Primary squamous cell carcinomas (PSCCs) arising in intracranial epidermoid cysts (IECs) are very rare, and their management and prognostic factors remain unclear. This study aimed to enunciate the clinical features and suggest a treatment protocol based on cases from the literature and the cases from our institution.The clinicoradiological data were obtained from nine patients with PSCCs arising in IECs, who underwent surgical treatment at Beijing Tiantan Hospital between July 2012 and June 2018. We also searched the PubMed database using the keywords “epidermoid cyst(s)” or “epidermoid tumor(s)” combined with “malignant” or “malignancy” or “intracranial” or “brain” or “squamous cell carcinoma” between 1960 and 2020. Risk factors for overall survival (OS) were evaluated in the pooled cohort.The mean age of our cohort was 51.2 ± 8.3 years (range: 39–61 years), which included eight males and one female. Gross total resection (GTR) was achieved in three patients, while non-GTR was achieved in six patients. Radiotherapy was administered to five patients. After a median follow-up of 16.7 ± 21.6 months (range: 3–72 months), eight patients died with a mean OS time of 9.75 ± 6.6 months (range: 3–23 months). In the literature between 1965 and 2020, 45 cases of PSCCs arising in IECs were identified in 23 males and 22 females with a mean age of 55.2 ± 12.4 years. GTR, non-GTR, and biopsy were achieved in six (13.3%), 36 (80%), and three (6.7%) cases, respectively. After a mean follow-up of 12.7 ± 13.4 months (range: 0.33–60 months), 54.1% (20/37) patients died, and recurrence occurred in 53.6% (15/28) patients. A multivariate analysis demonstrated that postoperative radiotherapy (p = 0.002) was the only factor that favored OS. The Kaplan–Meier analysis showed that, compared with no radiotherapy , radiotherapy  had significantly prolonged OS (p = 0.0011), and GTR could not improve OS (p = 0.5826), compared with non-GTR. The 1-year OS of patients with or without radiotherapy was 72.5% or 18.2%, respectively.Malignant transformation of IEC into PSCC was prevalent in elderly patients, with slight male predominance. GTR of previous benign IECs is recommended. For remnant benign IECs, close follow-up should be performed. Postoperative radiotherapy for PSCCs could bring survival benefit. GTR of these malignant intracranial tumors is difficult when they involve important brain structures. Future studies with larger cohorts are necessary to verify our findings. Intracranial epidermoid cysts (IECs) are benign tumors that typically develop in the cerebellopontine angle (CPA) region . The IECNine patients who underwent surgical treatment were pathologically confirmed to have PSCCs at Beijing Tiantan Hospital between July 2012 and June 2018. According to Hamlat (Then we searched the PubMed database using the keywords “epidermoid cyst(s)” or “epidermoid tumor(s)” combined with “malignant” or “malignancy” or “intracranial” or “brain” or “squamous cell carcinoma” between 1960 and 2020. A total of 45 articles in English language that met the Garcia criteria were included.The risk factors for overall survival (OS) were evaluated with univariate and multivariate Cox regression analyses of a total of 46 cases (including 37 cases from the literature and nine cases from our center). The survival curves were performed by the Kaplan–Meier method. Analyses were performed using SPSS Statistical Package software , with the significance set at p < 0.05.The cases from our institute included eight males and one female with a mean age 51.2 ± 8.3 years (range: 39–61 years). The duration of symptoms ranged from 2 to 12 months with a mean length of 9.33 ± 3.74 months. The preoperative symptoms included headache (n = 3), facial numbness (n = 3), trouble coughing (n = 3), diplopia (n = 2), limb weakness (n = 2), facial paralysis (n = 2), tinnitus (n = 1), ataxia (n = 1), ptosis (n = 1), hearing loss (n = 1), and vomiting (n = 1). Five patients experienced natural malignant transformation of benign intracranial ECs, and four patients suffered malignant transformation of remnant benign IECs. The interval from EC was 3–28 years Table 1.A total of 45 patients  diagnosed with intracranial PSCCs were identified between June 1960 and July 2020 Table 2.The mean follow-up in the pooled cohort (46 cases) was 13.5 ± 15.1 months. The results of univariate Cox regression analysis  showed that compared with no postoperative radiotherapy, postoperative radiotherapy had significantly prolonged OS . Other factors, including sex , age , surgical history , tumor location , GTR , peritumoral edema , and chemotherapy  were not significant. Then we added sex, age, GTR, and radiotherapy to the multivariate Cox regression analysis. Radiotherapy  was the only protective factor Table 4.A 61-year-old male presented with dizziness and hearing loss for over 1 year and developed left hemiparesis for 2 weeks. Neurological examination revealed dysfunction of right cranial nerve VIII and weakness of left limb. A T2-weighted MRI showed a heterogeneous signal of the lesion located at the right CPA region. Diffusion-weighted imaging (DWI) revealed a high-signal-intensity lesion located in the right CPA cistern, prepontine cistern, and right cisterna ambiens and low signal intensity in the right pons. A T1-weighted gadolinium-enhanced MRI showed an intra-axial ring-like enhanced mass in the right brainstem, while the high-signal-intensity lesion on DWI showed no enhancement. Peritumoral edema was also observed  and Special Fund of the Pediatric Medical Coordinated Development Center of Beijing Hospitals Authority (XTYB201822).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Although hypoxia has been shown to promote keratinocyte migration and reepithelialization, the underlying molecular mechanisms remain largely unknown. ADAM17, a member of the metalloproteinase superfamily, has been implicated in a variety of cellular behaviors such as proliferation, adhesion, and migration. ADAM17 is known to promote cancer cell migration under hypoxia, and whether or how ADAM17 plays a role in hypoxia-induced keratinocyte migration has not been identified. Here, we found that ADAM17 expression and activity were significantly promoted in keratinocytes under hypoxic condition, inhibition of ADAM17 by TAPI-2, or silencing of ADAM17 using small interfering RNA which suppressed the hypoxia-induced migration of keratinocytes significantly, indicating a pivotal role for ADAM17 in keratinocyte migration. Further, we showed that p38/MAPK was activated by hypoxia. SB203580, an inhibitor of p38/MAPK, significantly attenuated the upregulation of ADAM17 as well as the migration of keratinocytes induced by hypoxia. Activation of p38/MAPK by MKK6 (Glu) overexpression, however, had adverse effects. Taken together, our study demonstrated that hypoxia-induced keratinocyte migration requires the p38/MAPK-ADAM17 signal axis, which sheds new light on the regulatory mechanisms of keratinocyte migration. Our study might also help in developing therapeutic strategies to facilitate wound healing in vivo, where cells are migrated in a hypoxic microenvironment. Wound healing, an important physiological process that restores the integrity of skin after trauma, consists of a sequence of well-characterized stages governed by sequential and overlapping phases, in which reepithelialization is the key stage depending upon keratinocyte migration from the wound margins . HypoxiaκB, and specificity protein 1(Sp1), in which gene expression is involved in the transcription factors mentioned [In response to hypoxia, such as hypoxia-inducible factor (HIF), nuclear factor (NF)-entioned . Involveentioned . ADAMs aentioned . It has entioned , adhesioentioned , and migentioned . Resultsentioned ; in addientioned  and hypoentioned . These sImportantly, our previous research is consistent with others that show the role of p38/MAPK signaling in hypoxia-induced keratinocyte migration , 20. P382 incubator, and 95% humidity.HaCaT cells were purchased from the cell bank of the Chinese Academy of Sciences in Beijing, China. Cells were cultured in RPMI 1640 medium  containing 10% fetal bovine serum , 100 U/ml penicillin , and 100 mg/ml streptomycin .The cells were then put in 37°C, 5% CO2, 5% CO2, and 94% N2 were produced utilizing an oxygen control incubator . The p38/MAPK inhibitor SB203580 (Beyotime) (5 μmol/l) was added to these cultures and incubated at 37°C for 30 minutes before hypoxia treatment. In addition the ADAM17 inhibitor, TAPI-2 (40 μM) was added to the cultures and incubated at 37°C for 12 hours before hypoxia treatment.Hypoxic conditions of 1% Oμl lysis buffer on ice, and homogenized. The lysates were sonicated for 4 seconds and centrifuged at 14000 rpm for 15 min at 4°C. Protein concentrations were observed by the BCA protein assay kit (Beyotime). Cell lysates containing 20 μg of proteins were electrophoresed on 10% SDS-PAGE gels and transferred to a PVDF membrane (Millipore). The membranes were blocked in 5% bovine serum albumin (BSA) for 2 hours at room temperature. The following primary antibodies were used for detecting protein expression: anti-ADAM17 , anti-p38-HRP , and anti-phospho-p38 at Thr180/Tyr182 . Horseradish peroxidase-conjugated IgG was used as a secondary antibody, and β-actin  was used as a loading control. The immunocomplexes were imaged using an enhanced chemiluminescence detection kit (Amersham Pharmacia) and a ChemiDoc imaging system . The images were quantified with the Quantity One 4.1 software . Each experiment was repeated three times.Phosphate-buffered saline (PBS) was used to wash cells, harvested in 70–200 On-target siRNA specific to ADAM17 (siADAM17) and negative control siRNA (NC-siRNA) were obtained from GenePharma (China). The ADAM17 target sequences are shown in supplemental Table MKK6 (Glu) recombinant adenovirus vector and negative vector were produced by Shanghai GeneChem (China). Their transgenic expression in HaCaT cells was determined using Western blotting.The cell scratch wound assay is a straightforward and economical method to study keratinocyte migration collectively in vitro . HaCaT c5/ml. Twenty-four-well plates received overnight incubation in 1 ml RPMI 1640 media which contained 10% FBS. The keratinocytes were pretreated and exposed to hypoxia. Time-lapse imaging was performed with a Zeiss imaging system  that was equipped with a CO2- and temperature-controlled chamber and acquired the image each at 5 min. The time-lapse images were analyzed using NIH ImageJ software.HaCaT cells were seeded at a density of 0.5 × 103/well in 96-well plates in RPMI 1640 medium supplemented with 10% FBS. Cell proliferation was determined by Cell Counting Kit-8 (CCK-8)  assays according to the manufacturer's instructions. After inoculating the cell suspension in a 96-well plate, the plate was preincubated for the indicated time in a normoxia- or hypoxia-humidified incubator . CCK-8 solution (10 μl) was added to each well of the plate, and then, the plate was incubated for 1 hour. Finally, the absorbance was measured at 450 nm using a microplate reader . The experiment was repeated three times.HaCaT cells were seeded at 2 × 10α) was used. A total of 5 × 103 HaCat cells per well were seeded in a 96-well plate. Twenty-four hours after seeding and then incubating with ADAM17 fluorescently labelled substrate for 30 minutes at 37°C, the relative fluorescence at 520 nm (excitation: 490 nm) was monitored as baseline activity. Afterwards, the cells were treated with an inhibitor and/or stimulant and the activity was monitored at 37°C for one hour. The activity of the unstimulated/stimulated cells was normalized to their baseline activity.For activity assay, a SensoLyte 520 TACE activity assay kit  with the fluorogenic peptide OXLTM 520/5-FAM . In all cases, the data are presented as the mean ± SEM. Supernatant collection was performed in triplicate for each cell line, and the results from the control and vector control keratinocyte cell lines were separated for presentation. The results were analyzed by Student's t-test using GraphPad Prism .To assess the production of ADAM17 substrates, keratinocytes were incubated for different times with medium containing no exogenous EGFR ligands and then collected. To assess TNF-t-test. Statistical was significance set at ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, and statistical analyses were performed using GraphPad Prism software. P < 0.05 was considered statistically significant.All data obtained in the study are expressed as the mean ± SEM. The results were compared by a one-way analysis of variance, followed by the Student-Newman-Keuls 2) [μm2, 42.50 μm2, and 95.63 μm2, which occupied 25.36%, 43.93%, and 91.64% of the scratched area, respectively, while keratinocytes were under a normoxia area over only 14.01 μm2, 24.94 μm2, and 78.75 μm2, which occupied 15.75%, 26.98%, and 80.92%, respectively  . To asseectively . No diffectively . Thus, cectively . These r2) and normoxia (21% O2) for the indicated time. Western blot results found that the mature protein of ADAM17 was increased significantly under hypoxia   [2) compared to that in normoxic condition (21% O2)  . To asse Figures . Transweer cells . The aboGrowing evidence has demonstrated that the ADAM family could be a potential target in wound healing. The ADAM family consists of 22 members; importantly, only ADAM17 is thought to be a key regulator in the repair of skin . After aα or p38-β MAPK attenuated wound healing [α family ligands, which activate EGF receptor signaling [It has been shown that p38/MAPK signaling plays a promotional role in the wound healing process. An in vitro study demonstrated that a mutant in p38- healing . However healing . In addiignaling . Thus, wIn conclusion, our findings demonstrated that ADAM17 expression and activity were promoted by hypoxia through a p38/MAPK signaling, which account for the induced migration of keratinocytes under hypoxic condition. Our study shed new light on the regulatory mechanisms in keratinocyte migration that may help develop a therapeutic strategies to facilitate wound healing in vivo, where guided cell migration in hypoxic microenvironment is needed.


D) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled  antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K In recent years, antibodies have found wide-spread application for diagnostics as well as therapy, and currently constitute the fastest growing group of medication with sales volumes up to USD 100 billion in 2018) )c enzyme ,58. AlthIn summary, the combination of bioaffinity and mass spectrometry technologies bears high potential for the development of “hybrid” tools of affinity and chemical structure determination of biomolecular interactions. Although biosensor technologies alone have principal limitations for providing chemical structure information of protein–ligand interactions, mass spectrometry technologies using selective shielding and protection by proteolytic excision and extraction constitute a broad molecular platform for the analysis of biomolecular interactions. The PROTEX-MS technology has been shown to be well applicable to different types of antibodies ; it is feasible for combination with different biosensor techniques, and can be combined with other techniques, such as selective chemical modification ,76,77,78Myoglobin, native heme protein and apoprotein from equine heart (purity ≥90% by SDS-PAGE) and human MG, essentially salt-free, lyophilized powder were obtained from Sigma-Aldrich, Hamburg, Germany. Stock solutions of MG of 1 mg/mL were prepared in PBS at a 1:40 ratio of enzyme to protein. Interleukin-8 (IL8) and a murine monoclonal antibody (IgG1) from a hybridoma cell line were obtained from Sigma-Aldrich. Human recombinant TNF-α (soluble fraction (77–233)) was obtained from Abcam, Wiesbaden, Germany. Recombinant lysosomal enzyme samples and antibodies were obtained from Sigma-Aldrich and Sanofi-Genzyme, Frankfurt, Germany. Recombinant Human C-Met protein/Fc Chimera was obtained from Sino Biological . The protein comprised the extracellular domain of human C-Met (Met-1 to Thr-932) fused to a C-terminal poly-His-tagged Fc domain of human IgG1.DNA aptamers (CLN0003 and CLN0004) were prepared by IDT Integrated Technologies  .2PO4 containing 35 mM NaCl (pH 7.4) was diluted with 40 µL 100 mM ammonium hydrogen carbonate, and mixed with a 10 mM solution of dithiotreitol . After incubation for 15 min at 95 °C and cooling to 20 °C, 6 µL of 100 mM iodoacetamide (IAA) in water was added and incubated for 60 min in the dark. Subsequently, a 3 µL aliquot of DTT solution (100 mM) was added and the reaction mixture incubated for further 30 min in the dark. Protein reduction and alkylation prior to proteolytic digestion was performed at standard conditions as previously reported ,40,49,60Standard conditions for digestion at atmospheric pressure were employed with a 1 µL aliquot of a solution (0.5 mg/mL) of trypsin  added to an aliquot of 20 µg protein(s), and the reaction mixture incubated for 18 h at 37 °C. High pressure digestion was performed with a Barocycler 2320GTX instrument . Proteins in the high pressure cell were set to undergo pressure cycles of 50 s at 20 kpsi, and subsequent 10 s pressure release to atmospheric pressure. Typically, 90–120 cycles were executed at 37 °C for complete digestion. Prolonged times and increased pressure were applied for proteins showing slow unfolding (such as myoglobins), for which digestion times of 120–400 cycles were used. Epitope determinations using proteolytic extraction with tryptic digestion mixtures were performed both following high pressure and atmospheric pressure digestion. For C-Met protein, proteolytic digestion was performed both at atmospheric pressure conditions and at high pressure. Aliquots of 10 µg C-Met in 15 µL ammonium hydrogen carbonate  were subjected to reduction and alkylation prior to proteolytic digestion, as previously described .Immobilization of antibodies and aptamers on CNBr-activated 4B Sepharose columns was performed according to a similar procedure for all affinity pairs studied. Matrix preparation was performed by incubation in 0.1 M HCl for 15 min, followed by washing with coupling buffer . Samples (30–100 µg of antibody) were incubated by shaking for 2–3 h. Antibody columns were washed alternatively with washing buffer  and blocking buffer . Column preparation was completed by incubation with blocking buffer for 2–18 h.Aptamer immobilization was performed in the same manner as for direct antibody immobilization. The aptamer sequences carried a 5’-amino C6 linker that allows the identical immobilization procedure as for antibodies. Samples of 75 µg of aptamers were immobilized, and affinity columns blocked with 0.2 M Ethanolamine + 0.5 M NaCl, pH 8.3, followed by triplicate washing with washing buffer  and were resuspended in ammonium hydrogen carbonate buffer, pH 7.5. Affinity columns were stored at 4 °C until use.2SO4: H2O2, 2:1). This step was repeated three times, and the cleaned chip incubated overnight at room temperature with shaking in a solution of 16-mercaptohexadodecanoic acid in chloroform and then dried under N2. SPR chip immobilization was performed with an Ametek-Reichert 2Ch7500 SPR instrument . Preparation of activated SAM chips was performed with a mixture of 200 mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS), followed by injection of 10 to 50 µg antibody or aptamer in 250 µL 10 mM sodium acetate, pH 5.5. Remaining free NHS reagent was blocked with 1 M ethanolamine. For SPR chip immobilization, chip surfaces were washed three times with alternating water and 70% aqueous ethanol and then immersed for 15 s in Piranha solution (HTwo strategies were used to immobilize antibodies on the chip surface: (i), direct, random immobilization by surface activation with a mixture of EDC (40 mg/mL) and NHS (10 mg/mL). After immobilization of 40 µg antibody, the chip is blocked with 1 M ethanolamine (pH 8.5) in water at 25 µL/min for 10 min; (ii), in a second approach, oriented antibody immobilization is performed by surface activation with EDC (40 mg/mL) and NHS (10 mg/mL) at a flow rate of 25 µL/min for 8 min, followed by addition of 50 µg protein G  immobilized at 25 µL/min for 10 min, and additional capping with 1 M ethanolamine (pH 8.5) for 10 min. Subsequently, 1–5 µg antibody  is incubated with protein-G at 10 µL/min for 25 min. Using the same flow rate, 30 mM dimethylpimelidate (DMP) in 200 mM sodium borate (pH 9) is flushed over the antibody–protein G complex, and capping performed with 1 M ethanolamine (pH 8.5).4 and 150 mM NaCl, pH 7.5) for 10 min. Patients sera upon ERT were stored refrigerated until use, and centrifuged at 4000× g prior to application. For affinity capture of IgG antibodies, 2.5 mL of serum was injected on the cartridge at a flow rate of 0.5 mL/min. The flow through was monitored at 280 nm with a UV/VIS detector and collected. In order to block remaining protein G, 500 µL of diluted human IgG fraction was injected on the column.A POROS G sensor cartridge containing covalently immobilized protein G was also used. In this case, antibodies from patient sera were captured on the column and crosslinked as described above. Prior to epitope extraction, the column was equilibrated with buffer . Standard SPR gold chips (9 × 9 × 0.3 mm) , were coated with self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid. The SPR cell consisting of two channels (one of which containing the immobilized sample) provides the convenient determination of SPR sensorgrams, e.g., using the TraceDrawer 1.7.1 software from Ridgeview Instruments . Epitope determination from affinity columns was performed by proteolytic epitope extraction MS. Following the mass spectrometric characterization of proteolytic digestion mixtures, peptides are incubated with the antibody-bound or aptamer-bound affinity column for 2–3 h. Supernatant unbound peptides are removed with washing buffer and subsequently analyzed by MALDI-MS and compared to the initial digestion mixtures; washing is typically continued until the mass spectra show no residual background signal . Following mass spectrometric background analysis, the affinity columns are subjected to slight acidification (0.01% TFA) and epitope(s) peptide(s) eluted . After re-equilibration with washing buffer, the column is stored at 4 °C. Collected fractions are centrifuged in vacuo (Eppendorf Concentrator) until the volume is reduced to 10–30 µL. MALDI-MS Determination of elution fractions was performed using DHB as a matrix. Direct proteolytic epitope extraction on the SPR chip was performed with running buffer set to 50 mM ammonium bicarbonate buffer , and a 20 µg aliquot of the tryptic digestion sample injected over the immobilized antibody/aptamer. Subsequently the chip is flushed with buffer (60–120 min) and fractions collected over a period of 10–20 min. Elution is performed by injection of 0.1% TFA in water, and each fraction concentrated to 10–30 µL prior to MALDI-MS.v/v] acetonitrile: 0.1% TFA in MilliQ-H2O was used. Epitope peptide fractions and synthetic peptides were analyzed with a 20 mg/mL 2,5-DHB matrix solution in 50:50 acetonitrile: 0.1% TFA matrix. Typically, 0.5 µL of sample is spotted on the stainless steel MALDI sample target and mixed on the target with 0.5 µL of matrix. MALDI-TOF-MS was performed with a Bruker Autoflex-III Smartbeam . For protein analyses a 50 mg/mL SDHB matrix solution  in 50:50 [Peptide syntheses (Fmoc-SPPS) were carried out with an automated Applied Biosystems peptide synthesizer ABI-433. Epitope peptides were synthesized on preloaded PS-PHB resins with the corresponding amino acids at the C-terminus. Standard syntheses of 0.1 mmol for each peptide is performed using Fmoc protected amino acids. Side chain protection and removal of peptides from the resin are carried out by incubating the resin for 1 h in 5 mL of 94% TFA (with 3% TIS and 3% water). The crude peptides precipitated into 20 mL diethyl ether are centrifuged and the diethyl ether decanted. Purification of peptides was performed by HPLC using a RP-C18 column (250 × 4.6 mm), and characterization for molecular homogeneity is carried out by ESI-MS and/or MALDI-MS.The 23aa αGal epitope peptide (309–332) and the IL8 epitopes (12–20 and 55–60) were synthesized as described above ,40, whil


