed (limiting the resolution in the images), some samples need to be treated with staining to produce specific coloration of particular substances, and in turn, some samples can only be observed with the implementation of a cryogenic device (Cryo-LM) . TherebyIn particular, LM can be distinguished under two major classes , stereom2 and 0.065 to 0.100 mm, for area and diameter, respectively) than block freeze concentration , but in all cases, the ice crystals presented a shape of solid prisms or plate ice morphologies [Trachurus japonicus). The histological structure results indicated damage by numerous intracellular and extracellular ice crystals (between 50 to 100 μm) in muscle cells of frozen muscle specimens and small interstices between cells of freeze-thawed muscle specimens, i.e., there is no presence of ice crystals after thawing process. Moreover, Du et al. [Thus, these microscopy techniques (LM and Cryo-LM) allow the observation of changes in ice crystals during the freeze-thaw process, presenting the advantage that the sample preparation is not complicated, and thus, LM and Cryo-LM provide microscale data on frozen food structures and lyophilized and thawed samples . Hence, hologies . Likewishologies  focused u et al.  studied u et al.  studied Thus, In the last 40 years, electron microscopes have emerged as a powerful tool for the characterization of a wide range of materials, since these microscopes present a high versatility and extremely high spatial resolution, and thus, electron microscopy is a valuable tool for many applications, including food areas, where the understanding of structure in various foods has improved greatly, and thus, new foods have been developed . In partSpecifically, electron microscopy corresponds to different techniques, and each technique has been used with great success for freezing-related evaluations, including transmission electron microscopy (TEM), scanning electron microscopy (SEM), cryo-SEM, and environmental scanning electron microscopy (ESEM).In TEM, it is possible to obtain refined internal information from the sample as they are penetrated by short wavelength electron beams and form a highly magnified and detailed image in a vacuum environment. Hence, it provides powerful magnification and valuable information on surface, shape, size, and structure features .Thereby, TEM is used to analyze the effects of ice crystals on the internal structure of frozen foods instead of trying to observe them directly; for example, Zhang et al.  investigIn another TEM observation, Meziani et al.  studied However, this technique had some limitations, since the observed sample must be very thin  because the electron beam can be easily scattered, and in addition, the sample preparation is quite a complex and tedious procedure. Furthermore, the samples are limited to those that are electron transparent, capable of tolerating the vacuum chamber, and small enough to fit in the chamber .SEM obtains images through electron high-energy beam scanning, magnifying by using electrons, allowing a wide range of magnifications, and it can achieve a depth of field approximately 500 times greater with the object magnification from 10 to 300,000 times. Thereby, SEM has been a very attractive and powerful electron microscope for food scientists, since it allows the study of the internal and surface characteristics of food materials. Thus, some advantages include a simple sample preparation, a wide magnification range, a large depth of field, and the images are a representation of electronic data, and hence, it allows a better analysis and quantification of images. In addition, it combines the characteristics of optical microscopy and TEM. However, a clear disadvantage is the differentiation of agglomerated structures, i.e., SEM (and image resolution) depends on the interaction between the sample and electron beam, but an important point, the agglomeration of nanoparticles and their particular differentiation in a sample are not clearly appreciated and total dehydration is required for biological materials .Specifically, Ban et al.  used SEMAnother method used for the evaluation of frozen and freeze-dried foods is cryo-SEM. With a special cooling system, cryo-SEM supports the observation of frozen samples, allowing direct observation without a thawing step. Additionally, it provides an interesting alternative, since the unfrozen samples can be placed under the SEM equipment and can be gradually frozen, and thus, the sample is frozen and kept in the microscope at low temperature, allowing the investigation of the internal three-dimensional structure of wet samples. Cryo-SEM provides rapid physical fixation, avoiding the risk that other artifacts can be added in the preparation procedure, leading to chemical fixation, structural collapse, or cell contraction as sometimes occurs in typical preparation methods. However, as well as the SEM method, the main drawback corresponds to its high cost of acquisition and maintenance of instruments . This isIn food terms, cryo-SEM allows the ice crystals’ investigation in hairtail samples under three different frozen treatments, where the results showed ice crystals with an equivalent diameter close to 12.6 μm, 3.6 μm, and 1.2 μm for −20 °C, −80 °C, and −196 °C, respectively, with more small and regular ice crystals in samples frozen at −196 C in relation to the other freezing treatments . MoreoveFurthermore, Guo et al.  analyzedDue to the SEM limitations , ESEM was established to enhance the ice crystals’ visualization, and thus, ESEM differs from conventional SEM mainly by the presence of a gas in the sample chamber. The presence of a gas or environment around the sample is the inspiration for the term ‘‘environmental’’, and it can play two main roles . In the In a recently published review, Pach and Verdagues  explain Thereby, the use of optical and electron microscopy, as well as other techniques for the measurement, evaluation, and control of ice crystallization, and analysis of the characteristics of ice crystals before, during, or after the freezing process, has been addressed