Introduction: The development of adjacent-segment disease is a recognized consequence of lumbar fusion surgery. Posterior dynamic stabilization, or motion preservation, techniques have been developed which theoretically decrease stress on adjacent segments following fusion. This study presents the experience of using a hybrid dynamic stabilization and fusion construct for degenerative lumbar spine pathology in place of rigid arthrodesis.Methods: A clinical cohort investigation was conducted of 66 consecutive patients  who underwent posterior lumbar instrumentation with the Dynesys Transition Optima (DTO) implant  hybrid dynamic stabilization and fusion system over a 10-year period. The median length of follow-up was five years. DTO consists of pedicle screw fixation coupled to a rigid rod as well as a flexible longitudinal connecting system. All patients had symptoms of back pain and neurogenic claudication refractory to non-surgical treatment. Patients underwent lumbar arthrodesis surgery in which the hybrid system was used for stabilization instead of arthrodesis of the stenotic adjacent level.Results: Indications for DTO instrumentation were primary degenerative disc disease (n = 52) and failed back surgery syndrome (n = 14). The most common dynamically stabilized and fused segments were L3-L4 (n = 37) and L5-S1 (n = 33), respectively. Thirty-eight patients (56%) underwent decompression at the dynamically stabilized level, and 57 patients (86%) had an interbody device placed at the level of arthrodesis. Complications during the follow-up period included a single case of screw breakage and a single case of pseudoarthrosis. Ten patients (15%) subsequently underwent conversion of the dynamic stabilization portion of their DTO instrumentation to rigid spinal arthrodesis.Conclusion: The DTO system represents a novel hybrid dynamic stabilization and fusion construct. This 10-year experience found the device to be highly effective as well as safe. The technique may serve as an alternative to multilevel arthrodesis. Implantation of a motion-preserving dynamic stabilization device immediately adjacent to a fused level instead of extending a rigid construct may reduce the subsequent development of adjacent-segment disease in this patient population. Arthrodesis has been commonly utilized for symptomatic degenerative conditions of the lumbar spine, such as spinal stenosis, degenerative disc disease, and spondylolisthesis. The operation has shown satisfactory clinical results and can be especially therapeutic when coupled with nerve decompression for lower back pain relief . Further® Dynamic Stabilization System , which utilizes pedicle screws interconnected with flexible polyethylene terephthalate (PET) cords and polycarbonate urethane spacers in order to preserve motion in the lumbar spine.Dynamic stabilization has emerged in recent years as a surgical alternative to lumbar spinal fusion for the treatment of spinal stenosis. Although biomechanical models have shown dynamic stabilization to minimize pressures at, as well as adjacent to, the level of spinal instrumentation, the incidence of ASD after dynamic stabilization remains -8. Still® system [The Dynesys Transition Optima™ (DTO) , a unique system, enables hybrid rigid to dynamic stabilization. This transition system allows for arthrodesis of critically unstable vertebrae in combination with the dynamic stabilization of adjacent, moderately degenerated segments . Revisio® system -12. Few ® system . With ovA total of 66 patients, average age 53 years (range: 25-76 years), underwent lumbar spinal surgery with the DTO hybrid system Figures -2. The mAll surgeries were performed by the senior author after obtaining informed consent from each subject. The DTO system was utilized to achieve a solid arthrodesis at the level of rigid instrumentation and was not being used as an adjunct to fusion at the level that was dynamically stabilized. Surgeries were performed with the patient in a prone position under general anesthesia using midline lumbar incisions. As necessary, surgical decompression of the cauda equina and nerve roots was accomplished via discectomy, laminectomy, and/or facetectomy before instrumentation. A laminectomy was performed at the fused level in all cases. A full laminectomy was not performed at all levels of dynamic stabilization. Bilateral facetectomies were only performed in cases in which bilateral stenosis was present. At the level of dynamic stabilization, a discectomy was performed if there was a large disc herniation present.The DTO implant combines the Dynesys Neutralization System and the Optima Spinal System. The Dynesys implant consists of polyethylene-terephthalate (PET) cords, polycarbonate urethane spacers, and titanium alloy pedicle screws. The spacers are placed bilaterally between the pedicle screw heads to withstand compressive loads. The cords are run through the hollow core of the spacers and stabilize the construct by a tensile preload. The Dynesys/DTO system is currently approved by the United States Food and Drug Administration (US FDA) as an adjunct to spinal fusion. Its use for this study was in an off-label manner.Patients are placed in a prone position with care taken to preserve the natural lordosis of the lumbar spine. A standard midline lumbar incision over the spinous processes of the vertebrae is used with a bilateral subperiosteal muscle separation approach. All pedicle screws are placed under real-time fluoroscopy to ensure appropriate placement. Correct screw placement is absolutely necessary for optimal functioning of the Dynesys system. A spacer template is used to determine the correct position of the screws. Pedicle screws are placed lateral to the facet joints to avoid facet disruption. A spacer template is used to determine the correct position of the screws relative to one another, to allow for optimal placement of the spacer device. A pedicle probe is used to create a channel for the screw under fluoroscopic guidance. Every attempt is made to use the largest diameter and longest screw possible according to the patient’s anatomy. The pedicle screw is advanced in a lateral-to-medial trajectory until the head of the screw is at the synovium of the joint.Following pedicle screw placement, a pedicle distance gauge is placed between the screw heads in the center of the holes to measure the appropriate spacer length. The distance (spacer length) is measured with a slight distraction force. The PET cord is then advanced through the first screw with at least 10 mm of cord remaining outside the screw head. The anti-torque instrument is placed onto the screw head. The set-screw driver is applied to the screw head and tightened until the torque-limiting driver snaps. The cord is pushed through the appropriately sized spacer, and the spacer is placed against the first screw head. The cord is then inserted through the second screw. The cord guide is placed on the guide pin and screw, and the cord-tensioning instrument is placed on top of the cord guide. Care is taken to keep the cord, spacer, and screws in absolute proper alignment.The cord-tensioning instrument is used to pull the spacer into proper position. The set-screw is inserted into the cord guide using the set-screw starter. The set-screw driver is attached to the torque-limiting driver and then engaged with the set-screw. While maintaining tension on the cord, the set-screw is tightened until the torque-limiting driver snaps. The procedure is repeated for the contralateral side and can be repeated for adjacent levels if needed. When the system is fully tensioned, the cords are cut, leaving at least 10 mm of cord extending from the screw heads.Dynesys screws (6.25 mm in diameter and 45 to 55 mm in length) were placed under real-time fluoroscopy in a lateral-to-medial trajectory at the base of the transverse process as to avoid facet damage . Longer Instrumented fusion was performed by placing screws bilaterally into the pedicles with a bony autograft between segments. Optima screws (6.0 to 7.0 mm in diameter and 45 to 55 mm in length) were attached posteriorly with a standard 6 mm titanium rod . The DynThe authors independently reviewed the prospectively maintained database of all dynamic stabilization procedures performed between 2005 and 2015 at the University of Pittsburgh Medical Center. Medical records were reviewed for procedure characterizing information, such as underlying diagnoses, prior surgeries, frequencies of interbody fusion and decompression, levels of instrumentation, infectious and non-infectious complications, and subsequent spinal surgeries. Approval for the study was received from the University of Pittsburgh’s institutional review board  required additional lumbar spine surgery after being implanted with DTO. There were 10 patients (15%) who underwent conversion of the dynamic stabilization portion of their DTO instrumentation to rigid spinal arthrodesis for new symptoms or persistence of their preoperative symptomatology. The indications for conversion of dynamic stabilization to fusion are included in Table In the past 20 years, the range of spine stabilization therapies has been augmented by the advent of motion preservation systems that attempt to decrease the incidence of ASD after rigid fixation . These sThe more recent development of the DTO hybrid system can be utilized for the rigid stabilization of multilevel spinal degeneration while allowing for a limited degree of motion in the adjacent dynamically stabilized segments. DTO implantation is a good alternative to rigid fixation in the carefully selected patient, being ideal for those patients with critical spinal instability and adjacent level pathology of lesser severity that requires decompression but not necessarily rigid fixation. Overall, data regarding this hybrid system is limited, but some small retrospective reviews indicate that DTO implants can alleviate back pain with ASD reported in 10.0% and 12.5% of patients at five-year and mean eight-month follow-ups, respectively -14. Lee,There is an abundance of literature regarding traditional, non-hybrid Dynesys instrumentation and its associated complications -27. InfeThe overall incidence of hardware failure was exceedingly low. In the current study, we found only a single case of subsequent screw breakage during follow-up imaging. However, three cases of interbody cage migration were observed on follow-up imaging. This incidence of interbody cage migration is somewhat higher than our rate in standard arthrodesis cases. While a small sample size may reflect the error of undersizing the cage in these three cases, there may also be a relationship to the fact that the rigid rod is directly connected to a motion preservation system. Further monitoring will be required to ensure that the rates of arthrodesis with the DTO system are equivalent to those of standard methods.This study represents a long-term cohort evaluation of a unique hybrid lumbar dynamic stabilization to fusion system. The DTO system was found to be a safe and viable technology that allows for the coupling of arthrodesis with dynamic stabilization at adjacent levels in the lumbar spine. The incidence of wound infection and implant failure is no higher than with conventional arthrodesis systems. There were no cases of dynamic stabilization implant failure or screw loosening. The present series supports the efficacy, safety, and reliability of this hybrid posterior fixation system as an alternative to multilevel lumbar arthrodesis. Careful patient selection is still required for those patients who would benefit from arthrodesis rather than dynamic stabilization at adjacent levels of pathology.


