The measurement of total serum IgE is made in some cases to investigate if there is the occurrence of allergic processes. However, does not identify specific antibodies. When it is high may indicate an allergic process, or the ability to initiate some. However, increased IgE levels may also indicate other problems such as parasitic diseases, IgE myeloma, hyper-IgE syndrome and some stages of HIV infection. Therefore, to make a diagnosis of allergy is interesting to use other allergy tests, such as specific IgE (RAST) or prick-test. We surveyed data from patients who underwent the examination of serum total IgE to know which allergic diseases that most affect the examination, which gender and age group most affected, and also calculated an average of IgE in each pathology or group of pathologies.A retrospective observational study of the period 2010 to 2013 was conducted, we analyzed the key data from patient files of an Allergy and Immunology’s public ambulatory service in the city of Campina Grande, who had undergone the examination of serum total IgE.Among the 100% investigated (n = 100), 52% were male and 48% female. 61% of range 0-10 years, 10% of 10-20 years old, 13% of 20-30 years old, 9% of 30-40 years old, 3% of 40-50 years old, 4% of 50-60 year old. As pathologies, 27% had allergic rhinitis averaging 379.33 kU / L of total IgE, 18% had Bronchial Asthma averaging 223.89 kU / L of IgE, 11% had the association Allergic Rhinitis and Bronchial Asthma averaging 453.85 KU / L of total IgE, 8% had Atopic Dermatitis with an average of 931.6 KU / L of IgE, 9% had Food Allergy averaging 199.89 KU / L of IgE, and 27% other diseases or associations.We conclude that in this service there is an equivalence between the number of men and women who have changes in examination of IgE. We have also seen that children and adolescents are the most feature changes and perform the test. Furthermore, we showed that the majority of patients with high IgE levels has allergic rhinitis, although the values are higher in patients with atopic dermatitis.


The mechanisms whereby bile acid reflux may accelerate the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett’s patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA. The mechanisms of the progression from BE to EA are not fully understood. Bile acids may contribute to the progression from BE to EA1342O2 production is mediated by activation of NADPH oxidase (NOX) NOX5-S and a bile acid receptor TGR5 in EA FLO-1 cells1213Bile acids have been reported to cause DNA damage−11M TDCA for 24 hours. DNA damage was examined by a Comet Assay. This assay is based on the ability of denatured cleaved DNA fragments to migrate out of the cell under the influence of an electric potential, whereas undamaged supercoiled DNA remains within the confines of the cell membrane. The DNA damage is quantitated by measuring the tail moment, which is defined as the product of the tail length and the fraction of total DNA in the tail (tail moment = tail length x % of DNA in the tail). 15To investigate whether TDCA causes DNA damage, FLO-1 cells, a human Barrett’s adenocarcinoma cell line derived from human Barrett’s esophageal adenocarcinoma, were incubated with 10We have previously shown that TGR5 mediates bile acid-induced increase in NOX5-S expression. Therefore, we examined the role of TGR5 in TDCA-induced DNA damage. We used TGR5 siRNA, which had been shown by us to effectively knock down TGR52O2 production in FLO-1 cells and a Barrett’s cell line BAR-TWe have shown that TDCA increases NOX5-S expression and HThen we used NOX5 siRNA, which had been shown by us to effectively knock down NOX5-SWe have reported that cyclic AMP response element binding protein (CREB) is responsible for acid-induced expression of NOX5-S in SEG1 cells (a possible lung carcinoma cell line)Next we examine the role of CREB in TDCA-induced NOX5-S expression and DNA damage. We used CREB siRNA, which had been shown by us to effectively knock down CREBThere is increasing evidence that bile acids may contribute to the progression from