Pediococcus acidilactici M76 (PA-M76) for lactic acid fermentation of black raspberry extract (BRE). PA-M76 showed outstanding probiotic properties with high tolerance in acidic GIT environments, broad antimicrobial activity, and high adhesion capability in the intestinal tract of Caenorhabditis elegans. PA-M76 treatment resulted in significant increases of pro-inflammatory cytokine mRNA expression in macrophages, indicating that PA-M76 elicits an effective immune response. When PA-M76 was used for lactic acid fermentation of BRE, an EPS yield of 1.62 g/L was obtained under optimal conditions. Lactic acid fermentation of BRE by PA-M76 did not significantly affect the total anthocyanin and flavonoid content, except for a significant increase in total polyphenol content compared to non-fermented BRE (NfBRE). However, fBRE exhibited increased DPPH radical scavenging activity, linoleic acid peroxidation inhibition rate, and ABTS scavenging activity of fBRE compared to NfBRE. Among the 28 compounds identified in the GC-MS analysis, esters were present as the major groups. The total concentration of volatile compounds was higher in fBRE than that in NfBRE. However, the undesirable flavor of terpenes decreased. PA-M76 might be useful for preparing functionally enhanced fermented beverages with a higher antioxidant activity of EPS and enhanced flavors.This study aimed to determine the probiotic potential of  Although various exopolysaccharides (EPS) have been applied in the food industry as food additives for natural viscosifiers, emulsifiers, stabilizers, texturizers, and mouthfeel agents, they have also received great attention for human health because of their antitumor, anti-ulcer, antioxidant, blood glucose-regulating, and UV radiation-protecting activities . MicroorMany LAB strains considered as probiotics are effective against diverse gastrointestinal (GI) exertions by adhering to the gut mucosa and exerting beneficial effects on human health . For useRubus coreanus Miquel, BR), which is grown worldwide, is dark purple in color. Its natural colorant properties are used as a base for products such as ice-cream and sorbet. BR extract (BRE) can also be mixed with many beverages to enhance flavor and balance sweetness and tartness. However, the demand for BR has increased in other ways because of the expanded nutritional knowledge on anticancer, antibacterial, and antioxidant activities /mL) were incubated at 37 °C for 3 h, and the resistance to low pH was assessed by measuring the survival of bacterial colony counts on MRS agar plates. Bile tolerance was determined by incubating LAB cells in MRS broth containing 0.3, 1.0, and 3.0% (w/v) bile salt  at 37 °C for 24 h. The surviving bacterial cells on MRS agar were enumerated and the survival rate was calculated as the percentage of colonies grown on MRS agar.The in vitro survival ability of PA-M76 under conditions stimulating the GIT was determined using the method described by Oh and Jung (2015), with slight modifications. The active strains grown in MRS were centrifuged  cell-free supernatants of lactobacilli strains were dropped. The diameter of each clear zone was determined after incubation at 37 °C for 12 h.Antimicrobial activity was examined using a paper disc assay according to Chiu et al. (2008). g, 5 min) and washed three times with sterilized PBS (pH 7.2). The cells were resuspended and diluted in sterilized PBS to reach an optical density (OD) of 0.5, at 600 nm (A0), and 1.5 mL of the suspension was mixed with an equal volume of n-hexadecane  in duplicate, followed by thorough vortex-mixing. The phases were allowed to separate for 1 h at room temperature, after which the aqueous phase was carefully removed and absorbance at 600 nm was measured (A1). The surface hydrophobicity (%) was calculated as follows: H% = (1 − A1/A0) × 100. The hydrophobicity was determined in triplicate. Cells with a hydrophobic index greater than 70% were arbitrarily classified as hydrophobic. For the autoaggregation assay, cells were grown for 18 h at 37 °C in MRS broth and washed three times with sterilized PBS (pH 7.2), resuspended, and diluted in sterilized PBS to reach an OD600 of 0.5 (A0). Four-milliliter aliquots of the cell suspensions were vortexed for 10 s, and autoaggregation was determined during 2 h of incubation at room temperature (At) in triplicate. The autoaggregation (%) was calculated as follows: A% = (1 − A1/A0) × 100.Bacterial adhesion to hydrocarbons was determined according to the method described by Rosenberg (1984), with slight modifications as follows. After cultivation in MRS broth at 37 °C for 24 h, the obtained fresh cells were centrifuged II; fem-1(hc17)IV strain was used in this study, which was routinely maintained on nematode growth medium (NGM) plates seeded with E. coli OP50. First, PA-M76 was sub-cultured (~1 × 109 CFU/mL) three times before use. After exposing C. elegans to PA-M76 on NGM plates containing nystatin for 5 days, 10 worms were randomly picked, washed twice with M9 buffer, and placed on brain-heart infusion (BHI) plates containing kanamycin and streptomycin. Then, these plates were exposed to gentamycin (5 μL of a 25 μg/mL solution) for 5 min. Next, the worms were washed three times with M9 buffer and then pulverized using a pestle  in a 1.5 mL Eppendorf tube containing M9 buffer supplemented with 1% Triton X-100. After serial dilution in M9 buffer, the worm lysate was plated on MRS agar (pH 5.0), incubated for 48 h at 37 °C, and then counted for live bacterial cells. The results were compared with those of LGG used as a positive control.Colonization of the testines . The C. 2 at 37 °C in a humidified incubator. For cytokine analysis, heat-treated LAB  were added at concentrations of 1 × 107 CFU/well in RAW 264.7 cells after which the cells were plated at a density of 5 × 105 cells onto a 96-well plate and cultured for 2 h. As a positive control for cell stimulation, 1 μg/mL lipopolysaccharide from E. coli  was also used. After incubation for 24 h, RAW 264.7 cells with heat-killed LAB or LPS were harvested, and the cell culture medium was collected for real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay.The murine macrophage cell line RAW 264.7 was purchased from the Korean Cell Line Bank  and was cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution . The cells were incubated under 5% CO5 cells/well for 2 h and subsequently treated with heat-killed LAB or LPS (1 µg/mL) for a further 24 h. Cell viability was then measured using the EZ-Cytox Assay Kit  according to the manufacturer’s instructions. LPS was used as a positive control.Cells were cultured in 96-well plates at 5 × 10GAPDH), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and IL-12. The primer sequences were as follows (5′-3′): GAPDH , TNF-α , IL-1β , IL-6 , and IL-12 .Following each indicated treatment, total RNA was extracted from stimulated RAW 264.7 cells using the NucleoSpin RNA Kit , and cDNA was synthesized from RNA (2 μg) using the ReverTra Ace qPCR RT Master Mix . PCR was performed using a CFX96 Touch Real-Time PCR Detection System  with the SYBR Green Realtime PCR Master Mix  in a 20 μL reaction volume. The target genes for the PCR were as follows: glyceraldehyde-3-phosphate dehydrogenase (w/v) sugar at room temperature for 30 days at 20 °C. After soaking, the black raspberries were strongly pressed for 30 min. The obtained BRE was immediately diluted up to 20, 30, and 40° Brix with distilled water and then pasteurized for 15 min at 75 °C. The sterilized BRE was adjusted to pH 4–9 with 0.1 M sodium bicarbonate and 0.5 M citric acid . The strain PA-M76 was cultured in MRS broth for 24 h at 37 °C and then collected by centrifugation  at 10,000× g for 15 min and washed twice with sterilized water. PA-M76 (v/v) was inoculated with BRE, and fermentation was conducted at 25–30 °C for 3–15 days. The fermented BRE (fBRE) samples were collected aseptically every 24 h for further analysis. The obtained fBRE was stored at −80 °C until further analysis.Organically grown BR were harvested from Gochang province in South Korea, quickly rinsed with tap water, and soaked in a jar with 40% (w/v), and then the samples were centrifuged at 8000× g for 20 min . Cell biomass was determined by measuring the weight of the precipitated pellet after drying in a hot air oven at 90 °C to a constant weight. Crude EPS were retrieved by ethanol precipitation after mixing three volumes of cold ethanol and stirring vigorously. The precipitated crude EPS formed overnight at 4 °C were collected by centrifugation and dried as described above. The remaining ethanol was dried in a vacuum concentrator. The dry weights of the cell biomass and EPS were expressed in g/L.The obtained fBRE samples (20 mL) were boiled for 20 min to inactivate EPS-degrading enzymes and then quickly cooled to room temperature. Trichloroacetic acid was added to the fBRE samples at a final concentration of 4% (520nm − A700nm) pH 1.0 − (A520nm − A700nm) pH 4.5, MW: molecular weight of cyanidin-3-glucoside = 449.2, ε: molecular absorptivity = 26,900, DF: dilution factor = 50. The total phenolic content in fBRE was determined according to the Folin–Ciocalteu method described by Ju et al. (2009). Gallic acid  was used as a standard and each fBRE was diluted 50-fold with distilled water. Twenty-five microliters of diluted fBRE was transferred to a 1.75 mL tube to which 75 µL of 2 N Folin–Ciocalteu’s phenol reagent  was added, and the mixture was maintained for 5 min at room temperature. This was followed by the addition of 200 µL of 7.5% sodium carbonate  solution and 700 µL of distilled water, and the mixture was incubated for 60 min at room temperature after which the absorbance was measured at 765 nm. The results were expressed as milligrams of gallic acid equivalent (GAE) per gram of fBRE. The total flavonoid content was determined using the modified Meda method (2005). Each fBRE was diluted 50-fold with distilled water. Five hundred microliters of diluted fBRE were transferred to a 1.75 mL tube to which 30 µL of 5% sodium nitrite  was added, and the mixture was incubated at room temperature for 5 min. The pre-reacted sample was added to 30 µL of 10% aluminum chloride , and maintained at room temperature for 6 min. Finally, 200 µL of 1 M sodium hydroxide  was added to the maintained sample after which absorbance was measured at 510 nm. The total flavonoid content was determined using a catechin standard and the results were expressed as milligrams of catechin equivalent (CE) per gram of fBRE.The total anthocyanin content in fBRE was determined using the pH-differential method described by Giusti and Wrolstad (2001) in duplicate, with slight modifications. Briefly, samples were diluted sequentially up to 50-fold with 0.025 M potassium chloride buffer  or 0.4 M sodium acetate  solution, and the absorbance