Pollen released by allergenic members of the botanically unrelated families of Asteraceae and Cupressaceae represent potent elicitors of respiratory allergies in regions where these plants are present. As main allergen sources the Asteraceae species ragweed and mugwort, as well as the Cupressaceae species, cypress, mountain cedar, and Japanese cedar have been identified. The major allergens of all species belong to the pectate lyase enzyme family. Thus, we thought to investigate cross-reactivity pattern as well as sensitization capacities of pectate lyase pollen allergens in cohorts from distinct geographic regions.The clinically relevant pectate lyase pollen allergens Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1 were purified from aqueous pollen extracts, and patients´ sensitization pattern of cohorts from Austria, Canada, Italy, and Japan were determined by IgE ELISA and cross-inhibition experiments. Moreover, we performed microarray experiments and established a mouse model of sensitization.In ELISA and ELISA inhibition experiments specific sensitization pattern were discovered for each geographic region, which reflected the natural allergen exposure of the patients. We found significant cross-reactivity within Asteraceae and Cupressaceae pectate lyase pollen allergens, which was however limited between the orders. Animal experiments showed that immunization with Asteraceae allergens mainly induced antibodies reactive within the order, the same was observed for the Cupressaceae allergens. Cross-reactivity between orders was minimal. Moreover, Amb a 1, Art v 6, and Cry j 1 showed in general higher immunogenicity.We could cluster pectate lyase allergens in four categories, Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1, respectively, at which each category has the potential to sensitize predisposed individuals. The sensitization pattern of different cohorts correlated with pollen exposure, which should be considered for future allergy diagnosis and therapy. The complex interplay between the allergen source, the allergen itself, the circumstances of exposure, and host immune factors are some aspects to be considered. For inhalant allergens, the airway mucosa seems to be the logical site of allergen entry; however, alternative models also suggest a very early mode of allergic sensitization for airborne allergens via the penetration of disrupted or injured skin ), t17 (Japanese cedar Cryptomeria japonica), w6 (mugwort Artemisia vulgaris), or ImmunoCAP ISAC nCup a 1 were used  , ethics committee number 106/CE/2005, Rome, Italy; Japanese samples: Research was approved by the ethics committee at Juntendo University, Tokyo, Japan). Informed written consent was obtained from all subjects included in the study.Ambrosia artemisiifolia (formerly A. eliator) (batch number 02051201), Artemisia vulgaris (batch number 010111102), Cupressus arizonica (batch number 054709201), Juniperus ashei (batch number 042009207), and Cryptomeria japonica pollen (batch number 064309701) were purchased from Allergon AB . Cupressaceae pollen extracts were prepared by incubation with 20 mM Tris HCl, pH 7.4–8 over night at 4°C. Extraction of Astereaceae pollen was performed with 100 mM Tris HCl pH 9.5 for 2 hours at room temperature. Extracts were then centrifuged at 4°C for 20 min at 15.000 x g. The centrifugation step was repeated with the collected supernatant. Resulting supernatant was concentrated by ultra-filtration using a 30 kDa MWCO filter .Ambrosia artemisiifolia and Artemisia vulgaris extracts were thereafter directly applied on a 5 ml Q Sepharose column . After loading and washing the column was eluted using a pH followed by a sodium chloride gradient. Fractions containing purified protein were pooled and dialyzed against 20 mM sodium phosphate pH 7.4. Other pollen extracts  were precipitated with ammonium sulfate at 80% saturation and thereafter re-suspended in extraction buffers , followed by size exclusion chromatography using a Superdex 75 10/300 GL . Purity of the proteins was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Pollen extracts were filtered with a 0.45 μm syringe filter . Amino acid analysis was performed using the Pico Tag method  as described .Tryptic digests of purified natural allergens were performed with a ProteoExtract Trypsin Digestion kit . Peptides were separated by reversed phase-capillary HPLC and analyzed with an ESI-QTOF mass spectrometer  .NUNC Maxisorp plates  were coated with allergen (200 ng/well in 50μl of PBS pH 7.4) overnight at 4°C. Plates were blocked with TBS pH 7.4, 0.05% (v/v) Tween, 0.5% (w/v) BSA and incubated with patients’ sera diluted either 1:8 or 1:3, respectively, followed by an incubation step overnight at 4°C. For inhibition experiments, patients’ sera (dilution 1:10 or 1:3) were pre-incubated with 20 μg/ml antigen overnight at 4°C, thereafter 50 μl aliquots were transferred to the antigen-coated ELISA plate and incubated overnight at 4°C. Bound IgE was detected using alkaline phosphatase-conjugated monoclonal anti-human IgE antibodies  incubated for 1h at 37°C and 1h at 4°C. After washing a colorimetric detection was performed using 10 mM 4-nitrophenyl phosphate  as substrate. Plates were measured at OD 405/492 nm. Specific serum IgE levels were determined based on a quantitative sandwich ELISA using goat anti-human IgE  as catching antibody and different concentrations of purified natural human IgE . Values were calculated based on a regression curve obtained with the IgE standard and signals were considered positive when exceeding three times SD of background controls. For inhibition assays, sera were pre-incubated overnight with 20 μg/ml of purified allergen, and results are presented as percentage of inhibition comparing incubated with buffer-treated samples.A/L. Results from ISAC testing were collected during routine diagnostic testing and results used under the ethical committee approvals at IDI-IRCCS, Rome, Italy, ethics committee numbers 106/CE/2005 and 63/CE/2007.Immuno solid-phase allergen chip (ISAC) assays were performed as reported in previous studies . ISAC 105 cells were added to each well and stimulation with 20 μg/ml protein antigen in 100 μl MEM  medium  supplemented with 1% fetal bovine serum (FCS), 2 mM L-glutamine, 250 U/ml penicillin, 0.25 mg/ml streptomycin, 1 mM Na-pyruvate, 50 mM Hepes solution, 1x non-essential amino acids , and 2 μM β-mercaptoethanol (Sigma-Aldrich), at 37°C with 7% CO2. Antigen-specific IL-2 secreting splenic lymphocytes were analyzed by ELISpot assay using appropriate matched-pair antibodies  as described elsewhere [Female BALB/c mice  were purchased at 8 to 10 weeks of age and were used for experiments 7 days after arrival. Animals were immunized subcutaneously with 5 μg allergen adsorbed to Alu-Gel-S  given as two 50 μl injections administered bilaterally in the lumbar regions. Mice were boosted on days 14, 28, and 42. Sera were collected on days 0 and 49. Per group, 5 animals were tested, at total 25 animals were included in the study. No adverse events have been observed. Animal experiments were conducted in accordance with EU guidelines 86/609/EWG and national legal regulations (TVG 2012). This study was approved by the Austrian Ministry of Science, Ref. II/3b (Gentechnik und Tierversuche), permission number BMWF-66.012/0011-II/10b/2010. Additional details on animal welfare are given in the Methods Section of the Online Supporting Information . Antigenlsewhere . Each me4 cells / well were incubated in RPMI 1640, 5% FCS, 4 mM L-glutamine, 2 mM Na-pyruvate, 10 mM Hepes, 100 U/ml penicillin, 0.1 mg/ml streptomycin  and 100 μM β-mercaptoethanol (Sigma-Aldrich), overnight at 37°C with 7% CO2. Thereafter, cells were passively sensitized with murine immune sera at a dilution of 1:20, followed by 1 h incubation at 37°C, 7% CO2. After a washing step with Tyrode´s buffer (Sigma-Aldrich) supplemented with 0.1% w/v NaHCO3, 0.1% bovine serum albumin, pH 7.2 , antigen was added to a final concentration of 300 ng/ml and mediator release was determined by enzymatic cleavage of the fluorogenic substrate 4-methyl-umbelliferyl-N-acetyl-b-glucosaminide (Sigma Aldrich). The measurement was performed at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Relative β-hexosaminidase activity is expressed as a percentage of the total enzyme activity of Triton X-100-treated cells. All experiments were performed in duplicates.IgE triggered mediator release assays were performed using the cell line RBL-2H3 (ATCC number CRL-2256) as described elsewhere [P-values < .05 were considered statistically significant.Statistical analysis was performed with either Graphpad Prism 6 or PASW Statistics 18 using appropriate test systems as indicated directly in the results section and in the figure legends. Five clinically relevant allergenic pectate lyases  were purified from aqueous pollen extracts. Homogeneity of each preparation was verified by SDS-PAGE , proteinThe percentage of IgE positive sera and the mean IgE reactivity to different pectate lyase allergens were determined by ELISA using serum samples from four different geographic regions  . In AustFor inhibition ELISA, sera were pre-incubated with each of the five pectate lyase allergens Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1, respectively. Thereafter, IgE binding of the pre-incubated sera was tested using the allergens, which were found to be the strongest IgE binders in a distinct cohort as determined by direct ELISA experiments . In brieFor the Austrian patients we found that Amb a 1 inhibited IgE binding to coated Amb a 1 by 96.2%, whereas Art v 6 inhibited IgE binding by 18.8%, and the cypress allergens demonstrated very low levels of antibody inhibition (0–4.9%). However, when Art v 6 was coated, Amb a 1 could inhibit IgE binding only by 67.4%, Art v 6 by 90.6%, and the cypress allergens by 8.3–18.5%.For the Canadian cohort we found that only Amb a 1 pre-incubation was able to fully inhibit IgE binding to Amb a 1 (98.8%) whereas all other pectate lyases inhibited IgE binding to Amb a 1 between 0–4.2%. When Art v 6 was coated and the inhibitions were performed with the 5 candidate allergens, we found that Amb a 1 (81.6%) and Art v 6 (97.3%) strongly inhibited IgE binding; inhibitions with Cupressaceae allergens ranged from 1.4–10%.When analyzing the cohorts of Central Italy and Japan, all three members of the cypress pectate lyases were used for coating. For Italian patients we saw that the highly homologous proteins Cup a 1 and Jun a 1 could inhibit IgE binding to themselves by 78.6 and 88.5%, to each other by 70.2 and 83.5%. Cry j 1 inhibited IgE binding to Cup a 1 by 51.8%, Amb a 1 by 41.2%, and Art v 6 by 18.6%. IgE binding to coated Jun a 1 was inhibited by Cry j 1 with 22.4%, by Amb a 1 by 37.9%, and by Art v 6 by 5.2%. The IgE binding to coated Cry j 1 was fully inhibited with Cry j 1 as well as Jun a 1, Cup a 1 inhibition resulted was 87.2%, whereas Amb a 1 and Art v 6 inhibited binding to 13.9 and 16%, respectively. However, Italian sera showed very weak IgE binding to Cry j 1.For Japanese patients the IgE signals towards coated Cup a 1 as well as Jun a 1 were generally very low. Cup a 1 could inhibit IgE binding to itself with 82.3%, Jun a 1 with 59.1%, Cry j 1 with 44.3%, Amb a 1 with 23.4%, and Art v 6 with 12.2%. IgE binding to Jun a 1 was inhibited by Cup a 1 with 72.2%, with Jun a 1 itself by 9.6%, a value that is very likely a result of the extremely low overall IgE binding to this allergen, with Cry j 1 by 28.8%, and between 0–3.9 with Amb a 1 and Art v 6, respectively. The more interesting part was the IgE reactivity towards Cry j 1. Here IgE signals were higher and the Japanese sera showed inhibition rates of 89.4% with Cry j 1 itself, 15.2% with Cup a 1, 1.6% with Jun a 1, 0% with Amb a 1, and 0.6% with Art v 6.Chamaecyparis obtusa), which