by many authors, with the aim of understanding the relationship between microstructure and ice crystals in food quality. Additionally, the different investigations discussed novel technologies for the control of the ice crystals’ formation based on ultrasound, high pressures, electromagnetic fields, and the use of biological proteins by inducing or suppressing crystallization, to assist or accelerate the processes of freezing, showing great potential in obtaining a higher quality in frozen foods with smaller and evenly distributed ice crystals.Hence, As previously mentioned, optical and electron microscopies are widely used visual technologies for measurement and characterization of ice crystals and ice formation rate, as well as monitoring the entire freezing process. Hence, these technologies have also been used to perform a structural examination of frozen foods, including evaluating microstructural damage to cells and tissues, observing solute redistribution, establishing the degree of food heterogeneity, and important correlations between the microstructure and the texture of the mouth . NonetheIn the same way, computed tomography and infrared (IR) analysis have been used to study the freezing process and to monitor the state of water during freezing. IR spectroscopy has proven to be a good and reliable method for detecting ice nucleation and for studying crystal growth because the IR absorption spectra of ice and liquid water are very different. These techniques, being non-invasive methods, do not introduce processing artifacts and allow repeated observation of the same sample as it undergoes controlled changes during processing . MeanwhiAnother example is the study reported by Zhao and Takhar , the 3D In summary, the techniques used to observe and measure ice morphology have been summarized in Freezing has achieved resounding success in relation to food preservation, since it manages to preserve the nutritional elements of different foods. The types of frozen foods range from raw commodities such as fruits, vegetables, and meats to processed foods such as dairy desserts and frozen dinners. The structure of the ice phase in frozen foods is important at both the microscopic and macroscopic levels. The size and number of ice crystals determine sensory properties such as ‘hardness’ or ‘iciness’ in foods that are consumed without thawing, but they cause negative changes in many foods that are perceived in food quality when it is thawing. Additionally, a good control of the freezing rate parameter allows a high frozen food quality, since this parameter determines the size and shape of ice crystals, including mechanical stresses in the frozen foods, where, as the theory mentions, a fast freezing rate generates a small ice crystal size, while a slow freezing rate generates a large ice crystal size. Thus, a food with a cellular structure can be affected by the ice crystal size, since the cellular structure/ice crystal size ratio is a critical measure with respect to the texture and water holding capability, among other food characteristics. Hence, large ice crystals puncture the cellular structure (cell membranes), provoking loss of the intracellular fluid during thawing, and thus, the texture is unappetizing and mushy . Meanwhile, in ice cream, small round ice crystals allow a soft and creamy texture. Therefore, depending on the food, the freezing rate parameter can have different (positive and negative) effects on the (micro)structure of the food due to the formation of ice crystals.The purpose of this review was to show an overview of applications related to different microscopic observation techniques and the different uses in the frozen food industry. With the development and availability of microscopy, spectroscopy, and computer vision, new methods have appeared for the acquisition, direct monitoring, and study of ice crystals, microscopic frozen food structures, and associated processes. Thereby, as stereomicroscopes and compound microscopes present a low/regular resolution and the sample can be destroyed by the preparation procedure, novel optical and electron microscopies have been developed to obtain more complete sample information. Furthermore, spectral imaging and online techniques have shown auspicious results on the ice crystal behavior. More importantly, the emergence of the latter makes in situ detection a reality. However, the most appropriate technique to be used will depend on the sample property of the studied sample and its nature. This review shows that these imaging techniques can be used to find the relationships between food processing conditions and the morphological changes of its components, allowing higher quality products and improving associated processes. However, there are two important limitations in some of these methods, mainly they require special equipment (cryo-section) or cooled sample holders (Peltier) to keep the sample at temperatures below the freezing point during the measurement, as well as the availability of a cryostat to cut the sections of the frozen samples. Some of these innovative technologies are in development or at the laboratory stage, while for others the biggest obstacle is the high capital cost.Nevertheless, as these technologies provide sample images at different angles, more and more combinations will appear from these observation techniques which will allow more information for a better understanding of ice formation mechanisms. For this reason, non-destructive and online measurement will provide real information of the studied sample and will be an inevitable trend in the future. These non-destructive and online techniques have great future potential, since they allow costs to be reduced by keeping food samples unchanged when tested for their ice morphology in a production line using computational simulation models and automatic control systems. On the other hand, an important challenge is to couple the control parameters with artificial intelligence that allows prediction of the final conditions of ice morphology and therefore the final quality of frozen foods.