of the mixture was measured at 530 and 700 nm using a UV-Vis spectrometer. The difference in absorbance between pH values and wavelengths was calculated (mg/L) using the following formula: (A × MW × DF × 1000)/(ε × 1), A: Absorbance of the diluted sample =  as a free radical. Briefly, a 0.2 mM DPPH solution was prepared with methanol. Each sample was diluted with distilled water and then 100 µL of the diluted sample was mixed with 400 µL of prepared DPPH solution in a 1.75 mL tube. The mixture was covered with aluminum foil to protect it from light and maintained at 37 °C for 30 min. After the reaction, the absorbance of the resulting solution was measured at 517 nm against a blank. Ascorbic acid was used as a standard, and the percentage of DPPH quenched was calculated every minute as follows: % DPPH quenched = [1 − (Abs (sample)/(Abs (control) × 100].v/v) ethanol, 0.1 mL of 30% (w/v) ammonium thiocyanate, and 0.1 mL of 0.02 M ferrous chloride, dissolved in 1 M HCl. After 3 min, the absorbance of the oxidized samples was measured spectrophotometrically at 500 nm. Ascorbic acid was used as the standard and the inhibition degree of linoleic acid autoxidation percentage was calculated as follows: [(100 − sample Abs)/negative control Abs] × 100.The lipid peroxidation inhibitory activity of BRE and fBRE was also measured according to the method described by Osawa and Namiki , with so+) scavenging capacity of BRE and fBRE was measured using the Antioxidant Assay Kit CS0790 , following the manufacturer’s instructions. Trolox  was used as the antioxidant standard. The scavenging activity was expressed as the Trolox equivalent.The radical cation   were added to 20 mL glass vials . The SPME fiber needle was inserted into the vial, the volatile components were adsorbed on the autosampler for 30 min, and desorption was conducted using a GC-MS  injection port at 220 °C for 5 min. Divinylbenzene/carboxen/polydimethy siloxanelsiloxane  was used as the SPME fiber. Each adsorbed volatile compound was analyzed using an HP-5 MS column . The mobile phase gas was helium, and the flow rate was maintained at 1.0 mL/min. The oven temperature was maintained at 40 °C for 1 min and then increased to 250 °C at a rate of 20 °C/min and maintained for 3 min. The inlet temperature was 220 °C, and GC-MS was performed in the splitless mode with an MS ionization voltage of 70 eV, a source temperature of 230 °C, and an interface temperature of 280 °C. Mass spectra of each peak component separated by GC-MS were confirmed with those of the Mass Hunter database library . The volatile components were quantified by comparing the peak area of 2-methyl-3-heptanone and the peak area of the volatile flavor component identified.p < 0.05.Data are expressed as the mean ± standard deviation (SD). Statistical significance was determined by one-way analysis of variance (ANOVA) using SPSS software version 21 , followed by Duncan’s test. Differences were considered statistically significant at p < 0.05) after 3 h of incubation at 37 °C under acidic conditions (pH 2.5), but showed a survival rate of 71.7%. The level was similar to the survival levels of PS and LGG, in which the control and reference probiotic strains exhibited survival percentages >70% 3 h after exposure to simulated intestinal juice (p > 0.05). Considering that the low pH of 1.5 to 3.0 in the human stomach may increase to 4.5 during ingestion, PA-M76 may achieve a higher survival ability under low pH in the stomach. Compared to our strain, other Pediococcus strains could retain their ability at pH 3.0–4.0, but strains displayed high viability loss at lower pH. Furthermore, PA-M76 strains were highly resistant to bile stress. No differences in the cell counts were observed when PA-M76 strains were incubated for 24 h with bile salts ranging from 0.3 to 3.0% (p < 0.05). The human intestine contains the relevant physiological concentrations of bile salts, ranging from 0.3 to 0.5%, and bile salts are harmful to living cells because they damage the structure of the cell membrane [To affect the GI, any potential probiotic strain should exhibit tolerance to acid and bile salts. After being subjected to gastric acidity (low pH) and intestine conditions , the ability of probiotic strains to survive in adequate numbers is important for food industry applications . In this to 3.0% . In contmembrane . In the membrane , those wP. acidilactici M76 in simulated gastric juices and bile salt. Different small letters (a–c) in the same column indicate significant differences of values among three different LAB strains (p < 0.05). PA-M76, P. acidilactici M76; PP-33316, P. pentosaceus (ATCC 33316); LGG, Lactobacillus rhamnosus GG (ATCC 53103). Viable cell numbers were counted on MRS agar after incubation at 37 °C for 48 h.Regarding results of the tests for pH, temperature, NaCl and glucose tolerance, numbers indicates that the test strain can grow on the MRS broth with the respective conditions. Survival rates (%) of p < 0.05), whereas PP showed lower hydrophobicity and autoaggregation compared to the other two strains. Recently, as an alternative to in vitro models, such as propagated human intestinal cell lines, a C. elegans surrogate in vivo model was successfully used as a simple, rapid, and economic model system to study bacteria–host interactions in the gut, as the intestinal cells of C. elegans are similar in structure to those in humans [C. elegans model system was used to investigate the ability of PA-M76 to attach to the intestinal tract. As shown in C. elegans intestine with a cell count of over 5.0 log CFU/mL per worm for 5 days. Unexpectedly, LGG, used as the control strain in this study, showed poor attachment in the gut environment at 1 CFU/mL per worm, although LGG has been shown to bind to enterocytes in a previous study [Lactobacillus sp. were selected as potential probiotic bacteria among 2000 LAB strains isolated from infant feces because of their remarkably high colonization ability on the C. elegans intestine of over 4.3 CFU/mL per worm after 5 days [Pediococcus strains exhibited relatively high GIT colonization (>3.5 log CFU/mL per worm), whereas other Lactobacillus, Streptococcus, and Weissella sp. could not colonize the intestinal tract of C. elegans [The ability to adhere to the intestinal epithelial surface and colonize the GIT is one of the main desirable characteristics of probiotics . To deten humans . In thisus study . Four st elegans . Moreove elegans .IL-1β, IL-6, IL-12, and TNF-α, which may be produced by the activated macrophages, were determined qRT-PCR. Our results showed that the production of four cytokines in RAW 264.7 cells was significantly increased by treatment with LAB strains . Additionally, the expression of TNF-α by PA-M76 was almost similar to that by LGG. To date, various probiotic LAB strains have been shown to enhance nonspecific cellular immune responses, including activation of macrophages, natural killer (NK) cells, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines [Lactobacillus and Bifidobacterium sp., but only a few have reported the immunomodulatory activities of Pediococcus sp., such as P. pentosaceus NB-17, originated from Japanese traditional vegetable pickles [P. parvulus 2.6, isolated from cider [P. pentosaceus OZF, derived from human breast milk [P. pentosaceus L1, isolated from Chinese fermented vegetables [P. acidilactici. Our study showed that the treatment of PA-M76 induced the expression of cytokine genes in RAW 264.7 cells, and, to our knowledge, this is the first study to report the effects of P. acidilactici on immunomodulatory function.Intestinal macrophages are key players in mucosal immune responses to defense against injurious agents, such as pathogens and food antigens. Some LAB strains can activate innate immune cells by inducing or enhancing cytokine production . Probiot strains . Further pickles , P. parvom cider , P. pentast milk , and P. getables . MoreovePediococcus species, including Lactococcus, Leuconostoc species, Lactobacillus kefir, L. brevis L. fermentum, and Bifidobacterium bifidum, showed an almost 50% higher EPS production rate when the organisms were grown at a lower temperature of 25 °C than at 30 °C [p < 0.05). EPS production tended to decline after 72 h, suggesting enzymatic degradation caused by prolonged cultivation, but it was still maintained up to 1.04–1.08 g/L until the final stage of BRE fermentation. The cell biomass was highest at the initial stages on days 3 and 15 , but EPS production was highest on day 3 (1.62 g/L). As a result, maximum EPS production by PA-M76 was observed after three days of BRE fermentation. It seemed that extending the fermentation time resulted in decreased EPS content due to increased EPS degradation activity. To our knowledge, the volume of EPS produced under these optimized conditions in BRE is comparatively sufficient to prepare functional beverages because of its observed proliferative effect on RIN-m5F cells, cytoprotective activity against alloxan-induced cytotoxicity at 10 mg/mL concentration, and lipid-lowering effect at 10 mg/mL concentration. In conclusion, maximum EPS production was achieved up to 1.62 g/L under optimal conditions at pH 4.0, at 25 °C for 3 days with 30°Brix of BRE. This volume of EPS produced under the optimized conditions in fBRE would be sufficient to produce functionally active beverages with health-promoting effects.The BRE did not allow the growth of PA-M76 at a low initial inoculation cell density. The initial inoculation cell density of PA-M76 increased up to 8.2 log CFU/mL, which was maintained during fermentation (data not shown here). As shown in at 30 °C . The celat 30 °C . In thisp < 0.05) difference between fBRE and (99.9 mg CE/100 g) versus 81 mg CE/100 g in NfBRE and fBRE, respectively). However, a significant (p < 0.05) increase was only found at TP (379.3 mg GAE/100 g) compared to NfBRE (349.4 mg GAE/100 g). Lactic acid fermentation of berries is challenging owing to their high contents of acids and phenolic compounds. The abundant phenolic compounds in berries, including hydroxycinnamic, neochlorogenic, and chlorogenic acids, are usually hydrolyzed to quinic and caffeic acids, which are subsequently degraded to vinyl and ethyl catechols via the action of phenolic acid decarboxylases and reductase [L. plantarum, L. paracasei, and L. acidophilus showed an increase in the content of phenolic acids, total anthocyanins, and flavanols [We evaluated the contents of phenolic compounds such as total anthocyanins (TA), total polyphenols (TP), and total flavonoids (TF), of fBRE fermented under the optimized conditions with high EPS production, as described above. As shown in eductase . Howeverlavanols . The effP. acidilactici M76. First, the antioxidant activity was assayed by DPPH radical scavenging activity using the oxidant sp < 0.05) towards the stable radical DPPH, as shown in p < 0.05) was significantly higher than that of NfBRE, as shown in Three different methods were used to assay the antioxidant activity in vitro with NfBRE and fBRE after fermentation with During fermentation with PA-M76, a total of 28 volatile compounds were identified, as shown in To understand the interrelationships between volatile flavor compositions and fBRE depending on the fermentation stage, PCA analyses were performed. As shown in C. elegans intestinal tract in higher populations and exhibiting significant in vitro immunostimulatory activity compared to the conventional commercial starter LGG. The results of the current investigation revealed promising perspectives for the application of functional EPS-producing PA-M76 as a functional probiotic. Moreover, we also applied the EPS-producing strain PA-M76 to prepare functionally enhanced black raspberry beverages and showed how the antioxidant properties of black raspberry extract could be enhanced through lactic acid fermentation by this strain. Considering the great interest in natural materials with functional ingredients, the health-promoting effects of PA-M76 can be exploited for the bioprocessing of diverse food materials in the food industry.In this study, functional EPS-producing PA-M76 was shown to resist biological barriers, such as simulated gastric and pancreatic juices, particularly colonizing the surface of the


Other forms of experience-dependent plasticity, including stimulus selective response potentiation, were normal in MMP9−/− mice. Thus, MMP9 activity is dispensable for many forms of activity-dependent plasticity in the mouse visual system, but is obligatory for the plasticity engaged by cMD.The deletion of matrix metalloproteinase MMP9 is combined here with chronic monocular deprivation (cMD) to identify the contributions of this proteinase to plasticity in the visual system. Calcium imaging of supragranular neurons of the binocular region of primary visual cortex (V1b) of wild-type mice revealed that cMD initiated at eye opening significantly decreased the strength of deprived-eye visual responses to all stimulus contrasts and spatial frequencies. cMD did not change the selectivity of V1b neurons for the spatial frequency, but orientation selectivity was higher in low spatial frequency-tuned neurons, and orientation and direction selectivity were lower in high spatial frequency-tuned neurons. Constitutive deletion of MMP9 did not impact the stimulus selectivity of V1b neurons, including ocular preference and tuning for spatial frequency, orientation, and direction. However, MMP9 The shift in ocular preference in binocular neurons of the primary visual cortex is the canonical model of receptive field plasticity confined to a postnatal critical period. In mice, the critical period for ocular dominance plasticity peaks during the third postnatal week, after one week of binocular vision. During the critical period, MD induces an early decrease in excitatory drive onto parvalbumin-expressing interneurons, which is permissive for subsequent structural and functional changes in V1b ,3,4. An −/− mice [We have previously shown that the visual deficits induced by cMD can be reversed in adults by dark exposure followed by light reintroduction . Activat−/− mice ,18. Othe−/− mice .MMP9 is of particular interest, as MMP9 activity has been repeatedly shown to couple synaptic activity to structural plasticity in both mature and immature circuits. In the adult hippocampus, theta burst stimulation increases MMP9 activity, and MMP9 inhibition blocks the maintenance of synaptic potentiation and several forms of learning ,21,22,23−/− mice. However, the changes in neuronal response strength and selectivity induced by cMD in wild-type (WT) mice are absent in MMP9−/− mice.Here, we ask if deletion of MMP9 impacts neuronal response properties in binocular and chronically monocularly deprived adults. We find that visual response strength and selectivity is normal in adult MMP913 U/mL, 30 nl, The critical period for ocular dominance plasticity peaks during the third postnatal week in mice, one week after eye opening. To quantify the impact of a long-term asymmetry in the quality of vision across the two eyes initiated immediately after eye opening, we measured visually-evoked calcium transients in layer 2/3 neurons of awake adult C57BL/6J mice (WT) following chronic monocular deprivation from P14 until adulthood . Image acquisition was restricted to 150 to 250 μm from the brain surface to focus on neuronal cell bodies in layer 2/3 in the binocular region of the primary visual cortex (V1b) following targeted delivery of GCaMP6s ,100% contrast) in 12 directions were analyzed to identify the neuronal response preferences in normal-reared (NR) adult (>P90) wild type (WT) subjects B. The met-test, * p = 8.3 × 10−29 for contra, 26 ± 1% for NR, 26 ± 1% for cMD, p = 0.91 for ipsi, 365 and 201 neurons for NR and cMD, respectively, Monocular visual responses (ΔF/F) following cMD revealed the strength of response to the stimulation of the deprived, contralateral eye was significantly reduced but the strength of response to the stimulation of the non-deprived, ipsilateral eye was unchanged  adult (>P90) subjects were comparable to WTs  for > 40% or trials  for WTNR contra, WTNR ipsi, WTcMD deprived, and WTcMD non-deprived eye, respectively  for MMP9−/−NR contra, MMP9−/−NR ipsi, MMP9−/−cMD deprived, and MMP9−/−cMD non-deprived eye, respectively; Neither the genotype nor visual experience impacted the GCaMP6s expression or neuronal visual responsivity. In all cases, calcium imaging was performed in adult mice , at least 3 weeks after the delivery of GCaMP6s to the mouse V1b, following confirmation of high cytosolic expression excluded from the nucleus  − 29, R2 = 0.82) and comparable to NR WT, as predicted ) at contrasts ranging from 20–100%, and high contrast stimuli (100%) at spatial frequencies ranging from 0.05 to 0.4 cpd. In NR adult WTs, the magnitudes of evoked calcium transients were positively correlated with visual stimulus contrasts (y = 11 ln(x) − 7, R2 = 0.98; p = 0.02, n = 5 subjects; −/− mice, response magnitude and visual stimulus spatial frequency were also negatively correlated (y = −9 ln(x) + 18, R2 = 0.88) and comparable to WTs  + 22, R−/− mice, which was unchanged by cMD  and high SFs . In contrast, in MMP9−/− mice, no change in visual response strength (ΔF/F) was observed in neurons tuned to either low SF . Again, no change in orientation selectivity was observed following cMD in MMP9−/− mice  of WT NR, 15.2 ± 1.8% for RE of MMP9−/− NR, p = 0.49, Student’s t-test, n = 4 subjects, p = 0.74; 15.2 ± 0.9% RE of MMP9−/− NR, 15.3 ± 1.5% for dep of cMD, p = 0.98; Student’s t-test, n = 4 subjects, To examine the impact of experience-dependent development and plasticity upstream of V1, we traced the distribution of LGN axon terminals in the deprived and non-deprived mice. Intraocular injection of fluorophore-conjugated cholera toxin subunit B (CTB) revealed the expected eye-specific segregation in both normal-reared (NR) WT and MMP9−2 for dep, 395.0 ± 49.8/0.01 mm−2 for non, Paired t-test, * p = 0.048, n = 5 subjects 2 for dep, 0.37 ± 0.01 μm2 for non, Paired t-test, p = 0.15; intensity 46.7 ± 7.3 pixel for dep, 53.4 ± 3.8 pixel for non, Paired t-test, p = 0.51; n = 5 subjects; −/− mice .We quantified the number and size of the vesicular glutamate transporter (VGluT2) puncta in thalamic axons to assess the impact of cMD on the feedforward input from dLGN to layer 4 . cMD sig−/− mice. The analysis was limited to dendritic segments 75–100 microns from the pyramidal neuron cell body  in mouse V1, via a mechanism that is distinct from that engaged by monocular deprivation [p = 0.04, Student’s t-test, novel: 103.7 ± 8.4% of initial, p = 0.36, Student’s t-test, n = 6 subjects; p = 0.03, novel: 108.7 ± 5.8% of initial, p = 0.16, Student’s t-test, n = 5 subjects; −/− mice retained the ability to express robust SRP.Finally, we asked in the visual system of MMP9rivation ,49. Acco−/− mice, including eye specific segregation of RGC axons in the dLGN, the expression and maintenance of contralateral bias of visually-evoked responses, and robust stimulus selective response potentiation induced by repetitive visual stimulation. Thus, MMP9 is obligatory for the plasticity engaged by cMD at eye-opening, but is dispensable for many other forms of plasticity that refine and maintain the mouse visual system.We demonstrate that manipulations of postnatal visual experience disassociate MMP9-dependent and -independent forms of plasticity in the murine visual system. In WT mice, chronic monocular deprivation from eye opening to adulthood reduces the strength of deprived eye visually-evoked responses to all stimulus spatial frequencies and contrasts. Furthermore, cMD impacts the neuronal selectivity for orientation and direction, but not spatial frequency. Constitutive deletion of MMP9 did not impact the stimulus selectivity of V1b neurons, including ocular preference and tuning for spatial frequency, orientation, and direction. However, cMD did not impact the strength or the tuning of the neuronal responses evoked by deprived eye stimulation. Nonetheless, other forms of activity-dependent plasticity persist in MMP9Changes in visual response strength induced by cMD were limited to deprived eye responses, and therefore differed from the response to MD at the peak of the critical period, in which deprived eye depression is followed by an increase in the strength and selectivity of non-deprived eye responses ,35,50,51Tuning for SF is broader than tuning for orientation and direction ,57. AlthThe characterization of neuronal responses in mouse V1b as binocular has recently come under scrutiny ,60,61,62Although MMP9 activity is confined to excitatory synapses, it is differentially expressed across synapse classes in adult mouse V1b. The baseline MMP9 activity is high at cortico-cortical relative to thalamo-cortical synapses, which may contribute to differences in the rates of dendritic spine turnover ,69. Impo−/− mice) are normalized by MMP9 deletion [−/−, dendritic spine density is reduced and extant spines have a normal morphology [−/− mice, suggesting that visual experience may promote MMP9-sensitive synaptogenesis. Furthermore, in adult mouse V1b, levels of the MMP9 substrate aggrecan are decreased by dark exposure and subsequent light reintroduction, and blocked by the pharmacological inhibition of MMP9 [−/− mice [The targets of MMP9 that mediate the response to manipulations of visual experience have yet to be defined, but include a number of perisynaptic extracellular molecules that regulate synaptic plasticity ,70,71. Pdeletion ,77. In Mrphology ,47, sugg of MMP9 ,78. Simi−/− mice [−/− mice. Furthermore, cMD induced a significant decrease in the number of thalamocortical afferents on layer 4 pyramidal neuron cell bodies in WTs but not MMP9−/− mice.MMP9 expression peaks at P0 ,80, none−/− mice ,82. Stim−/− mice demonstrates that the underlying experience-dependent strengthening of contralateral-eye responses is independent of MMP9 activity [−/− mice [−/− mice to chronic MD initiated at eye opening suggests that the visual system cannot compensate for the absence of MMP9, and is consistent with the observation that MMP2 activity is not elevated in MMP9−/− mice [The presence of normal contralateral bias of neurons in V1b of MMP9activity ,61, consactivity ,83,84,85−/− mice ,49,86. T−/− mice ,87.