has been described as highly cross-reactivity with Jun a 1 and Cup a 1 [In summary, we conclude that Canadian patients are sensitized by Amb a 1, which shows cross-reactivity with Art v 6. Austrian patients are sensitized to both Amb a 1 and Art v 6. Patients from Central Italy are sensitized to cypress group 1 allergens, whereas sensitization of Japanese patients occurred mainly via Cry j 1. However, we also found some exclusive IgE to cypress allergens (mainly Cup a 1), which is most likely a result of sensitization to Cha o 1, the pectate lyase allergen from Japanese cypress  of IgE binding only with Cup a 1 and Cry j 1, whereas there was no correlation of IgE reactivity between Amb a 1 and Cup a 1 or Amb a 1 and Cry j 1  allergic patients, all from Italy. The age range was between 1 and 97 years old  and Art v 6 (2.4*104), however the recognition of Cup a 1, Jun a 1, and Cry j 1 was low (titers below 1*102). Art v 6 immunized animals had very high titers against Art v 6 (6*106), but the antibodies showed hardly any cross-reactivity . The murine IgG1 response triggered by Cup a 1 immunization was generally very low. The highest titers were induced against Cup a 1 itself (2.9*103), Jun a 1 (1.8*103), and Cry j 1(7.1*102), whereas almost no reactivity was observed with Amb a 1 and Art v 6 (titers of 31 and 17). Jun a 1 immunization led to a similar picture, the titers against Jun a 1 itself were 5.5*103, followed by 1.6*103 against Cup a 1, and 7.5*102 against Cry j 1. Amb a 1 and Art v 6 showed titers of 12 and 3, which resembles background values. Interestingly, the immunization with Cry j 1 led to high specific IgG levels (5*105), whereas the recognition of all other pectate lyases including Cup a 1 and Jun a 1 was very low (between 32 and 289 titer units). Though, IgG2a levels were low, the IgG2a binding pattern was interesting. The cross-reactivity was low between Astereaceae and Cupressaceae allergens, and also the cross-reactivity between Amb a 1 and Art v 6 was low. However, we found some cross-reactivity within the Cupressaceae allergens. Of note, the allergens which elicited robust IgG1 titers had hardly detectable IgG2a , whereas the allergens with low IgG1 titers had more IgG2a (Cup a 1 and Jun a 1).To further compare cross-reactivity pattern induced by Asteraceae and Cupressaceae allergens, mice were immunized four times by s.c. injection of Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1, respectively, all adsorbed to Alu-Gel-S. Antibody responses and cross-reactivity of IgG  were monBiological active IgE was detected by degranulation of rat basophilic leukemia cells (RBL 2H3) . Sera ofProliferation of splenocytes derived from immunized mice was induced by re-stimulation with pectate lyase allergens . We analDue to their broad distribution, Asteraceae as well as Cupressaceae species are among the most potent elicitors of pollen allergy in many regions including North America, Europe, and vast parts of Asia. Though not botanically related, the major allergens of these plant families have been identified as pectate lyases. In general, the most abundant proteins in allergenic pollen extracts represent the major allergens of that source . For pecTo identify cross-reactive pattern of pectate lyase pollen allergens, ragweed Amb a 1, mugwort Art v 6, cypress Cup a 1, mountain cedar Jun a 1, and Japanese cedar Cry j 1 were purified from aqueous pollen extracts. Sera from patients of four geographic regions  showing different exposure to pectate lyase allergens were collected and used for cross-reactivity profiling. As inclusion criterion for the study typical pollen allergy-associated symptoms and positive specific IgE values to the respective weed or tree pollen extracts and/or purified allergens were defined.www.gbif.org; www.pollenlibrary.com; eol.org) [In Austria, exposure to mugwort as well as ragweed has been reported, while in Canada, ragweed pollen has been described as the main source of pectate lyases allergens. Patients from the southern and central parts of the Italy are primarily exposed to cypress pollen, whereas in Japan, Japanese cedar pollen represents the main source of allergenic pectate lyases (eol.org) , 4, thoueol.org) .Direct ELISA experiments revealed that more Austrian patients reacted with Amb a 1; however mean IgE levels towards Art v 6 were higher than to Amb a 1. This was surprising since almost all Austrian sera had higher CAP values to ragweed than to mugwort, but may be explained by the low levels of Art v 6 in mugwort pollen extracts. Canadian sera reacted strongly with Amb a 1, followed by Art v 6, whereas cross-reactivity with cypress allergens was minimal. Italian sera showed almost equal specific IgE binding to all three Cupressaceae pectate lyases, whereas Japanese sera reacted significantly stronger with Cry j 1 than with any other pectate lyase allergen. These observations were confirmed by inhibition ELISA.For each region the prevalent allergens were used for coating . Thereafter, sera were pre-incubated with each of the allergens and residual IgE binding was determined. For Austrian patients we found that Amb a 1 most efficiently inhibited IgE binding to itself, whereas when Art v 6 was coated, Art v 6 was the most efficient inhibitor. In that case Amb a 1 could inhibit IgE binding to approx. 70%. Cupressaceae pectate lyases showed almost no inhibitory capacities. Therefore, we conclude that within the Austrian population we find both, sensitization to Amb a 1 as well as Art v 6. A recent study demonstrated that Amb a 1 and Art v 6 are widely cross-reactive in an Italian cohort. Both Amb a 1 as well as Art v 6 have the capacity to sensitize patients, and that IgE reactivity to the major mugwort pollen allergen Art v 1 can be used to distinguish between ragweed and mugwort sensitized individuals . We coulFor Canadian sera only Amb a 1 could effectively inhibit IgE binding to coated Amb a 1, whereas the inhibition to coated Art v 6 was almost equal with Art v 6 as well as Amb a 1. Almost no inhibition could be observed with the Cupressaceae pectate lyases. This indicates that Canadian patients are sensitized by Amb a 1 and cross-react to Art v 6.Chamaecyparis obtusa) pollen contains an allergenic pectate lyase (Cha o 1) showing high cross-reactivity with Jun a 1 and Cup a 1 [For Central Italy it has been reported that 62.9% of atopic patients showed sensitization to cypress pollen . We foun Cup a 1 . We founWe further investigated pectate lyase cross-reactivity in a large cohort from Italy, which are mainly exposed to cypress pollen as major allergen source by testing IgE reactivity to Amb a 1, Cup a 1, and Cry j 1. Thereby, we found essentially the same picture as with ELISA experiments. The vast majority of the patients had strong IgE levels against Cup a 1 which were to a certain degree cross-reactive with Cry j 1. Sensitization to Amb a 1 was rare and did not correlate with IgE reactivity to either Cup a 1 or Cry j 1.Despite the extremely low specific IgE levels of Italian patients towards Cry j 1 and Japanese patients towards Cup a 1 we could observe that IgE cross-reactivity was mainly restricted to botanical orders. We did not exclude this data because even the variable inhibition results of the two data sets reflected the picture observed in all the other inhibition ELISA experiments—a clustering of IgE cross-reactivity within plant orders, families, and sub-families.To get a more detailed picture about the sensitizing capacity of each pectate lyase pollen allergen, an animal model of allergic sensitization was established. When looking at IgG1, Amb a 1, Art v 6, and Cry j 1 showed high immunogenicity, whereas Cup a 1 and Jun a 1 were weak immunogens. The cross-reactivity of Amb a 1-induced IgG1 with Art v 6 was high, vice versa it was lower, indicating that antibody epitopes of Amb a 1 and Art v 6 are slightly divergent. Between Cup a 1 and Jun a 1 we found extremely high levels of cross-reactivity, whereas Cry j 1 had a rather independent antibody profile. Of note, despite low immunogenicity at the IgG1 level, Cup a 1 and Jun a 1 were most efficient in inducing allergen-specific IgG2a antibodies. At the IgE level the picture was slightly different. Amb a 1 induced high levels of biologically active IgE levels only against itself. Art v 6-specific IgE were slightly cross-reactive with Amb a 1. Cup a 1 induced cross-reactive IgE with Jun a 1 and—though weaker—this was also observed vice versa, and Cry j 1 induced a very strong IgE response only against itself. In proliferation assays we found essentially the same pattern for cross-reactivity as we saw in ELISA or microarray experiments; however cross-reactivity was limited in general.In summary, we could cluster our pectate lyase allergens in four categories, which were Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1. We found hardly any cross-reactivity between Asteraceae as well as Cupressaceae pectate lyases. However, within the plant orders the allergens were cross-reactive. We could further demonstrate that members of each allergen category can induce sensitization in humans, which raises the question on how to simplify the diagnosis and therapy for pectate lyase sensitized patients. Based on our findings, we consider that it will be necessary to include a member of each of the four categories for effective diagnosis and, considering individual exposure pattern, also for therapeutic applications. In the clinic, the allergenic potential of the minor mugwort pollen allergen Art v 6 seems to be dampened by the low expression levels in pollen extracts.S1 FileTable A. Demographic and diagnostic data of pectate lyase allergic patients included in the study. Table B. Demographic and diagnostic data of Italian pectate lyase allergic patients involved in the microarray survey.(DOCX)Click here for additional data file.


Allergen specific immunotherapy (ASIT) is the only disease modifying treatment of allergic diseases. A key element in the protective effect is thought to be induction of a specific IgG4 response leading to a decrease in the IgE-mediated allergic symptoms. The effect of ASIT can be ascertained by provocation or skin prick testing. IgE measurement by ImmunoCAP is a sensitive standard procedure for allergy diagnosis and this has recently been supplemented by a single allergen array (ISAC), which employs significantly lower amounts of allergen and may have different assay dynamics. 24 subjects suffering from allergic rhinitis due to grass pollen allergy were stratified on basis of sensitivity in a basophil activation test, to receive standard subcutaneous immunotherapy (n=18) or to an open control group (n=6). Allergen specific IgE and IgG4 was measured at baseline and when reaching maintenance dose by using both the quantitative ImmunoCAP for Phl p1 and Phl p5 and the chip-based semi-quantitative ISAC method . Data are presented as mean and 95% CI. ASIT treatment reduced self-reported symptoms from 10,7 to 5,5 . ASIT increased allergen specific IgG4 levels, both by ISAC   and ImmunoCAP . Specific IgE increased from 23,0 to 48,8 kU/l when measured by ImmunoCAP . Surprisingly, ISAC measurement of specific IgE decreased from 21,4 to 2,5 ISU  during updosing. No significant changes in IgG4 or IgE measurements were found in the control group during the treatment period. ImmunoCAP measurements indicate that IgG4 and IgE increase during ASIT updosing as previously reported. In contrast, when IgE in the same serum samples was measured by ISAC a decrease in IgE seems to be the result of successful ASIT. This may be the result of inhibition of IgE-binding in vitro in the ISAC assay, possibly reflecting functional IgE inhibition in vivo in the treated patients. ISAC measurements before and after ASIT may thus be useful in monitoring the clinical effect of allergen specific immunotherapy.