BE to EA12O2 production2O2 production via activation of NOX5-S and TGR5 may contribute to the DNA damage.We have previously shown that the NADPH oxidase NOX5-S is present in EA FLO-1 cells and mediates the bile acid-induced increase in HWe firstly examined the role of bile acid receptor TGR5 in bile acid-induced DNA damage. We found that TGR5 may mediate bile acid-induced DNA damage since 1) knockdown of TGR5 significantly decreased TDCA-induced increase in tail moment and H2AX phosphorylation in FLO-1 cells; 2) overexpression of TGR5 significantly increased TDCA-induced increase in tail moment and H2AX phosphorylation.Next we examined the role of NOX5-S in bile acid-induced DNA damage since we have shown that the NADPH oxidase isoform NOX5-S is present in FLO-1 EA cells and may be a source of reactive oxygen species152+-dependent and ubiquitous transcription factor and binds the consensus CRE DNA sequence TGACGTCA . The ability of CREB to activate transcription requires its phosphorylation on serine 13321In addition, we examined the role of cyclic AMP-response element-binding protein  in TDCA-induced DNA damage since we have found that CREB is responsible for acid-induced expression of NOX5-S. CREB is a Cain vitro cell line, which is a limitation of this study, our data imply that in Barrett’s esophagus bile acids present in the refluxate activate TGR5 and CREB, thereby upregulating NOX5-S. High levels of ROS derived from NOX5-S may cause DNA damage, thereby contributing to the progression from BE to EA.In conclusion, bile acid causes DNA damage via activation of TGR5, CREB and NOX5-S in FLO-1 cells. Although these data were obtained in an The human Barrett’s adenocarcinoma cell line FLO-1−11 M TDCA for 24 h. For diphenyleneiodonium (DPI) treatment, FLO-1 cells were pretreated with DPI (10−5 M), or culture medium (control) for 1 h and then treated with or without TDCA (10−11 M) for an additional 24 h. Finally, the culture medium and cells were collected for measurements.For taurodeoxycholic acid (TDCA) treatment, FLO-1 cells were incubated with 10The protocol for small interfering RNA (siRNA) transfection has been described by us in our previous publicationThe protocol for plasmid transfection was similar to that in our previous publication−11 M in culture medium  for 24 h, and then the culture medium and cells were collected for measurements.Twenty-four hours after transfection with siRNA or plasmid, cells were treated with or without TDCA 10Total RNA was extracted by Trizol reagent  and 1.5 μg of total RNAs were reversely transcribed by using a SuperScript First-Strand Synthesis System for reverse transcription-PCR .′-AAGACTCCATCACGGGGCTGCA-3′), NOX5-S antisense (5′-CCTTCAGCACCTTGGCCAGA-3′), 18S sense (5′-CGGACAGGATTGACAGATTGATAGC-3′), and 18S antisense (5′-TGCCAGAGTCTCGTTCGTTATCG-3′). Reactions were carried out in an Applied Biosystems StepOnePlus real-time PCR system for one cycle at 94 °C for 5 min; 40 cycles at 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s; 1 cycle at 94 °C for 1 min; and 1 cycle at 55 °C for 30 s.Real-time PCR was performed as we previously described112AX antibody , phospho-CREB (Ser133) antibody  and CREB antibody .Cells were lysed and Western blot analysis were carried out as we previously described 18The comet assay was done as we previously reportedHuman TGR5 and CREB siRNA were purchased from Santa Cruz biotechnology Inc., Dallas, Texas; human NOX5 siRNA from Ambion Inc. , and CREB siRNA from Upstate . DPI, Triton X- 100, phenylmethylsulfonyl fluoride, DMEM, antibiotics, and other reagents were purchased from Sigma-Aldrich.t test. Differences among multiple groups were tested using analysis of variance (ANOVA) and checked for significance using Fisher’s protected least significant difference test.Data is expressed as mean ± S.E. Statistical differences between two groups were determined by Student’s How to cite this article: Li, D. and Cao, W. Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett’s Esophageal Adenocarcinoma Cells. Sci. Rep.6, 31538; doi: 10.1038/srep31538 (2016).