The internal stratum (lamellar) of the equine hoof is composed of the interdigitated dermal and epidermal lamellae, forming the dermo-epidermal junction (DEJ). In cases of laminitis, the configuration of the lamellae can be affected, leading to changes in the position of the distal phalanx in chronic cases. Digital radiography (DR) is a routine diagnostic imaging technique used to detect, measure, and monitor such changes. The objective of the study was to compare the use of the transmural ultrasound technique and digital radiography (DR) to detect the hoof strata and structures within, as well as to measure the spatial relationship between the distal phalanx and the hoof capsule. This ultrasonographic transmural technique enables the determination and measurement of the ultrasonographic distal phalanx–hoof relationship. The transmural ultrasound allows for the definition of the normal appearance of the hoof tissues and internal structures. Determining such measurements and proportions could contribute to evaluations of the normal spatial distal phalanx–hoof relationship. The objective was to describe the hoof’s dermis and epidermis measurements using the transmural technique, in comparison with DR and anatomical sections. Sixty-two healthy hooves without digital radiographic abnormalities, made up of 30 anatomical pieces (phase 1) and 32 alive horse hooves (phase 2), and 16 sagittal sections of the first ones were used. The proximal and distal planes were compared, defining the following measurements: coronary band-extensor process, distal phalanx apex–hoof wall, sole dermis, middle stratum, parietal dermis, lamellar layer, the sublamellar dermis, and ratios between them. Most of the measurements were consistent among methods. Some showed measurement differences and a minority was impossible to determine. The transmural technique allowed for the observation and replication of measurements of the hoof structures, described with other diagnostic aids. It also allowed for the implementation of new measurements that would help to determine the spatial location of the distal phalanx. Additionally, it contributes to the visualization of normal findings, which will be useful to compare variations in the different phases of laminitis. The transmural ultrasound technique  was able to describe the normal appearance of the medium layer , the dermo-epidermal junction (DEJ) and the sublamellar dermis of the hoof in the parietal region . The parDigital radiography has been used to describe the normal position of the distal phalanx ,15,16,17More recently, transmural ultrasound has been used to distinguish between all these layers, highlighting the importance of this diagnostic imaging modality . While tDetermining the anatomical relationship between the distal phalanx and the hoof capsule is diagnostically valuable, and radiographic evaluation is routinely used to estimate distances, angulations, and proportions to determine normality and deviations ,15,16,17Therefore, the objective of this study is to determine the diagnostic value of transmural ultrasonography in comparison to digital radiography and anatomical sections in estimations of normal measurements and ratios of the hoof’s dermis and epidermis, as well as the ratios of the lamellar layer and the sublamellar dermis with reference to the parietal dermis. The study aims to estimate the spatial relationship of the soft tissues and the distal phalanx, which could be useful in identifying and managing the conditions that affect these structures.This research was approved by the Ethics Committee for Animal Experimentation of the Universidad de Antioquia, Colombia . A total of 62 healthy hooves of thoracic limbs (TL) and pelvic limbs (PL) from Colombian creole horses were distributed in two study phases. In phase 1, 30 hooves collected in a slaughterhouse were disarticulated from the fetlock joint, transported fresh for 4 h, and finally frozen at −21 °C for 150 days. In phase 2, 32 hooves of eight healthy mares without lameness and with an average age, weight, and body condition score  of 6.9 ±The hooves were selected based on a negative lameness inspection by assessing the lameness at walk and light trot using hoof testers, and percussion was also carried out in all horses, including on horses that were the donors of the pieces. ® HFX90V , with 1.2 mAs—66 kv settings. The location and normal anatomy of the distal phalanx were determined by measuring angles and other relevant factors , with relevance to laminitis [All hooves were also evaluated using digital radiography to rule out any alterations in internal and external structures. Lateral–medial projections were taken using a portable veterinary X-ray equipment, generator MINXRAYaminitis ,15,16,17The measurements evaluated in the sagittal plane sections of the anatomical pieces , digitalWith a ultrasonographic sagittal view, the distal interphalangeal joint (DIPJ) and the extensor process  were ideThe coronary band-extensor process (A), the distal phalanx apex–hoof wall (B), and the sole dermis (S) distances were also determined in a ultrasonographic sagittal view, with the exception of distance A, which was determined using the transverse view  at the lIn the latero-medial radiographs, the contrast-adjusted technique permitted the differentiation of the soft tissue from the others with a radiolucent and radiodense appearances, respectively ,18,19. HTo obtain ultrasonographic measurements, all hooves were prepared and submerged in water or ice and water in order to differentiate the middle layer (tubular), the internal layer (lamellar), and the sublamellar dermis, as well as the transitions between them. This methodology and the findings were described in a companion article titled Transmural Ultrasonography in the Evaluation of Horse Hoof Internal Structures: Comparative Qualitative Findings—Part 1 [The sagittal plane sections were generated from 16 hooves in anatomical