Inhibitory neurons have a large diversity , howeverVasoactive intestinal peptide-positive (VIP+) interneurons are one of the major inhibitory cell types in the cortex , and theSST+ cell mediate inhibition across columns and allow contextual visual processing in V1.We also note that PV+ cell-mediating inhibition across columns is critical for regulating the responses over small spatial scales: e.g. induced by non-preferred stimulus (orientation).


Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S.Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H Connexins (Cxs), a protein family with 21 members in humans, share some important functional and structural characteristics. Thus, Cxs are catalogued as transmembrane proteins, containing four transmembranous domains, two extracellular loops, one cytoplasmic loop and both cytoplasmic N- and C-terminal domains facing the cytoplasm. To constitute a gap junction (GJ) channel, six Cx subunits first assemble into a hemichannel (HC), also known as connexon that docks to another HC located in a neighbouring cell to finally form an intercellular GJ channel , whereas. found a possible phosphorylation site at the Cx26 N-terminal region using HeLa cells as exogenous expression system, but phosphorylation of Cx26 in cells that endogenously express this protein, like in liver cells, has not be detected yet [Cxs have multiple phosphorylation sites, e.g. 21 of these sites have been described for Cx43 , 8. The cted yet , 14. Thected yet , 16.Post-translational phosphorylation of Cxs can regulate channel properties of both HCs and GJs . AdditioThe half-life of Cx43 has been reported to be about 5 h in liver hepatocytes and 1.3 h in the heart , 23. Cx4Gating kinetics of GJ channels can be modulated in a rapid and reversible manner by direct phosphorylation of Cxs, which changes the extent of GJIC. GJs can thereby undergo conformational changes in order to modify channel gating as described in the “ball and chain”, “cork gating” or “particle-receptor” models . The pho2+/calmodulin-dependent kinase II (CaMKII), but also the tyrosine kinase Src is able to phosphorylate Cxs [In general, the phosphorylation of Cxs can occur through serine/threonine kinases or tyrosine kinases. Among the serine/threonine kinases that phosphorylate Cxs are e.g. protein kinase C (PKC), MAP kinase (MAPK), cAMP-dependent protein kinase A (PKA), casein kinase (CK), p34cdc2, protein kinase G (PKG), Calate Cxs , 18. Somlate Cxs . Reporte. have shown that elevated levels of cAMP enhanced the membrane trafficking of Cx43 and could trigger a rapid GJ assembly at the cell surface [. [. have found that Cx43 is a relatively poor substrate of PKA compared to PKC, but the phosphorylation at Ser364 by PKA seems to be important for subsequent phosphorylation of Cx43 by PKC [In a variety of cell populations, an increase of cAMP concentration is correlated with an upregulation of GJIC, but there are also reports showing that cAMP increases had an inhibitory effect or no effects upon GJIC . For Cx4 surface . The incrface [. . The trerface [. . It is trface [. . While Srface [. , Ser365 rface [. . Shah et3 by PKC . In addi3 by PKC . The PKA3 by PKC , 36, 37.. They demonstrated that the dopamine induced uncoupling of AII amacrine cells resulted from PKA activation of protein phosphatase 2A (PPA2) and subsequent dephosphorylation of Cx35 [A negative regulation by PKA was reported for GJs formed by Cx35. In HeLa cells expressing Cx35, the application of a PKA activator reduced GJIC, while a PKA inhibitor increased GJIC . In vitr of Cx35 . These r. demonstrated that lipopolysaccharide (LPS) and hypoxia/reoxygenation (H/R) reduced GJIC of wild-type microvascular endothelial cells but not in Cx40 null cells, and this was associated with a decreased PKA specific phosphorylation of Cx40 [A role for PKA in the regulation of GJIC was also reported for Cx40. Administration of cAMP resulted in a mobility shift of Cx40 on western blot and increased the permeability of GJs formed by Cx40 in transfected SKHep1 cells . Moreove of Cx40 .AKT dependent phosphorylation of Cx43 at Ser373 increased GJ size and communication by preventing the binding of ZO-1 to Cx43 . Phospho. showed that Ser368 phosphorylation by PKC was accompanied by a loss of Cx43 from GJs, an increased ubiquitination of Cx43 and reduced GJIC [., who found an increased localization of Cx43 in the membrane and enhanced GJIC when PKC mediated Ser368 phosphorylation of Cx43 was inhibited [A PKC dependent phosphorylation of Cxs has been shown to affect GJIC in a negative manner decreasing GJ conductance or reducing dye coupling , 46. Theced GJIC . This wanhibited , 53. Thenhibited . They denhibited .The role of Ser368 phosphorylation of Cx43 on the electrical conductance during myocardial ischemia does not appear to be sufficiently clear. A study by Axelsen and colleagues demonstrated that Ser368 became dephosphorylated during ischemia in isolated rat hearts and that was associated with a similar decrease in electrical coupling . A preseThe phosphorylation of Cx32 at Ser233 in a PKC dependent manner has been described in freshly isolated hepatocytes , 37, 57.Cx35 , that is expressed in neurons, can be phosphorylated by PKG . In vitr2+/calmodulin kinase II plays a critical role in the regulation of Ca2+ homeostasis, transcription and apoptosis, as well as in ischemic heart diseases. In cardiac diseases, such as heart failure or myocardial ischemia and infarction, an increased expression or activation of CaMKII has been shown [2+-induced uncoupling of GJs. The GJ inhibition by Ca2+-CaM can be prevented by CaM blockers or CaM antagonists [., in purified liver GJs and in hepatocytes [2+ levels that induced the activation of CaMKII. Therefore, the enhancement of the GJ conductance could be mediated by a phosphorylation of Cx36 by CaMKII [Caen shown . Calmoduagonists . A co-loagonists . Huang aagonists . Dephospagonists . Phosphoagonists . Cx32 caatocytes . An inteatocytes . They idatocytes . An incry CaMKII , 66. Alsy CaMKII . Some ofy CaMKII . It remay CaMKII .. [. reported that Cx45.6  is phosphorylated by CK2 in vitro at Ser363, but not by CK1 and they also observed that this phosphorylation event led to destabilization and degradation of Cx45.6 [Phosphorylation of Cx43 by CK1 at Ser325, Ser328 and Ser330 has been reported to promote GJ assembly . As ment. . On the f Cx45.6 .Cdk5 is involved in developmental processes of the brain. Cdk5 directly phosphorylates Cx43 at Ser279 and Ser282 and thereby preventing the membrane targeting of Cx43 and promoting its proteasome dependent degradation . Cx43 exPhosphorylation of Cxs during cell cycle progression can be regulated by p34cdc2 playing an important role for the initiation of mitosis . The redXenopus oocytes and primary lens epithelial cells [Serine residues 255, 262, 279/282 of Cx43 are target sites for MAPK phosphorylation , 72–74. al cells . In thesal cells .Activation of the oncogene and tyrosine kinase Src leads to acute down-regulation of GJIC . Src can. found a phosphorylation at Tyr243 by the epidermal growth factor receptor tyrosine kinase [Additionally, an indirect influence of Src on GJIC via Src effector kinases has been shown, e.g. for MAPK, PI3K/Akt and PKC signaling pathways . Chronice kinase .Despite the large number of kinases that phosphorylate Cxs, only PKA, PKC, MAPK and Akt activity have been found to regulate the HC activity , 83–88. A few studies have shown that also neuronal HC activity can be controlled by phosphorylation of Cxs. Mitropoulou and colleagues could demonstrate that HCs formed by Cx35 can be regulated by cAMP/PKA. They injected two different fish Cx35 RNAs (from perch and skate) into Xenopus oocytes to investigate dopamine induced PKA signaling on HC functions. The incubation with cAMP caused a dose-dependent inhibition of voltage-activated HC currents only in the presence of perch but not of skate Cx35 . Amino aRecently it has been reported that AKT is capable of regulating HCs, specifically those formed by Cx43 . PreviouXenopus oocytes expressing recombinant Cx43 showed that fluorescent dye (5(6)-carboxyfluorescein) uptake via HCs was increased by different blockers of PKC activity  [It is well documented that inhibition of PKC increases the Cx43-HC activity , 84, 86.ythrine) . The amiythrine) , 83, 84.ythrine) , 83, 84.ythrine) . Other sIn vitro studies showed that MAPK phosphorylated Ser255, Ser262, Ser279, and Ser282 of Cx43 , 74. Sub2S) [2) [Changes in the redox potential are probably the broadest control mechanism of ion channels under physiological and pathological conditions . Several2S) , and the2S) [2) , to our NO is a gaseous transmitter that is formed by the action of an enzyme family known as nitric oxide synthases (NOS) . There a. demonstrated that the increased risk of arrhythmias in response to increased cardiac oxidative stress is associated with a downregulation of Cx43. Oxidative stress triggered the S-nitrosylation of caveolin 1 (Cav1) by enhanced binding of Cav1 to eNOS, which resulted in the dissociation of S-nitrosylated Cav1 from cSrc/Cav1 complexes and subsequent phosphorylation of cSrc. Activated cSrc finally competed with Cx43 and led to a dislocation of Cx43 from ZO-1 disrupting the GJs and increasing their internalization [In the heart, Kirca et al. suggested that reduced mitochondrial Cx43 is associated with a change of NOS isoforms leading to reduced NOS activity and - thereby - to reduced S-nitrosylation of several proteins. However, a direct S-nitrosylation of Cx43 by NO was not shown in this study , but thilization .. demonstrated that NO in the long term increased the insertion of Cx40 into the cell membrane via activation of cGMP/cAMP dependent kinases [Endothelial cells mainly express Cx37, Cx40 and Cx43 . These c kinases  therefor kinases .Because NO is a highly reactive molecule eNOS is expected to be in very close proximity to Cxs present in endothelial cells. This was, in fact, observed for at least Cx37 and Cx40 , 119, anBy the mid-nineties it was shown in astrocytes that NO produced by iNOS after LPS exposure, decreased GJIC in a peroxynitrite anion (ONOO-) dependent manner . Later oIn the case of neurons, seminal experiments performed by Rörig and Sutor, demonstrated that a NO/cGMP dependent mechanism decreased GJIC between neurons within the neocortex . In the The retina is a tissue that enables the transduction of light into information to be processed by the brain . GJs areHCs participate in several physiological and pathological processes . For tha. [Not only Cxs but also pannexin channels can be regulated via NO. An inhibitory effect of NO in a cGMP/PKG dependent way was demonstrated by Poornima et al. . They sh. .. the application of CO donors  to the bath solution inhibited Cx46 HC currents in Xenopus laevis oocytes [50 of about 3.4 μM , making Cx46- and Cx43-HCs to excellent sensors of physiological and pathological changes in CO concentration [CO is a toxic molecule because it binds with high affinity to hemoglobin , 145. Ho oocytes . This efntration . This stntration . So far,2S from L-cysteine: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) [2S reacts with cysteine residues to form SSH or persulfide groups [2S has not been reported yet. However, it has been shown that H2S improved the expression of Cx43 in cardiomyocytes [2S decreases platelet aggregation probably by interfering with GJ channels, at least in part [Three enzymes are responsible for the formation of the gaseous transmitter H (3-MST) . Sulfhyde groups . The modmyocytes , which i in part .Changes of environmental conditions, e.g. the presence of growth factors, cytokines, wounding, ischemic conditions, changes in the redox potential, but also of cellular processes as cell cycle progression often lead to post-translational modifications of Cxs. Thus, to respond to acute changes of environmental or cellular conditions cells have developed an appropriate mechanism to regulate their intercellular communication with neighboring cells or their communication with the extracellular space via HCs. The great amount of excellent studies and reports clearly demonstrate the importance of post-translational changes of Cxs for the regulation of HCs or intact GJ channels. In the case of phosphorylation, similar roles regarding the channel permeability have been described for different Cx types, but there are also some exceptions, too. The effect on channel permeability of HCs is consistent with that of GJs after phosphorylation of Cxs. However, the effect of NO on GJ channels or on HCs is not unitary. Therefore, it is not possible to establish a common pattern of action of NO on Cx HCs or GJ channels. Based on these reports it has been shown that not only the Cx type is important, but also the different intracellular pathways activated by NO in different types of cells. The complex situation of a certain cell type and its environment is critical for the net effect of post-translational modifications and will also depend on the metabolic cell status and kinase/phosphatase expression under a given condition. The abundance of literature demonstrate that post-translational modifications of Cxs are of great importance in modifying HC activity or GJIC and in future probably additional sites for post-translational modifications will be identified.