Damage to T cells of the immune system by reactive oxygen species may result in altered cell function or cell death and thereby potentially impact upon the efficacy of a subsequent immune response. Here, we assess the impact of the antioxidants Ebselen and N-acetyl cysteine on a range of biological markers in human T cells derived from a SENIEUR status donor. In addition, the impact of these antioxidants on different MAP kinase pathways in T cells from donors of different ages was also examined.T cell clones were derived from healthy 26, 45 and SENIEUR status 80 year old people and the impact of titrated concentrations of Ebselen or N-acetyl cysteine on their proliferation and in vitro lifespan, GSH:GSSG ratio as well as levels of oxidative DNA damage and on MAP kinase signaling pathways was examined.In this investigation neither Ebselen nor N-acetyl cysteine supplementation had any impact on the biological endpoints examined in the T cells derived from the SENIEUR status 80 year old donor. This is in contrast to the anti-immunosenescent effects of these antioxidants on T cells from donors of 26 or 45 years of age. The analysis of MAP kinases showed that pro-apoptotic pathways become activated in T cells with increasing in vitro age and that Ebselen or N-acetyl cysteine could decrease activation (phosphorylation) in T cells from 26 or 45 year old donors, but not from the SENIEUR status 80 year old donor.The results of this investigation demonstrate that the biological phenotype of SENIEUR status derived human T cells negates the anti-immunosenescence effects of Ebselen and also N-acetyl cysteine. The results highlight the importance of pre-antioxidant intervention evaluation to determine risk-benefit.The online version of this article (doi:10.1186/s12979-014-0017-5) contains supplementary material, which is available to authorized users. T cells need to undergo rapid clonal expansion upon antigenic stimulation to produce an immune response. Any factor that interferes with the ability of T cells to clonally expand may impact on the effectiveness of an immune response with the potential to render it sub-optimal.in vivo  , Phospho c-Jun , SAPK / JNK , p38 , p44/p42 [ERK1/2]  and Anti-β- Actin . All antibodies except Anti-β-Actin were purchased from Cell Signaling Technology, Boston, USA. Anti-β-Actin was purchased from Sigma-Aldrich. Secondary antibodies, conjugated to horseradish peroxidase (Dako) were used at 1:10,000 dilution and blots developed using ECL detection system and radiographic film . After the film development, quantification of the signal intensities of the bands in the Western blots was carried out using the Metamorph software . The sigThe results were tested for significance using paired two-sample type 2 Student’s t-tests assuming equal variances; p values are presented as appropriate.TCC samples were taken from the cultures at various time points during their lifespan and the effect of 30 μM Ebselen or 7.5 mM NAC on intracellular redox status (GSH:GSSG ratio) and total glutathione levels within the T cells were determined. Figure in vitro age” with 30 μM Ebselen , the GSH:GSSG ratio significantly decreased with increased time in culture  or NAC (7.5 mM) investigated in this study had any impact on total glutathione levels, at any of the three time points in TCCs derived from a healthy 80 year old donor compared to non-supplemented clones, as was the case for GSH:GSSG ratio. However, the levels of total glutathione were significantly lower in the 399-37 TCCs compared to levels in TCCs from both younger aged donors (Data not shown).The TCCs used in this study underwent apoptosis at the end of their lifespan after completing a finite number of PDs. This is in line with previous reports ,33.in vitro lifespan of TCCs was investigated by supplementing them with one of the either antioxidants until the end of their lifespan. The results presented in Table The effect of different concentrations of Ebselen 0, 10, 30 μM) or NAC  on the proliferative capacity and 0 μM or NAliquots of TCC samples were taken from culture at various time points and the effect of 30 μM of Ebselen or 7.5 mM of NAC on the levels of oxidative DNA damage within the T cells were determined. In control (non-supplemented) samples, levels of oxidative damage to DNA increased as a function of age, as measured by the modified endonuclease III (Endo III) and formamidopyrimidine DNA glycosylase (FPG) comet assays, in line with previously published findings .The results presented in Figure The levels of oxidative DNA damage significantly increased with time in culture in both supplemented and non-supplemented TCCs derived from a healthy 80 year old donor Table . This waThe impact of Ebselen or NAC supplementation on MAP kinase phosphorylation status and total protein levels was determined in TCC samples from healthy young (26 year old), middle aged (45 year old) and elderly (80 year old) donors.in vitro age (PD). In contrast, JNK, p38 and c-Jun phosphorylation levels were absent (or low) in young cells (Y) but greatly enhanced in late PD cells (O) from all donors. 