−/− mice. Together, this suggests that MMP9 is obligatory for the plasticity engaged by cMD from eye opening, but it is dispensable for other forms of plasticity that refine and maintain the mouse visual system.The contribution of MMP9 to specific forms of synaptic plasticity is predicted to emerge with age, in parallel with the maturation of critical MMP9 substrates. Interestingly, deprived-eye depression in response to MD during the CP persisted in the presence of an MMP9 inhibitor, but the subsequent non-deprived eye strengthening was absent . Non-dep−/−  mice were purchased from Jackson Laboratory . Equal numbers of adult  males and females were used. The animals were raised in a 12 h light/dark cycle. All procedures conformed to the guidelines of the University of Maryland Institutional Animal Care and Use Committee. Experiments were performed (or subjects were sacrificed) 6 h into the light phase of a 12:12 h light/dark cycle.C57BL/6J and MMP92. The margins of the upper and lower lids of one eye were trimmed and sutured together using a 5-0 suture kit with polyglycolic acid . Subjects were returned to their home cage after recovery at 37 °C for 1–2 h. Subjects were disqualified in the event of suture opening.Chronic monocular deprivation was performed at eye opening (P14). Subjects were anesthetized with 2.5% isoflurane in 100% O2 and perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. The brain was post-fixed in 4% PFA for 24 h, followed by 30% sucrose for 24 h, and cryo-protectant solution for 24 h  ethylene glycol, 3 mM sodium azide, 0.64 M sodium phosphate, pH 7.4). Coronal sections (40 μm) were cut on a Leica freezing microtome (Model SM 2000R). Sections were blocked with 4% normal goat serum (NGS) containing 0.4% TritonX-100 and 0.1% Tween-20 in 1X PBS for 1 h. The primary antibody mouse anti-vesicular glutamate transporter 2  was presented in a blocking solution for 18 h, followed by the secondary antibody goat anti-mouse IgG Alexa-488 conjugated . Images were acquired on a Zeiss LSM 710 confocal microscope with a 40× lens . VGluT2 puncta were analyzed using a single Z-section image after the threshold function (auto threshold + 20) was applied, and were then identified by size exclusion (0.15–2.0 μm2) using the “analyze particles” function in Fiji (NIH).Subjects were anesthetized with 4% isoflurane in OGolgi staining was performed with FD Rapid GolgiStain Kit , as per the manufacturer’s instructions. The brains were immersed in solution A + B for 7 days at room temperature, and then transferred to solution C for 3 days at 4 °C. Coronal sections (100 μm) were made with a Leica VT100S vibrating microtome and mounted on gelatin-coated slides (FD Neuro Technologies). Neurolucida  with an Olympus BX61 light microscope was used to trace the morphologies of the dendrites and spines. Using a 40× lens , the arbors of basolateral dendrites of layer 4 neurons were traced, followed by sholl analysis in a Neuroexplorer  to identify the region 75 to 100 μm from the soma, which were then traced using a 100× lens . Dendritic protrusions with diameters ≥ 50% than the dendritic shaft diameter were classified as spines, and counted in Neuroexplorer.2. Fluorescently-labeled cholera toxin subunit B solutions  were injected into the temporal region of each eye with a Hamilton syringe [v/v) ethylene glycol, 3 mM sodium azide, 0.64 M sodium phosphate, pH 7.4) for 24 h prior to sectioning. Coronal sections (40 μm) were made on a Leica  freezing microtome (Model SM 2000R), and then stained with a nuclear marker, DAPI .Subjects were anesthetized with 2.5% isoflurane in 100% O syringe . SubjectConfocal images were acquired on a Zeiss LSM 710 confocal microscope with a 10× lens . Maximal intensity projections (MIP) of z-stack images  were used for the analysis. Three successive sections through the middle of dLGN were used to quantify the areas of contra- and ipsi-lateral projection defined by fluorescence thresholding (autothreshold + 20) in Fiji. dLGN occupancy was calculated as the ratio of each projection to the total dLGN area.13 U/mL) injected via a Hamilton syringe attached to a Microsyringe Pump Controller (World Precision Instruments) at a rate of 100 nl/min with a total volume of 30 nl.For two-photon calcium imaging, GCaMP6s was expressed in V1b  using adeno-associated virus (AAV) purchased from Addgene  to a 5 mm diameter coverslip) was implanted following a method described by Goldey et al. . The gap0) for 40% or more of trials during either contra or ipsi eye stimulation, the neuron was defined as visually responsive. If this criterion was met in response to stimulation of both eyes, the neuron was designated binocular. Using this criterion, the majority of visually-responsive neurons were binocular in both genotypes. Raising the threshold for the definition of visually responsive did not change the % of neurons defined as binocular  controlled by ThorImageLS software with a 16× NA 0.8 water immersion objective lens  was used to acquire the time lapse fluorescence images. A Chameleon Vision Ti: Sapphire laser  was tuned to 940 nm for excitation, and the emitted photons were directed through a 525/50 nm bandpass filter onto a GaAsP photomultiplier tube. The field of view was 370 × 370 μm (512 × 512 pixels) at 150 to 250 μm from the brain surface. The images were acquired at 30 Hz by bidirectional scanning. Before measuring responses to the visual stimuli, the pattern of the GCaMP signal was evaluated to confirm the absence of toxic expression effects . The neuinocular D.2) for 2.5 s interleaved with 2.5 s intensity-matched grey scale) delivered by a 23” display  28 cm in front of the eyes. To measure the strength of the visual responses across contrasts and spatial frequencies, visual stimuli were delivered at a range of contrasts  at 0.05 cycle/degree or a range of spatial frequencies  at 100% contrast to the eye contralateral to the cranial imaging window. Movement artifacts were corrected using the Suite2P package [0)/F0, where F corresponds to the fluorescence intensity at a given time point and F0 corresponds to mean fluorescent intensity during 1 s before the visual stimulus onset.For ocular dominance measurements, subjects received monocular visual stimuli controlled by the PsychToolBox plugin in MATLAB, (random order of five repeats of 12 orientations of square grating drifting at 1 Hz) at 0.05 cycle/degree (cpd), 100% contrast  for 40% or more of trials during either contra or ipsi eye stimulation, the neuron was defined as visually responsive. An ocular dominance score (ODS) of visually responsive neurons was defined as (C − I)/(C + I), where C and I represent the ΔF/F values at a preferred direction delivered to the contra and ipsi eye, respectively. ODSs were binned into five categories: 1 = 1.00–0.60, 2 = 0.59–0.20, 3 = 0.19–−0.19, 4 = −0.2–−0.59, and 5 = −0.60–−1.00. Contralateral bias index (CBI) was calculated as {(N1−N5) + (N2−N4) + Ntot}/2Ntot, where Ntot is the total number of neurons and N1-5 are the numbers of neurons in bins 1 through 5, respectively.If a mean ΔF value during visual stimulation at any direction exceeded 3 × STD of baseline (F2) for 2.5 s with 2.5 s intensity-matched grey scale) to the eye contralateral to the imaging window at a range of spatial frequencies . Orientation selectivity and direction selectivity were calculated at the neuron’s peak spatial frequency. The direction vector, dirR, was defined as dirR = (ΣR(θ) * cosθ/ΣR(θ), ΣR(θ) * sinθ/ΣR(θ)), where R(θ) represents the response (ΔF/F) at the stimulation direction, θ. Direction selectivity was calculated using the direction circular variance, dirCV = 1 − |dirR|, in order to detect small differences . The orientation vector, R was defined as R = (ΣR(θ) * cos2θ/ΣR(θ), ΣR(θ) * sin2θ/ΣR(θ)). The orientation selectivity was calculated using the orientation circular variance, CV = 1 − |R|, in order to detect small differences [All visually responsive neurons were analyzed. Responses were acquired to visual stimulation . After recovery of the righting reflex, animals were administered buprenorphine  for post-surgical analgesia and returned to their home cage. Awake, head fixed recordings began more than 7 days after implantation. Activity was evoked through passive viewing of 200 × 1 s trials of square-wave gratings , via MATLAB with Psychtoolbox extensions [2) for spontaneous responses. The initial visual stimulation was presented for 200 s at a single orientation, then 24 h later, the familiar and novel stimuli were presented for 200 s, respectively [Adult mice were anesthetized with 2.5% isoflurane in 100% Otensions ,96, and ectively . t-test and Mann−Whitney test were used to determine the significance between two independent experimental groups, and a paired Student’s t-test was used for two measurements within the same subjects. A one-way ANOVA was used to determine significance between three or more independent experimental groups, as well as repeated measures ANOVA for more than two measures within the same subjects, followed by a Tukey−Kramer post hoc when ANOVA was p < 0.05 (JASP). A Kolmogorov−Smirnov (KS) test was used to determine the significance between the distribution of two independent data sets.An unpaired two-tailed Student’s −/− mice were also completely insensitive to plasticity engaged by cMD. Other forms of experience-dependent plasticity, including stimulus selective response potentiation, were normal in MMP9−/− mice. Thus, the MMP9 activity was dispensable for many aspects of maturation of the visual system, but was obligatory for the plasticity engaged by cMD.The aim of this study was to identify the contributions of the matrix metalloproteinase MMP9 to postnatal experience-dependent plasticity in the murine visual system. Chronic monocular deprivation initiated at eye opening significantly decreased the strength of the deprived-eye visual responses to all stimulus contrasts and spatial frequencies. Constitutive deletion of MMP9 did not impact the stimulus selectivity of V1b neurons, including ocular preference, as well as tuning for spatial frequency, orientation, and direction. MMP9