Hazardous bacterial strain  and protein stability of Italian Riesling ice wine were evaluated in terms of different amounts of lysozyme, SO2, polyphenols, and wine pH by single-factor experiments. Subsequently, a quadratic rotation-orthogonal composite design with four variables was conducted to establish the multiple linear regression model that demonstrated the influence of different treatments on synthesis score between LAB inhibition and protein stability of ice wine. The results showed that, synthesis score can be influenced by lysozyme and SO2 concentrations on an extremely significant level (P < 0.01). Furthermore, the lysozyme-combined antibacterial system, which is specially designed for ice wine aging, was optimized step by step by response surface methodology and ridge analysis. As a result, the optimal proportion should be control in ice wine as follows: 179.31 mg L−1 lysozyme, 177.14 mg L−1 SO2, 0.60 g L−1 polyphenols, and 4.01 ice wine pH. Based on this system, the normalized synthesis score between LAB inhibition and protein stability can reach the highest point 0.920. Finally, by the experiments of verification and comparison, it was indicated that lysozyme-combined antibacterial system, which was a practical and prospective method to reduce SO2 concentration and effectively prevent contamination from hazardous LAB, can be used to stabilize ice wine during aging process.For the purpose of SO L-malic acid to form L-lactic acid which provides the elegant and round taste during malolactic fermentation (MLF)  are the most important microorganisms initially present in vineyards and thrive throughout fermentation until packaging  is always regarded as a conventional and effective preservative which is necessary for LAB inhibition and MLF control in winemaking process  that damages bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan . (Charter and Lagarde 2 concentration (>500 mg L−1) is added during aging progress  is a historic and major wine region in China. It is characterized by year-round dry and cold climate, fewer pest and disease problems, and Italian Riesling grapes, one of the primary white varieties in this region, are rich in fruity and elegant aromas  at full maturity  and in good sanitary conditions. The wine was made with a traditional ice-wine-processing protocol: grapes were pressed in a pneumatic press and 80 mg L−1 sulfur dioxide and 35 mg L−1 pectinolytic enzyme  were added afterward. The grape juice was then settled at 10°C for 12 h. The alcoholic fermentation was induced by inoculating Saccharomyces cerevisiae  and occurred at controlled temperature (10–12°C). At the end of alcoholic fermentation, 50 L of wine samples were immediately transported to the laboratory and stored at 0°C for further use. All wine samples were filter-sterilized through 0.65 μm pore size of polycarbonate membrane of Tong-Wang TWML04 membrane filter  before experiments. Experimental bottles were pregassed with food-grade nitrogen gas before filling to prevent microbial contamination and oxygen entrance.Italian Riesling ice wine was supplied by Qilian winery . The grapes were harvested in 2012 vintage (November 3Lactobacillus plantarum VT12 and Oenococcus oeni VP41) used in this work were, respectively, purchased from Martin Vialatte Oenologie and Lallemand Oenology, France. Potassium metabisulfite and potassium hydrogen tartrate were purchased by Kemiou Chemical Reagent Co. . In order to simulate different winemaking scenarios, Oligomeric Proanthocyanidin (OPC), which was obtained from JF-Natural industry , was used to adjust the concentration of total polyphenols in ice wine. Folin–Ciocalteu reagent was supplied by Sigma-Aldrich chemical Co. . Deionized water (<18 MW resistance) was obtained from a Milli-Q Element water purification system .Lysozyme chloride was supplied by Asahi Kasei Corporation , while the LAB  and alcohol degree (11.2–13.8% v/v) combined with the residual sugars (188.2 g L−1) denoted the proper fermentative condition. , wine pH 4.27, total polyphenols 0.41 g L−1, and total sulfur dioxide 49.2 mg L−1 (<60.0 mg L−1) were detected as basic indexes after AF.The principle physicochemical parameters in ice wine were detected at just the end of alcoholic fermentation, according to the China national quality standard of vine and wine (GB/T 15038 – 2006). These parameters included alcohol degree, residual sugar, total acids, volatile acids, total sulfur dioxide, L. plantarum VT12 and O. oeni VP41 mixed by 1:1 inoculation ratio) were enumerated under 37°C in liquid MRS medium which included 50 mg L−1 pimaricin and the initial pH was adjusted to 5.0 with a concentrated (6 molL−1) hydrochloric acid solution . The total LAB colonies in ice wine samples were calculated with the formula 1 stated below:LAB colonies quick-counting method was carried out to modify plate colonies counting according to De-qing Zhou  was measured by Thermo GENESYS™ 10S UV-Vis spectrophotometer . Wines were considered to be stable if the difference in absorbance did not exceed 0.2 units. All the treatments were made in triplicate.The level of protein stability was determined by heat test  which can affect the LAB inhibition and protein stability with common influences were studied, respectively, by single-factor experiments. Then, the quadratic rotation-orthogonal composite design with four factors at five levels was used to establish the multiple regression model that expressed the influence of different treatments on the synthesis score between LAB inhibition and protein stability.First of all, four variables  To understand the influence of SO2, its concentration was adjusted to 50 − 250 mg L−1 by potassium metabisulfite addition; (3) To understand the influence of polyphenols, 500 mL ice wine samples were supplemented with OPC to reach total polyphenols content at 0.4 g L−1, 0.5 g L−1, 0.6 g L−1, 0.7 g L−1, and 0.8 g L−1; (4) 3.0 − 5.0 wine pH was studied with adjustment of potassium hydrogen tartrate. All samples were stored 5 days at 25°C for LAB development and then conducted for LAB inhibition and protein stability. Each sample was made in triplicate.(1) To understand the influence of lysozyme, 0 mg LThe quadratic rotation-orthogonal composite design Table is a com540 (for protein stability) and log10 CFU mL−1 (for LAB inhibition). Conversion functions are as follows.Min–max normalization is a statistical method, which is used for linear transformation of the original data and makes the result value mapped into the range of 0−1. In this way, the restriction of raw data units can be removed, and the raw data are converted into pure dimensionless value, which facilitates the indicators of different units or magnitudes that can be compared and weighted  and ΔOD540 as b = 40% (secondary index). In formula 3, lv was the dimension of LAB inhibition and le was the dimension of protein stability. Letters “a” and “b” stand for the two weights.Synthesis score of the antibacterial system was obtained by weighting log10 CFU mLP < 0.05. Subsequently, SAS 9.0 was used to generate response surfaces and contour plots for the relationships between two variables. In order to modify the multiple regression model (formula 4), ridge analysis was applied to determine the optimal combination for lysozyme-combination antibacterial system.The multiple regression model and multiple-way analysis of variance (ANOVA) were analyzed by SAS 9.0 software . Tukey's test was carried out to express group differences at significance level −1 lysozyme addition in approximately 25 days. However, spontaneous LAB activity was completely eliminated in 20 days when lysozyme concentration was increased to 300 mg L−1.Lysozyme-induced inhibition of spontaneous LAB in ice wine was illustrated in Fig.L. plantarum VT12 and O. oeni VP41, which are the typical and numerous LAB strains for ice wine making, was completely eliminated by 200–300 mg L−1 lysozyme in 25–30 days  of −1 lysozyme. However, 150 mg L−1 lysozyme significantly affected LAB activity (log10 CFU mL−1), which reduced by 57.3% as comparing with 100 mg L−1 lysozyme. Therefore, LAB growth can be effectively inhibited by 150 mg L−1–200 mg L−1 lysozyme addition. The protein stability of ice wine was enhanced as the amount of lysozyme was increasing. For instance, ΔOD540 kept relatively stable at the level of 50–100 mg L−1 lysozyme and then increased dramatically when the addition was increasing from 100 mg L−1 to 200 mg L−1 until the highest point at 0.147 with 200 mg L−1 lysozyme. However, ΔOD540 decreased slightly at the range of 200–250 mg L−1 lysozyme. Figure−1 lysozyme can significantly affect the protein stability of Italian Riesling ice wine.Base on Fig.2 concentrations. Figure2 concentration increased, 33.9% LAB activity was inhibited by 50–150 mg L−1 SO2 addition. LAB inhibition at 200 mg L−1 SO2 content was about twice higher than that at 150 mg L−1 SO2. This result indicated that 150–250 mg L−1 SO2 can significantly affect LAB growth in Italian Riesling ice wine. With the same gradient of SO2, heat test was conducted to detect the protein stability. Figure540 decreased as SO2 was increased from 0 mg L−1 to 150 mg L−1. A considerable decrease was observed when SO2 concentration was 100–150 mg L−1, which could be a hazardous level to the protein stability. But above 150 mg L−1 SO2 seems moderate for the change in the protein stability.Same methods and LAB strains were used to monitor the changes of LAB activity under different SO2 showed no negative effects on the fermentative process, providing even a better sensory perception when compared with the wine having only SO2 addition , SO2 contents (x2), polyphenol contents (x3), and pH-value (x4). LAB inhibition (log10 CFU mL−1) was fixed as y1 and ΔOD540, the index of protein stability was fixed as y2. Quadratic rotation-orthogonal composited design Table was carrSAS 9.0 was used to carry out experimental data processing for multiple linear regressions. A quadratic polynomial regression equation (formula 4) with key controlling factors was established.FLf = 2.04 < F0.05  = 2.8655, was not significant at α = 0.05,while, total regression FTr = 18.11 > F0.05  = 2.209, was significant at α = 0.05. Thus, the regression model was logically fitted into experimental data. It demonstrated that the multiple regression model not only reflected the practical treatments accurately but also can be used to evaluate the influences of lysozyme-combined antibacterial system on both LAB inhibition and protein stability during ice wine aging.ANOVA results are shown in TableP < 0.01) by lysozyme and SO2 addition, whereas the influence of polyphenols and ice wine pH was not significant. Furthermore, only the interaction between polyphenols and pH was significant (P < 0.05). According to the numerical magnitude of standardized regression coefficient, the sequence of influence of the four factors on synthesis score was SO2 > lysozyme > polyphenols > pH.On the other hand, synthesis score was significantly affected .2 contents, respectively, the synthesis score initially increased and then decreased according to Fig.−1 lysozyme and 160–200 mg L−1 SO2. Excess SO2 addition (above 200 mg L−1) resulted in pH reduction and inhibited the activity of lysozyme  weakened the LAB inhibition. Based on Fig.