pieces (phase 1) to permit replication of the previously mentioned measurements and evaluate the parietal dermis and its components, which were also obtained with the transmural ultrasound.In addition, ultrasonographic ratios were evaluated with the aim of reinforcing the identification of the normal position of the distal phalanx inside the hoof. The evaluated ratios were the parietal dermis: coronary band-extensor process (D1:A and D2:A); the lamellar layer:parietal dermis (L:D) and the sublamellar dermis:parietal dermis (C:D) at the proximal and distal levels; the apex–hoof wall:coronary band-extensor process (B:A) and the sole dermis:coronary band-extensor process (S:A).Descriptive statistics were first calculated as measures of central tendency , mean (average), median) and measures of dispersion , coefficient of variation (CV)). Non-normally distributed data were analyzed using the median. In phase 1, we compared the distances A, D1, D2, B, and S, obtained from transmural ultrasonography, digital radiography, and anatomical sections using the nonparametric Friedman test. We also compared the measurements of the parietal dermis and its components (the lamellar layer and the sublamellar dermis) obtained from transmural ultrasonography and anatomical sections using the nonparametric Wilcoxon test. In phase 2, we compared the distance measurements of A, D1, D2, B, and S obtained from transmural ultrasonography and digital radiography using the Wilcoxon test.®, version 16.1  considering a statistical significance level of p ≤ 0.05.All data were analyzed using STATAThe measurements obtained from digital radiography (DR), transmural ultrasound (US), and sagittal sections (AN) in phase 1 are presented in In In In the ultrasonographic ratios evaluation of the TL , the appIn the ultrasonographic ratios evaluation of the PL , the appUltrasonography is a widely used technique for the diagnosis of lameness in horses . SeveralIn this study, the transmural ultrasonographic technique was compared with radiology and anatomical sections, obtaining a qualitative description of the previously mentioned structures. However, in addition to the ultrasonographic anatomy, it is important to define the distances between the internal structures of the digit to establish the spatial relationship or location of the third phalanx with relevance to the adjacent structures. Therefore, this study aimed to obtain quantitative information based on measurements and ratios in anatomical pieces and alive horse hooves.The obtention of larger radiographic measurements when compared with macroscopic anatomical measurements (millimeter ruler) in anatomical sections and MRI has also been reported ,19. The When comparing the measurements of the parietal dermis (D) and its layers (L and C) between the ultrasonography and the anatomical sections, several factors could have influenced the results. For instance, the anatomical pieces were not individually packed in plastic bags to prevent desiccation  and undeWhile the stereomicroscope or distance of the camera lens were not effective in determining configuration changes in the tissues and the limits between L-C and D-P, which were described in the qualitative findings of the transmural technique , histoloThese observations are supported by the fact that the distance D was not statistically similar between ultrasonography and anatomical sections in Distances A, B, and S were previously described in radiography and can also be measured by ultrasonography. These measurements are of great importance in establishing the positioning of the third phalanx in conditions that affect its support, such as chronic laminitis ,42,43,44The measurements made towards the distal level of the parietal surface of the distal phalanx are also smaller when compared to previous radiographic studies . A similThe transmural technique used in this study required different approaches for measuring distances A, B, and S. Distance A was determined using a transverse view at the level of the coronary band  as a diaHowever, due to the interaction between ultrasound and the distal phalanx, it is impossible to detect its inclination and to estimate the ratios associated with palmar cortical length ,19,44,46The ratios of the parietal dermis and the layers that compose it (L:D and C:D) would increase in cases of symmetrical distal displacement or “sinking” ,40,41. IThe information provided here may be a starting point for the diagnosis of chronic laminitis by ultrasonography. Although it exceeded the stated objectives, incidental findings  were repThe changes in the positioning of the distal phalanx were better explained in the anatomical pieces of a horse subjected to euthanasia due to chronic laminitis . The incThe ultrasonographic measurements of the spaces, layers, and spatial relationship of the internal structures of the hoof, as determined in this study, can provide several advantages in similar cases of chronic laminitis. By using this technique, clinicians can obtain detailed and accurate information about the condition of the hoof, which can aid in diagnosis and treatment planning. Therefore, ultrasonography can be considered a valuable tool in the management of chronic laminitis. To further validate the usefulness of these measurements, it is recommended that future studies be conducted using MRI and histological sections of normal horses, as well as natural or induced clinical cases of laminitis. This will help to objectively determine the suitability of these measures for clinical use and improve our understanding of chronic laminitis.The transmural ultrasonographic technique used in this study allowed for the observation and replication of measurements of the internal structures of the hoof that have been described by other diagnostic aids. Additionally, this technique provided new measurements that can aid in determining the spatial location of the distal phalanx within the hoof.