DTR/+ mice and treatment with the CSF1R antagonist BLZ945) had no effect on the pattern of GER regression. Together, these findings suggest that macrophages are recruited into the GER region after initiation of developmental PCD, but that they are not essential for GER regression during cochlear remodeling.Programmed cell death (PCD) plays a critical role in the development and maturation of the cochlea. Significant remodeling occurs among cells of the greater epithelial ridge (GER) of Kölliker’s organ, leading to tissue regression and formation of the inner sulcus. In mice, this event normally occurs between postnatal days 5–15 (P5-15) and is regulated by thyroid hormone (T3). During this developmental time period, the cochlea also contains a large population of macrophages. Macrophages are frequently involved in the phagocytic clearance of dead cells, both during development and after injury, but the role of macrophages in the developing cochlea is unknown. This study examined the link between developmental cell death in the GER and the recruitment of macrophages into this region. Cell death in the basal GER begins at P5 and enhanced numbers of macrophages were observed at P7. This pattern of macrophage recruitment was unchanged in mice that were genetically deficient for CX3CR1, the receptor for fractalkine (a known macrophage chemoattractant). We found that injection of T3 at P0 and P1 caused GER cell death to begin at P3, and this premature PCD was accompanied by earlier recruitment of macrophages. We further found that depletion of macrophages from the developing cochlea (using CX3CR1 The cochlea of the inner ear detects sound vibrations and transmits information to the auditory brainstem. Cochlear development is a complex process, and any defects in developmental patterning can lead to congenital hearing loss . The devβ gene (THRB) have been linked to hearing loss (l-thyronine) at P0 and P1 induces premature GER remodeling  mice with wild type C57BL/6 mice. Similarly, we generated CX3CR1DTR/+ mice by breeding CX3CR1-null (CX3CR1DTR/DTR) mice with wild type C57BL/6 mice. All mice were housed in the animal facilities at Washington University, School of Medicine, and were maintained on a 12-h/day-night light cycle with open access to food and water. All experimental protocols involving animals were approved by the Animal Studies Committee of the Washington University School of Medicine, in Saint Louis, MO.DTR mice , of bothpromoter . We geneTreatment: At P0 and P1, mouse pups were subcutaneously injected with 1.5 μg of T3  (10 μL) or saline (control), in order to induce premature apoptosis and GER remodeling  and CX3CR1-DTR mice (DTR heterozygous) was performed by Transnetyx , following previously described methods , combined with mouse anti-Neurofilament  antibodies, and apoptotic cells were labeled using rabbit anti-cleaved caspase-3 antibody . To avoid non-specific binding of the antibodies, samples were preincubated in a blocking solution consisting of 5% normal horse serum/0.2% Triton X-100 in PBS for 1 h at room temperature. Samples were then incubated overnight with primary antibodies prepared in PBS with 2% normal horse serum and 0.2% Triton X-100 at room temperature. Samples were washed 3x for 5 min each in PBS, pH 7.4 at room temperature and then labeled with secondary antibodies, conjugated with Alexa-488, Alexa-568 or 555, and Alexa 647, . Secondaries were prepared in PBS with 2% normal horse serum and 0.2% Triton X-100. Samples were incubated in secondaries for 2–3 h at room temperature. The secondary solution also contained DAPI, in order to counterstain cell nuclei . All samples were washed 3x for 5 min each in PBS, pH 7.4 at room temperature, mounted on glass slides in glycerol: PBS (9:1), and coverslipped.Animals were euthanized either by quick decapitation or deep anesthesia . Temporal bones were isolated and fixed overnight with 4% paraformaldehyde (in PBS) at 4°C. After fixation, cochleae were isolated and washed 3x for 5 min each in PBS, pH 7.4 at room temperature. All specimens, except those collected at P5, were then incubated for ∼24 h in 10% EDTA in PBS, pH 7.4 at room temperature. Specimens were thoroughly washed 3x for 5 min each in PBS, pH 7.4 at room temperature and were then dissected into whole mount samples. Other samples were prepared as frozen mid-modiolar sections (30 µm thickness). Those cochleae were incubated in 10% EDTA in PBS, pH 7.4 at room temperature for 3–5 days and then washed 3x for 5 min each in PBS, pH 7.4 at room temperature. Cochlear whole mounts and mid-modiolar sections were immunolabel for GFP-expressing macrophages , neurons were labeled with mouse monoclonal anti-Cellular imaging and analyses: Fluorescence images were obtained using an LSM 700 confocal microscope (Zeiss). For cochlear whole mounts and mid modiolar sections, Z-series images were obtained using the following objective lenses: ×5 (∼40-micron z-step-size), 20x (1 or 2 -micron z-step-size), or 63x (0.5 or 1.0-micron z-step-size). Images were processed and analyzed using Volocity 3D image analysis software  and Fiji (ImageJ2.0)  and Adobe illustrator CS5.1.Auditory brainstem responses (ABR): The hearing of 1-month old CX3CR1-GFP mice  was assessed by quantifying thresholds for auditory brainstem responses (ABR). Prior studies have shown that CX3CR1GFP/GFP mice have normal ABR thresholds  at a repetition rate of 20 per second. The responses were amplified 100,00x, filtered (100 Hz–3 kHz), and averaged using BioSig software . Stimuli were presented in 5 dB steps from 5 to 100 dB SPL, in decreasing order, to the right ear. At each level, up to 1,000 responses were averaged. The lowest sound level at which a recognizable and reproducible wave 1 and 2 complex was noted, and considered threshold. If hearing threshold was not present at 100 dB SPL , the threshold value was assigned as 100 dB for graphing and statistical purposes.resholds . Mice weMacrophage, pyknotic nuclei and hair cell counts: Quantification was performed from 20X images, using Fiji software (ImageJ2.0) . For counts of macrophages, hair cells and pyknotic nuclei, 2-3 areas from the apical and basal GER regions were selected. The Cell Counter plug-in was used for all counts. Macrophages were identified by strong GFP and CD45 labeling. Pyknotic nuclei were identified by small size and intense DAPI nuclear labeling, and apoptosis was verified by immunolabeling for cleaved caspase-3. All macrophages, pyknotic nuclei and hair cells counts were obtained manually, within the designed area of complete z-stack using Fiji software.Statistical analysis: All data analysis and statistical tests were carried out using GraphPad Prism version 6.0d. Data are presented as mean ± SD. Student’s t-tests or analyses of variance (ANOVA), followed by post hoc tests, were applied as appropriate. Results were considered statistically significant when p < 0.05.GFP/+ mice, which express GFP in macrophages, microglia, monocytes and related cells   and CX3CR1GFP/GFP mice (in which both copies of CX3CR1 had been replaced with the gene for GFP). Cochleae were collected from mice of both genotypes at P5, P7, P10 and P13 and processed as whole mounts. Samples were immunolabeled for GFP , cleaved caspase-3 (to identify apoptotic cells), and stained with DAPI. These specimens displayed similar patterns of cell death and macrophage recruitment to those described above  . CX3CL1 talkine) . To inveed above . Apoptoted above , and proed above . Macrophed above . MacrophGFP/GFP , and theFP mice . These r. Both hypothyroidism and mutation of the thyroid hormone receptor β (THRB) gene delay the onset of GER remodeling and result in subsequent hearing loss . We collected cochleae from both T3-treated and saline-injected (control) mice at various developmental time points. Whole mount specimens were labeled with anti-GFP , and DAPI. Both T3-and saline-treated cochleae showed a base-to-apex pattern of GER remodeling. However, treatment with T3 resulted in a significant increase in cell death at P3, which was followed by early recruitment of macrophages into the GER region at P5 , we observed significantly elevated hearing thresholds in the T3-treated animals, when compared with saline-treated animals  mice. We observed similar patterns of apoptosis and macrophage recruitment in both CX3CR1GFP/GFP and CX3CR1GFP/+ (control) mice  mice , suggestol) mice , and thiIn order to determine the relationship between GER cell death and macrophage recruitment, we studied whether early induction of apoptosis by T3 hormone affected the recruitment of macrophages into the GER. Similar to DTR/+ mice with DT (GFP/+ mice with the CSF1R antagonist BLZ945 (A prior study reported that short-term depletion of macrophages during cochlear development leads to hearing impairment, which was attributed to the presence of excessive glial cells, abnormal myelin formation, and defects in the stria vascularis . The pre with DT , and tret BLZ945 . Similart BLZ945 . We furtt BLZ945 . Similart BLZ945 . RecruitIn conclusion, the results of this study demonstrate that programmed cell death recruits macrophages in the GER of the developing cochlea and that macrophages engage in the phagocytic clearance of dead cells. However, elimination of macrophages does not affect the patterns of cell death and remodeling in the GER. Thus, although the developing cochlea contains a higher density of macrophages than are present in the mature cochlea, macrophages may not play an essential role in the development of the organ of Corti.