−1 and lysozyme was 125–250 mg L−1. It was due to the inhibition of lysozyme activity and even deactivation by protein convergence of excess polyphenols. On the contrary, because of low polyphenols (<0.6 g L−1), LAB reproduced dramatically, meanwhile, the antioxidant activity and stability of ice wine were affected simultaneously  induced more bonded SO2 and attenuated the inhibitory effects on LAB, while lysozyme activity was inhibited by low pH (below 3.5). On the other hand, high concentration of polyphenols strongly bonded with lysozyme and induced protein haze in white wine  and pH (3.5–4.5) in ice wine.According to Fig.Response surface methodology verified the conclusion of ANOVA in Tablex1, x2, …, xq) =  of the factor space  were monitored during 6 months aging in bottles. The results were shown on Table420) of the optimal group increased slightly and remained constant in 6 months. The total sugar concentration of the optimal group basically remained the same; in contrast, the total sugar concentration of the control group decreased by 8.9% at the end of the 6th month. The ice wine pH was maintained at 4.0 in the optimal group but pH of the control group slightly increased about 0.3. It was demonstrated that the ice wine had been totally spoiled. With SO2 concentration decreasing from 47.73 mg L−1 to 30.1 mg L−1, LAB activity in the control group intensified and resulted in a considerable increase (40.4%) in volatile acidity. In addition, the changes in alcoholic degree and polyphenol concentration in the two groups were not significant. Besides these, the lysozyme-combined antibacterial system did not lead to ice wine browning since color density in the optimal group was stabilized at 1.25 during storage. Furthermore, the change in ΔOD540 in the control group was irregular; comparatively, it was consistent in the optimal group. LAB activities were under control in the optimal group during the 6 months. However with a slight decrease, about 107 CFU mL−1 LAB colonies were clearly observed in the control group at the end of 6 months.Compared with various indexes on physical–chemical composition in control group, the turbidity (OD−1 (LAB inhibition) and ΔOD540 (protein stability), meanwhile, a multiple regression model for microbiological experiments was established. Afterward, with the response surface methodology and the ridge analysis, lysozyme-combined antibacterial system, which is special for ice wine, was developed and optimized. The main physical–chemical compounds of optimal groups and control groups were compared during 6 months of aging; the results indicated that lysozyme-combined antibacterial system positively influences physical–chemical compositions and stabilized Italian Riesling ice wine for a long time. In conclusion, as a new technique to reduce SO2, lysozyme-combined antibacterial system can be established to stabilize ice wine by adjusting four parameters before aging process. However, further details should be obtained regarding the evolutions of volatile compositions during aging. In addition, the correlated level between different phenolic compounds and lysozyme needs to be investigated as well.In the beginning, LAB counting method was modified for the practical maneuverability of quadratic rotation-orthogonal composited design. Min-max normalization was carried out to conduct data conversion and to evaluate the synthesis score of the two different indexes, namely, log10 CFU mLNone declared.


In the early spring of 2014, an unusually large amount of sea ice drifted from the Gulf of St. Lawrence, where it had been produced, towards the open Atlantic Ocean through the Cabot Strait, between Nova Scotia and Newfoundland, Canada. In early April, significant amounts of drift ice reached the Atlantic coast of mainland Nova Scotia. The ice floes persisted in those coastal waters for up to 16 days, depending on the location. During that time, the ice fragments caused extensive physical disturbance in rocky intertidal communities, removing high quantities of seaweeds and invertebrates. For example, at a location where the ice stayed for 9 days, the loss of macroalgal and invertebrate biomass was almost total. At a location where the ice stayed for 4 days, losses were lower, albeit still high overall. Such a magnitude of disturbance is not common on this coast, as sea ice had not reached the surveyed locations in the previous 4–5 years. We suggest that the frequency of ice scour events may help to predict intertidal community structure. This notion could be tested through multiannual surveys of ice conditions and biological communities along the Atlantic coast of Nova Scotia. Unlike most other temperate coastal systems, however, on the NW Atlantic coast, sea ice may affect considerably the survival of intertidal species and, consequently, the structure of biological communities.The NW Atlantic coast exhibits cold-temperate conditions. As with similar systems in other parts of the world, the distribution and abundance of rocky intertidal species are greatly influenced by latitudinal changes in temperature and pelagic food supply4, the movement of ice fragments because of tides, currents, winds, and waves can severely damage or remove intertidal organisms6. On many NW Atlantic shores from relatively enclosed bodies of water, such as gulfs or bays, sea ice readily develops on the sea surface every winter, causing a great deal of disturbance in rocky intertidal communities when ice fragments move around7. On the open Atlantic coast, however, ice does not form on the sea surface. Nonetheless, drift ice produced in enclosed bodies of water may still reach the open coast and cause damage there. Such is the case of the open Atlantic coast of Nova Scotia. Between mid-winter and early spring, sea ice produced in the large Gulf of St. Lawrence often drifts towards the Atlantic Ocean through the Cabot Strait, between Nova Scotia and Newfoundland (8 (Canadian Ice Service).While a stable ice coverage of intertidal habitats (the ice foot) prevents benthic organisms from experiencing very low temperatures during low tidesundland . The floFucus algae (Mytilus) and barnacles  in understory habitats  dominated the landscape, while, at the lowest intertidal elevations, kelp (mostlyLaminaria andSaccharina) formed a conspicuous canopy that covered smaller algae, such asC. crispus and coralline algae, and a diversity of small invertebrates , the intertidal zone underwent an almost total loss of organisms  of ice coverage , the observations herein described suggest that intertidal community structure may be influenced by latitude mediated by ice scour effects. We predict that communities from northern locations in this coastal range would remain in early successional stages, as such places receive drift ice from the Gulf of St. Lawrence mostly every year. Conversely, communities from southern locations in this coastal range might reach more mature stages because of sea ice failing to reach those places for a number of years. This notion is supported by the fact that, on Sober Island , which is located south of the southernmost reach of the sea ice in 2014 (Canadian Ice Service), intertidal communities were well developed and seaweeds extensively covered the rocky surface shortly after the 2014 ice season .I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This observation article provides a portrait of an ice scouring event on the Nova Scotia Atlantic shoreline. The article is very short yet explains and demonstrates well the devastating effects of ice scouring. The authors provide several pictures that efficiently illustrate the disturbed community. It would have been ideal if the authors had taken pictures 4 and 7 as well as 6 and 7 with a common guide mark  on the ground. However, the pictures are clear enough to appreciate ice scouring impacts on the benthic communities. Such impacts on macrobenthic communities from ice scouring are very common in the St. Lawrence estuary and gulf. I do agree that communities in the latter regions would remain in early successional stages on exposed substrates.All information provided in this communication is accurate. I consider the observations reported by this paper as interesting and useful.I would suggest the authors add the reference below into the paragraph 2, first sentence:Bergeron P, Bourget E (1986) Shore topography and spatial partitioning of crevice refuges by sessile epibenthos in an ice disturbed environment. Mar Ecol Prog Ser 28:129-145I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


There were no significant differences in average velocity between competitive and non-competitive players; however, the former reached a greater maximum velocity in the 10–20 m section. Average velocity in the 0–10 m section identified specificity among playing positions in competitive players. The forwards were the fastest followed by the central midfielders, the wide defenders/midfielders and the central defenders. No differences were found among the non-competitive players. Average velocity over the 0–10 meter section may be an important indicator when assigning a playing position for competitive players. These results support the use of more accurate systems, such as a laser system, to identify soccer players’ speed qualities (including maximum velocity) during short sprints.The purpose of this study was to determine the kinematic variables that identify the quality of velocity in soccer players at different competitive levels and playing positions. This study had two independent variables: 1) a competitive level (competitive and non-competitive players); and 2) a playing position, with four levels . Forty-two soccer players took part in a 30 m sprint-test, which was measured using a laser sensor-type 1 (LDM301-Jenoptik) at 2000 Hz. Absolute and relative times, average velocities and absolute and relative maximum velocities over 10 m sections were analyzed at 200 Hz with BioLaserSport The importance of sprinting is that it is utilized at key moments during a match, allowing a player to escape from his opponent and/or to reach a free zone to shoot on the goal or to make a decisive pass  the players covered between 9.9 – 32.5 m of the total distance (average of 19.3 ± 3.2 m) over the course of 17.3 ± 7.7 (range of 3 – 40) sprints (−1) varied by a playing position: wide midfielders (35.8 ± 13.4), forwards (attackers) (30.0 ± 12.0), wide defenders (29.5 ± 11.7), central midfielders (23.5 ± 12.2) and central defenders (17.3 ± 8.7) (During high intensity sprints (> 6.3 m·s sprints . During 3 ± 8.7) . In term3 ± 8.7) .Other studies focused on analyzing the differences in performance between elite and sub-elite soccer players and observed that the elite players reached faster times over 15 m, 25 m and 30 m sprint distances than did the sub-elite players . ComettiIn the last few years researchers have developed new technology to obtain accurate “real time” results for the variables related to the positions and velocity of soccer players on the field. As well as using multiple timing systems such as photoelectric cells, Global Positioning Satellite devices (GPS)  and the Based on the aforementioned articles, the present study considered specific sprint characteristics in a game, according to the field position and competitive level. The laser system used in this analysis is an innovation that could make it possible to identify differences in sprint velocities not detected with other customary techniques and may be able to guide coaches’ decision making. Therefore, the purpose of this study was to analyze whether using the 30 m sprint test, registered with an accurate laser system, could identify the different playing positions in soccer players at different competitive levels.The present study compared performance of soccer players from different playing positions and competitive levels in a 30 m sprint test to observe whether there was a difference in the quality of velocity among the different positions. Performance was assessed with a 30 m sprint test using a laser system capable of recording the soccer player’s position and velocity at a sampling frequency of 2000 Hz. Forty-two soccer players participated in the study and were divided into eight groups according to their competitive level (competitors and non-competitors) and playing position . Thirty dependent variables were defined and measured in the 0–10, 10–20, 20–30, 0–20, 0–30 and 10–30 m sections obtaining absolute and relative times, average and maximum absolute and relative velocities. We expected to find differences among the groups that would make it possible to define the variables that determine speed performance in soccer players. These data could help coaches to determine which velocity characteristics are most suitable for each playing position.A total of 42 university students from the Physical Activity and Sport Sciences program participated in this study. They were divided into two groups based on their performance level: competitive and non-competitive soccer players. The competitive group consisted of 21 soccer players who competed in the national level junior, first or second regional divisions. The non-competitive group consisted of 21 physically active subjects who played soccer 2–3 days a week. Each group was further divided into four subgroups according to their position on the field: 11 defenders, 13 wide defenders and wide midfielders, 11 central midfielders and 7 forwards . These sLDM301, Jenoptik, Germany) integrated into a Kinematic analysis system in real-time for the training and the sports competitions  laser calibrated according to the ISO standard with a range of 0.05 to 60 m, accuracy of ± 0.0015 m and a visualized minimal unit of 0.001 m. The data were recorded and processed with a calculus routine designed by the authors using the DASYLAB v. 10.0 software program .A laser sensor-type 1  expressed in s.RT: Interval time taken to cover each section relative to the time over the 30 m . RT (section) = (Tsection × 100)/ T0–30, expressed in percent.Vm: Average value for the velocity over each section , expressed in m·s−1.Vmax: maximum velocity reached in each section , expressed in m·s−1.RV: Percentage of velocity relative to maximum velocity reached over the 30 m . RV section = (Vmax section × 100)/ Vmax0–30, expressed in m·s−1.The tests were performed on a natural grass pitch on two consecutive days between 10 a.m. and 2 p.m. The meteorological conditions were similar with dry sunny weather, and there were similar conditions on the grass pitch on both days. The test started with a standard 15 min warm-up, which included continuous running, joint mobility, static and dynamic stretching of the lower and upper limbs and 3–4 series of 30 m runs at increasing velocity until a submaximal velocity was reached. The participant then stood at the starting line with the front foot on the line and the trunk behind it. Two posts were placed at each end of the line to make sure that the player’s chest did not cross the line before the start of the test. The starting signal was verbal. Each player performed three 30 m sprints with a 5 min rest period between runs. The laser beam had to hit the player’s back at a height of 1 m from the ground with the horizontality of the beam being controlled. Position data were recorded at a sampling frequency of 2000 Hz. Average data were calculated for each 10 records, obtaining plots and data for positions and velocities to 200 Hz in real time. The data were filtered at a frequency of 3 Hz with a analyzed  and its Kolmogorov-Smirnov Test and the coefficients of asymmetry and kurtosis were found. Subsequently the Levene’s test was applied to analyze the equality of the variances in the competitive level groups and by a playing position and to see if an ANOVA should be performed. Using a one-way ANOVA for the independent variables of the competitive level and playing position, the degree of significance was determined at p < 0.05 and η2, establishing from 0.2 a moderately consistent effect. Lastly, the effect of the interaction among the competitive level and playing positions was studied among the eight groups formed. The level of significance was set at p < 0.05. All the calculations were performed with the SPSS 18.0 program.A two-way ANOVA was applied using the level of competition (2 levels) and the playing position (4 levels) as factors along with 30 dependent variables. The normality of the variables was tested with the RT0–10 accounted for 42%, RT10–20 for 29% and RT20–30 for 22% of the total time. RV reached 84% in the first 10 m and 95% in 20 m. p < 0.05)  .p < 0.05; η2 = 0.21). Specificity was evident in the playing position in relation to average velocity in the 0–10 m section with the forwards being the fastest, followed by the central midfielders, wide defenders-midfielders and central defenders, in this order.p = N.S.). The results obtained from elite players by The 30 m test has often been used by authors to assess velocity of soccer players . The resThe data on times per section in the competitive players were T0–10: 1.82 ± 0.07 s, T0–20: 3.8 ± 0.1 s and T0–30: 4.28 ± 0.2 s and were consistent with those found by The most innovative aspect of the present study was the possibility of describing the velocity curves over the 30 m sprint with a laser system at a frequency of 200 Hz. −1 and 8.29 ± 0.28 m·s−1, p < 0.05, which was higher in the competitive players   . This sh effect) , and in  effect) . However effect) . It coulIn conclusion, among competitive soccer players, the forwards were the fastest followed by the central midfielders, the wide defenders/midfielders and then the central defenders. These differences were not found among the non-competitive players. Average velocity over the first 10 m could be an indicator to be taken into account when assigning playing positions for competitive soccer players. The 10 m test could be a good method for identifying forwards and central midfielders to determine which players will be most effective in each position. The competitive players reached higher maximum velocities in the 10–20 m section than did the non-competitive players. The use of an accurate system such as the laser system permitted recording of velocity variables and the position of the athletes in real time with a sampling frequency of 200 Hz and accuracy of ± 0.06 m, providing a more sensitive analysis of the velocity curves.This study provides data that can be used by coaches to make a more complete assessment of their players’ physical level profiles with regard to the quality of their velocity. This may help coaches to determine the strengths and weaknesses of their players to place training emphasis on improving the weaker variables. Moreover, the presentation of standardized variables could facilitate understanding of the results and the comparison of inter-subject and intra-subject data at different times of the year to record the percentage gains and losses in velocity experienced over the competitive season.


Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.We examined the thymoquinone induced inhibition of purified F F1 is composed of α3ß3γδε and Fo of ab2c10–14. ATP hydrolysis and synthesis occur on three catalytic sites in the F1 sector, whereas proton movement occurs through the membrane embedded Fo [o sector. Proton gradient-driven rotation of γ-subunit causes conformational changes in the α/β subunits which in turn results in ATP synthesis or hydrolysis depending on the direction of the proton gradient. This terminal enzyme of oxidative phosphorylation is also the smallest known biological nanomotor [ATP synthase is the principal energy generating enzyme in all organisms from bacteria to vertebrates through oxidative phosphorylation or photophosphorylation. This is a highly conserved enzyme with two sectors Fanomotor ,6.ATP synthase is an important molecular drug target for many diseases, like cancer, tuberculosis, obesity, and microbial infections ,8,9. TheA variety of natural and modified plant based molecules are known to induce either complete or partial inhibition of ATP synthase with potential resulting health benefits ,16,17,18Nigella sativa an annual flowering plant in the family Ranunculaceae  is a major phytochemical compound found in the medicinal plant culaceae . Thymoqu1Fo of E. coli ATP synthase [Thymoquinone with 99% purity (274666-5G) was purchased from Sigma-Aldrich Chemical Company. TQ is unstable in aqueous solution and is light sensitive therefore it was dissolved in DMSO and kept in dark . In ATPasynthase . All oth1 or membrane bound F1Fo was isolated from the wild-type pBWU13.4/DK8 E. coli strain [Purified Fi strain . Growth i strain . In this1 Fo bound E. coli membranes were prepared as in [4 pH 8.0, 2.5 mM MgSO4 before the experiments. F1 was purified as in [1 samples (100μl) were passed twice through 1-ml centrifuge columns (Sephadex G-50) equilibrated in 50mM TrisSO4 pH 8.0 to remove catalytic site bound-nucleotide. ATPase activity was measured in 1 ml ATPase cocktail containing 10 mM NaATP, 4 mM MgCl2, 50 mM TrisSO4, with pH 8.5 at 37°C. Reactions were initiated by the addition of 1 ml ATPase cocktail to purified F1 or membranes and stopped by the addition of SDS to 3.3% final concentration. Liberated Pi was measured as in [1 (20 μg protein), reaction time was 5–10 min. All reactions were found to be linear with respect to time and protein concentration. SDS-gel electrophoresis (10% acrylamide) and immunoblotting with rabbit polyclonal anti-F1-α and anti-F1-β antibodies was used to check the integrity and purity of protein  were preincubated with varied concentrations of thymoquinone for 1hour at room temperature, in 50 mM TrisSO4, pH 8.0 buffer. The volume of TQ added was in the range of 0–20μl in a total reaction volume of 550μl. Then 1 ml ATPase cocktail was added to measure the ATPase activity. Inhibitory exponential decay curves were generated using SigmaPlot 10.0. The best fit line and IC50 value for the curve was obtained using a single 3 parameter model. Statistical significance of the relationship between TQ concentration and enzyme activity was analyzed by linear regression. The range of absolute specific activity for membrane bound F1Fo was 13–20 and for purified F1 was 18–28 μmol/min/mg at 30°C for different preparations. These absolute values were used as 100% bench mark to calculate the relative ATPase activity.Membranes or purified F1 through 1ml centrifuge columns. In reversibility by dilution membranes were reacted with 150 μM concentration of thymoquinone for 60 min at room temperature. Then 50 mM TrisSO4 pH 8.0 buffer was added to reduce thymoquinone concentration to non-inhibitory levels and incubation continued for an additional 60 min at room temperature before ATPase assay. For purified F1, TQ inhibited enzyme was twice passed through 1 ml centrifuge columns before measuring the ATPase activity. Control samples without TQ were incubated for the same time periods as the samples with TQ.Reversibility was measured by dilution of the membranes and by passing the inhibited purified FE. coli ATP synthase [1 or membrane bound F1Fo ATP synthase with ~0.3% residual activity  and succinate (non-fermentable carbon source). 45 to 48% reduction in wild-type growth was observed in presence of 150 μM TQ. No growth inhibition of null strain (pUC118/DK8) by TQ was observed as this strain lacks ATP synthase. Limiting glucose assay contained 3mM glucose and OD595 was measured till no further growth occurred which takes about 20 hr time. Growth on succinate plate may take up to 72 hr.As shown in E. coli to examine the potential of ATP synthase as a molecular target.There is growing interest in the use of natural compounds as antimicrobial and antitumor agents individually or in combination with other such molecules ,44,45. S1 and membrane bound F1Fo ATP synthase with IC50 ~36.95μM  have been used for centuries in traditional medicine to treat many disease conditions, including bronchial asthma, dysentery, infections, and hypertension [o part of ATP synthase, as was the case with Streptococcus mutans, where inhibition of ATP synthase of S. mutans inhibited biofilm formation and acid production [Black seeds (rtension . So far rtension ,28,55. Artension ,28. It wrtension ,56. It ioduction . Also, Toduction . TQ was oduction . TQ was oduction ,57.E. coli cells corroborated the F1-ATPase inhibition by TQ (1-ATPase activity. These results demonstrate that TQ induced inhibition of microbial growth is through the inhibition of ATP synthase.TQ induced growth inhibition of on by TQ . Null stOur results suggest that dietary benefits of TQ in part may be linked to its inhibitory effects on ATP synthase. Inhibition of bacterial cell growth in the presence of phytochemicals like bioflavonoids ,24,34, a