The Hippo pathway plays a wide variety of roles in response to stress in the heart. Lats2, a component of the Hippo pathway, is phosphorylated by Mst1/2 and, in turn, phosphorylates YAP, causing inactivation of YAP. Lats2 stimulates apoptosis and negatively affects hypertrophy in cardiomyocytes. However, the role of Lats2 during cardiac stress is poorly understood in vivo. Lats2 is activated in the mouse heart in response to transverse aortic constriction (TAC). We used systemic Lats2 +/- mice to elucidate the role of endogenous Lats2. Cardiac hypertrophy and dysfunction induced by 4 weeks of TAC were attenuated in Lats2 +/- mice, and interstitial fibrosis and apoptosis were suppressed. Although TAC upregulated the Bcl-2 family proapoptotic (Bax and Bak) and anti-apoptotic (Bcl-2 and Bcl-xL) molecules in non-transgenic mice, TAC-induced upregulation of Bax and Bak was alleviated and that of Bcl-2 was enhanced in Lats2 +/- mice. TAC upregulated p53, but this upregulation was abolished in Lats2 +/- mice. Lats2-induced increases in apoptosis and decreases in survival in cardiomyocytes were inhibited by Pifithrin-α, a p53 inhibitor, suggesting that Lats2 stimulates apoptosis via a p53-dependent mechanism. In summary, Lats2 is activated by pressure overload, thereby promoting heart failure by stimulating p53-dependent mechanisms of cell death. Various mechanisms facilitate the progression of heart failure. Among these, cardiomyocyte death reduces the number of functional cardiomyocytes in the adult heart, which in turn plays an essential role in mediating the development of heart failure2.Heart failure remains the number one cause of death in developed countries and the number of patients is increasing, despite the remarkable progress in medical treatment, including effective coronary interventions for acute myocardial infarction patients3. The hippo pathway includes upstream kinases, including Mst1, Mst2, Lats1 and Lats2, and downstream nuclear transcription co-factors, including YAP and TAZ. Mst1 and Mst2 activated by environmental stress and other stimuli induce phosphorylation of Lats1 and Lats2, which in turn induce phosphorylation of YAP and TAZ. When YAP is phosphorylated at Ser127, it exits the nucleus and becomes inactivated. On the other hand, when YAP is unphosphorylated at Ser127, it is localized in the nucleus and stimulates the activity of transcription factors, including TEAD1, which in turn activates cell proliferation and cell survival5.The Hippo pathway is an evolutionarily conserved signaling mechanism that plays an important role in regulating organ size and tumorigenesis8. Lats2 is also activated by cardiac stress and stimulation of Mst1, in turn stimulating apoptosis and inhibiting cardiac hypertrophy10. On the other hand, YAP mediates cardiac hypertrophy, cardiomyocyte cell cycle reentry and cell survival, mediated through interaction with TEAD and FoxO transcription factors14.We have shown previously that Mst1 is activated by cardiac stress, including ischemia/reperfusion in the heart, inducing apoptosis and suppressing autophagy in cardiomyocytes9. Interestingly, cardiac-specific overexpression of dominant-negative Lats2 inhibits cardiomyocyte apoptosis and enhances cardiac hypertrophy in mice in response to pressure overload9. Nevertheless, how inhibition of Lats2 affects cardiac function during chronic pressure overload remains unclear. In addition, dominant negative Lats2 inhibits both Lats1 and Lats29 and protein overexpression could elicit unphysiological effects in the heart.We have shown previously that Lats2 plays an important role in mediating apoptosis and inhibiting hypertrophy in cardiomyocytes in vitroThus, the goals in this study were to examine the effects of a loss of Lats2 function, using systemic Lats2 heterozygous KO (Lats2 +/-) mice, and investigate the role of endogenous Lats2 in cardiac hypertrophy and heart failure during pressure overload.In order to investigate the role of endogenous Lats2, we studied the effects of a loss of Lats2 function. We used systemic heterozygous Lats2 KO (Lats2 +/-) mice because homozygous genetic deletion of Lats2 is embryonic lethal. Left ventricular (LV) weight/body weight (LVW/BW) at baseline did not differ significantly between Lats2 +/- and non-transgenic (NTG) mice at 5 months of age. LVW/BW was significantly greater in Lats2 +/- mice at 8 months of age than in NTG mice  and left ventricular weight/body weight (LVW/BW) were significantly increased after 2 and 4 weeks of TAC. Although there was no significant difference in HW/BW or LV/BW after 2 weeks of TAC between Lats2 +/- and NTG mice, both ratios were significantly smaller in Lats2 +/- mice than in NTG mice after 4 weeks of TAC , a specific p53 inhibitor18. PFT-α inhibited Lats2-induced upregulation of Bax and Bak in cultured cardiomyocytes . Systemic Lats2 KO mice are embryonic lethal, whereas Lats2 +/- mice were born in a Mendelian ratio and their phenotype was apparently normal at 5 months of age. Mice were housed in a temperature-controlled environment within a range of 21–23 °C with a 12-h light/dark cycle and were given free access to water and a standard diet. To collect heart samples, mice were euthanized by cervical dislocation. The numbers of animals used are shown in figure legends. Interventions were performed in a blind fashion. No animals were excluded from analyses. All procedures involving animals were performed in accordance with NIH guidelines and ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357) and protocols (PROTO9999000934 and PROTO201900140) approved by the ethics committee of Rutgers Biomedical and Health Sciences.All genetically modified mouse lines used in this study have been described previously13. Mice (12–14 weeks old) were subcutaneously injected with a small volume of bupivacaine, anesthetized with pentobarbital , and mechanically ventilated. Aortic constriction was performed by ligation of the transverse thoracic aorta between the innominate artery and left common carotid artery with a 27-gauge needle using a 7–0 braided polyester suture. After surgery, mice were allowed to recover in a Thermocare unit, followed by injection with Meloxicam-SR .The method for TAC has been described13. A 13 MHz linear ultrasound transducer was used. For all mouse experiments, data analysis was conducted in a blinded manner.Mice were anesthetized with 2.5% avertin . Echocardiography was performed using ultrasonography 13.Heart specimens were fixed with 4% paraformaldehyde and sectioned at 6 μm thickness. Cardiomyocyte size was evaluated using wheat germ agglutinin (WGA) staining. Fibrosis was evaluated with picrosirius red staining. Quantification of cell size and fibrosis was conducted using wheat germ agglutinin (WGA)-conjugated CF488 (Biotium) staining and the ImageJ program. Cardiomyocyte apoptosis was evaluated with TUNEL staining as described previously13. A cardiomyocyte-rich fraction was obtained by centrifugation through a discontinuous Percoll gradient. Cardiomyocytes were cultured in complete medium containing DMEM/F12 supplemented with 5% horse serum, 4 μg/mL transferrin, 0.7 ng/mL sodium selenite, 2 g/L bovine serum albumin, 3 mM pyruvate, 15 mM HEPES pH 7.1, 100 μM ascorbate, 100 mg/L ampicillin, 5 mg/L linoleic acid, and 100 μM 5-bromo-2’-deoxyuridine (Sigma) overnight. The medium was then changed to normal serum-free DMEM/F12 medium with penicillin/streptomycin. Culture dishes were coated with 0.3% gelatin. For treatment with adenoviruses, after changing the complete medium, Cardiomyocytes were transduced with the indicated adenoviruses in serum-free DMEM/F12 medium. The cell viability was evaluated with CellTiter-Blue (Promega) assays.Primary cultures of neonatal rat ventricular cardiomyocytes were prepared from approximately 100 pooled hearts of both male and female 1-day-old Hsd:WI Wistar rats 12. pBHGloxΔE1,3Cre plasmid was co-transfected with the pDC316 shuttle vector (Microbix) or pDCSilencer vector (Microbix) containing the gene of interest into HEK293 cells using Lipofectamine 2000 (Thermo Fisher).Recombinant adenovirus vectors for Lats2 overexpression were constructed, propagated, and tittered as previously describedCells and hearts were lysed with ice-cold lysis buffer composed of 10 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 50 mM NaF, and 10% glycerol containing protease inhibitor (Sigma-Aldrich) and 1 μM MG-132 (Sigma-Aldrich). The protein concentrations of samples were measured using a BCA protein assay kit (Thermo Fisher). The denatured protein samples (10–30 μg) were analyzed by Western blotting using the indicated antibodies. Signal intensities of bands were quantified with the ImageJ program.18S forward: AGGGGAGAGCGGGTAAGAGA.18S reverse: GGACAGGACTAGGCGGAACA.Bax forward: GAGCGGCTGCTTGTCTGGAT.Bax reverse: CAAGGCAGCAGGAAGCCTCA.Bad forward: AATGGCATCCGGACAAGGAC.Bad reverse: TGTTCCTGCTGGTGGAGGTA.P53 forward: ACAGTCGGATATGAGCATCG.P53 reverse: CCATGGAATTAGGTGACCCT.GAPDH forward: CTCCCACTCTTCCACCTTCG.GAPDH reverse: GCCTCTCTTGCTCAGTGTCC.Bcl2 forward: ATGCCTTTGTGGAACTATATGGC.Bcl2 reverse: GGTATGCACCCAGAGTGATGC.Bcl-xL forward: AGGCGATGAGTTTGAACTGC.Bcl-xL reverse: TGAAGCTGGGATGTTAGATCACT.Lats2 forward: ATTCCTGGTGCGTGTCTGTTT.Lats2 reverse: CTGTTTGGGAGATGGGAGGG.Total RNA was prepared from cultured cardiomyocytes using TRIzol (Thermo Fisher). cDNA was generated using 1,000 ng total RNA and PrimeScript RT master mix (Takara). qPCR was performed using TB Green Premix Ex Taq (Takara). Ribosomal protein 18S was used as an internal control. The oligonucleotide primers used to carry out the qPCRs of 18S, Bax, Bak and p53 and 18S are listed below:23. Cells were plated on 12-well plates and transfected with 1 μg of the indicated luciferase vectors using Fugene 6. Total DNA quantity was kept constant. Six hours after transfection, the cells were washed and transduced with the indicated adenovirus. The cells were lysed with Passive Lysis Buffer, and the transcriptional activity was determined using a luciferase assay system (Promega).p53-luciferase reporter has been describedAntibodies against the following proteins were used: GAPDH ), YAP , pS127-YAP , Lats2 , Bcl-2 , Bcl-xL , Bax , Bak , Bad , p53 , and cardiac troponin T . Anti-Lats2 Ser872 phospho-specific antibody and Anti-Lats2 Thr1041 phospho-specific antibody were obtained from Dr. Hiroshi Nojima’s laboratory (Osaka University).24 and pilot experiments. The effect size in this study was 1–5 with an alpha = 0.05 and power = 0.80. Microsoft Excel 2016 was used for Student’s t tests and GraphPad Prism 8 was used for ANOVA and Kruskal–Wallis tests. All quantified Western blot and cell viability data are shown as values relative to control.All values in graphs are expressed as the mean Supplementary Information 1.Supplementary Information 2.