An important challenge in the field of Parkinson’s disease (PD) is to develop disease modifying therapies capable of stalling or even halting disease progression. Coupled to this challenge is the need to identify disease biomarkers, in order to identify pre-symptomatic hallmarks of disease and monitor disease progression. The answer to these challenges lies in the elucidation of the molecular causes underlying PD, for which important leads are disease genes identified in studies investigating the underlying genetic causes of PD. LRRK2 and α-syn have been both linked to familial forms of PD as well as associated to sporadic PD. Another gene, microtubule associated protein tau (MAPT), has been genetically linked to a dominant form of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and genome-wide association studies report a strong association between MAPT and sporadic PD. Interestingly, LRRK2, α-syn, and tau are all phosphorylated proteins, and their phosphorylation patterns are linked to disease. In this review, we provide an overview of the evidence linking LRRK2, α-syn, and tau phosphorylation to PD pathology and focus on studies which have identified phosphatases responsible for dephosphorylation of pathology-related phosphorylations. We also discuss how the LRRK2, α-syn, and tau phosphatases may point to separate or cross-talking pathological pathways in PD. Finally, we will discuss how the study of phosphatases of dominant Parkinsonism proteins opens perspectives for targeting pathological phosphorylation events. Parkinson’s disease (PD) is an incurable disease of aging characterized by the progressive death of dopaminergic cells in the midbrain as well as by α-synuclein-rich intracytoplasmic depositions called Lewy bodies (LBs). Treatments which alleviate disease symptoms have been available for several decades; however, these do not halt disease progression. The development of disease-modifying therapies to replace the symptomatic treatments is therefore a major priority in the biomedical research field. Genetic studies of families with a history of PD (genetic linkage studies) as well as of PD patient groups compared to matched groups of healthy individuals (genetic association studies) have identified genes and genomic variants which contribute to the development of PD . These sMAPT gene mutations cause frontotemporal dementia (FTD) with Parkinsonism. Interestingly, these three dominant genes in Parkinsonism  have also been identified as risk factors for sporadic PD in genome-wide association studies  and mutations in leucine-rich repeat kinase type 2  are linked to autosomal-dominant forms of PD . Also, aes GWAS; . The domLRRK2, α-syn, and tau are all phosphorylated proteins, and their phosphorylation patterns are linked to disease . Early wThe involvement of α-syn in PD was initially identified through genetic linkage studies in a small number of families , includiFigure 1). The pY125 modification has been reported to be inversely correlated with PD-related pathology. Indeed, pY125 appears to protect brains against α-syn mediated toxicity, as this modification is reduced in aged human brain tissue and absent in brain tissue affected by Lewy body dementia  as well as at several tyrosines including Y125, Y133, and Y136 . The precise heterotrimeric composition of the holoenzyme guides PP2A to specific substrate sites. Accordingly, the testing of four different holoenzyme compositions shows that holoenzymes with regulatory subunits of the B family are more efficient at dephosphorylating α-syn-pS129 than those of the B′ and B′′ families. Interestingly, α-syn may function in a feedback loop with PP2A, with studies reporting that α-syn has the ability to activate PP2A activity  together with a scaffold subunit (Aα or Aβ) and a regulatory subunit . Accordingly, treatment of mice with the PME inhibitor eicosanoyl-5-hydroxytryptamide (EHT) increases PP2A methylation as well as decreased α-syn-pS129 levels in brain and a concurrent reduction in synuclein pathology . RelatedFigure 2). Several arguments underline the importance of LRRK2 for PD. First, LRRK2 is one of the most prevalent causes of monogenic PD. Furthermore, LRRK2 mutations are present in apparently sporadic cases of PD, with prevalences of 2% to up to 40% in certain population groups and LRRK2 was recently genetically associated to PD in several independent GWAS  domain  and anotin vitro and in COS-7 cells have suggested a role for protein kinase A as an upstream kinase of the S910–S935 sites . A first class of phosphosites in LRRK2 is the in vitro autophosphorylation site class. These sites appear after in vitro autophosphorylation by purified LRRK2; however, their presence in cells is not confirmed. While these sites offer opportunities to develop assays of LRRK2 kinase activity, their physiological relevance is unclear. Further work will be required to determine whether these sites occur in physiological systems under specific conditions of activation, or whether the appearance of these phosphorylations is an in vitro phenomenon. A second class of LRRK2 phosphorylation sites may be termed ‘cellular’ phosphorylation sites, including those sites of the ANK-LRR interdomain region introduced above, exemplified by the S935 site. Finally, the third and most recent class of LRRK2 phosphorylation sites is the class of cellular autophosphorylation sites, exemplified by the S1292 site, which are detected in cells and which also increase after in vitro autophosphorylation. The cellular sites (S935) and cellular autophosphorylation sites (S1292) are the most physiologically relevant and the comparison of both sites (summarized in Table 2) suggests that these may be useful indicators of LRRK2 activity or pathology. With a few exceptions, pathogenic mutants of LRRK2 display decreased phospho-S935 levels and increased phospho-S1292 levels. It remains to be confirmed whether these changes can be used as diagnostic or disease biomarkers, either individually or together. Interestingly, cellular treatment with kinase inhibitors leads to a reduction of both S935 and S1292. Therefore, both sites are also useful as pharmacodynamic marker to assess activity of kinase inhibitors in cellular and animal models.In sum, the emerging picture of LRRK2 phosphorylation is that LRRK2 is a highly phosphorylated protein where at least two, perhaps three, classes of phosphorylation sites can be discerned  is a main phosphatase of the LRRK2 ANK-LRR interdomain sites . The stuInterestingly, the effects of PP1 in LRRK2 phosphorylation could be confirmed in multiple cell types including HEK293T, SH-SY5Y neuroblastoma cells, mouse primary cortical neurons, U2OS osteosarcoma cells, NIH3T3 mouse fibroblast cells and A549 human lung cancer cells. This shows that PP1 is active as a LRRK2 phosphatase independent of the cell type tested, and it may be predicted that PP1 can dephosphorylate LRRK2 throughout the body.Similar to PP2A phosphatases, PP1 class phosphatases are holoenzymes which are composed of one catalytic subunit, responsible for catalyzing the actual dephosphorylation event, and one regulatory subunit, responsible for directing the holoenzyme to its specific substrates. There are more than 150 PP1 regulatory subunits reported, allowing several 100 possible holoenzyme compositions . This moThere is little data available on the phosphatases involved in the regulation of LRRK2 phosphosites outside of the ANK-LRR interdomain region, however, initial evidence suggests that other phosphatases are at play. Work done at the S1292 site shows that inhibitor induced dephosphorylation of LRRK2 at S1292 is insensitive to the phosphatase inhibitors calyculin A (mixed PP1 and PP2A inhibitor) and OA , corticobasal degeneration (CBD), and some cases of frontotemporal lobar dementia (FTLD). Although tau is mostly associated to dementias, the tau gene has also been identified as a risk factor for PD via genome wide association studies GWAS; . The genFigure 3; for reviews of tau phosphorylation, please refer to Tau can occur in six different splice isoforms ranging in size from 352 to 441 amino acids and at least 45 potential tau phosphorylation sites have been reported, including serine, threonine and tyrosine phosphorylation sites [see figure schematic representation of tau protein and the localization of phosphorylation sites, Tau phosphorylation is important for tau function/dysfunction as hyperphosphorylation of tau is generally correlated with the formation of tau protein aggregates which are major components of neurofibrillary tangles, one of the main neuropathological hallmarks of AD which is also observed in other tauopathies including PD. Phosphorylated tau also more specifically influences its affinity for microtubules and high phosphorylation levels of tau have been reported to negatively influence cytoskeleton, synaptic functions as well as cell viability . Recentlin vitro at S202, S262, and S356, but not S396 . Based on the knowledge of the phosphatases responsible for dephosphorylation of specific phosphosites of proteins, such as emerging knowledge of the composition of phosphatase holoenzymes dephosphorylating specific sites in α-syn, LRRK2, and tau, the specific modulation of phosphorylation levels of single phosphosites is theoretically possible. Further work, including further identification and characterization of specific phosphatase holoenzyme compositions for α-syn, LRRK2, and tau and development of modulators of phosphatase holoenzyme complexes with their specific substrates, is required to test the efficacy of this approach.Phosphatases are divided into different classes, including phosphoprotein phosphatases (PPP), metal-dependent protein phosphatases and protein tyrosine phosphatases (PTP). Phosphatases from the PPP class are responsible for the vast majority of dephosphorylations of central nervous system proteins. PPPs, such as PP1 and PP2A class phosphatases, have the characteristic of functioning as a holoenzyme composed of two or more subunits, including a catalytic subunit as well as regulatory subunits. Interestingly, there are few catalytic subunits in the PPP family and the regulatory subunits have therefore generally been thought to confer substrate specificity to the holoenzyme. Emerging structural evidence is supporting this view, namely that phosphatase holoenzymes of the PPP family associate in a structured way and that this structure determines substrate specificity of the dephosphorylation. Some examples are the interaction between PP1 and spinophilin, a complex found in neurons , and theThe three main proteins linked to Parkinsonism, α-syn, tau, and LRRK2 all display phosphorylations which are modified in disease. The phosphorylation changes are the result of a balance between activity of kinases and phosphatases. Emerging evidence points to phosphatases regulating different pathological phosphorylations in these three proteins, primarily PP1 for LRRK2 and PP2A for α-syn and tau. It appears now feasible to target phosphatases of α-syn, tau, and LRRK2 in order to alleviate pathology mediated by pathological phosphorylation of these disease proteins. In order for this approach to reach its full potential, additional research will be required further linking individual or clustered phosphorylation events in these three proteins to disease. Also, refined knowledge of the precise holoenzyme compositions for each pathological phosphorylation is necessary to develop highly specific phosphatase holoenzyme modulators.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.