30 μM Ebselen did not significantly alter the increase of p38 phosphorylation in late PD TCCs. There was a significant reduction in JNK and c-Jun phosphorylation in young and middle-aged donor TCCs on Ebselen supplementation. However, Ebselen did not result in a reduction in JNK or c-Jun phosphorylation in TCCs derived from a healthy 80 year old donor . Quantification of the signal intensities of the bands in the Western blots were performed for both supplemented and non-supplemented clones  TCCs with and without 7.5 mM NAC supplementation, with only ERK phosphorylated to any significant extent Figure B. PhosphThe total levels of JNK, p38 and ERK  or NAC (7.5 mM) in CD4itro age . The ROSin vitro age  did not significantly alter the lifespan, the proliferative capacity  with 30ty Table A and B, in vitro lifespan did not alter the longevity of TCCs derived from an 80 year old donor [in vitro lifespan under conditions of 20% O2 that may have compromised a range of intracellular systems. One piece of evidence in this regard is the measured increase in basal levels of oxidative DNA in TCCs from a healthy 80 year old donor, compared with basal levels in TCCs from healthy 26 or 45 year old donors  supplementation from the midpoint of their ld donor . In thatin vitro lifespan.A similar scenario is applicable in terms of the GSH:GSSG ratio. Intracellular redox status (as reflected in the GSH:GSSG ratio) is an important mechanism having an invaluable role as a mediator in apoptosis in many cell systems . PreviouAlthough ROS are generally thought of as harmful molecules, they do play an important role in T cell signalling events  includin2O2-induced p38 MAP kinase activation, c-Jun phosphorylation and JNK activation by Ebselen in a concentration-dependent manner [in vitro age with activation of JNK, p38 and c-Jun across all old TCCs irrespective of donor age  in TCCs from donors of different ages. JNK activation primarily results in apoptosis by the phosphorylation of c-Jun (serine 63), which is a component of the transcription factor complex AP-1 that binds to a specific DNA sequence in the AP-1 site  resultint manner . Furthert manner , selenitt manner  and taxot manner . The resin vitro age with no significant change in the levels of ERK phosphorylation in any of the age groups upon supplementation with either 30 μM Ebselen or 7.5 mM NAC, compared to non-supplemented TCCs  . Howeverin vivo antioxidant supplementation is to be attempted then careful pre-intervention evaluation should be undertaken to determine risk-benefit.The results of this study highlight a heterogeneous potential of Ebselen or NAC as anti-immunosenescent interventive strategies in human T cells. If


There have been numerous studies conducted to investigate the pullout force of pedicle screws in bone with different material properties. However, fewer studies have investigated the region of effect (RoE), stress distribution and contour pattern of the cancellous bone surrounding the pedicle screw.Screw pullout experiments were performed from two different foams and the corresponding reaction force was documented for the validation of a computational pedicle screw-foam model based on finite element (FE) methods. After validation, pullout simulations were performed on screw-bone models, with different bone material properties to model three different age groups . At maximum pullout force, the stress distribution and average magnitude of Von Mises stress were documented in the cancellous bone along the distance beyond the outer perimeter pedicle screw. The radius and volume of the RoE were predicted based on the stress distribution.3, 7.35 mm3 and 8.07 mm3, respectively.The screw pullout strengths and the load–displacement curves were comparable between the numerical simulation and experimental tests. The stress distribution of the simulated screw-bone vertebral unit showed that the radius and volume of the RoE varied with the bone material properties. The radii were 4.73 mm, 5.06 mm and 5.4 mm for bone properties of ages >75, 75 > ages >50 and ages <50 years old, respectively, and the corresponding volumes of the RoE were 6.67 mmThis study demonstrated that there existed a circular effective region surrounding the pedicle screw for stabilization and that this region was sensitive to the bone material characteristics of cancellous bone. The proper amount of injection cement for augmentation could be estimated based on the RoE in the treatment of osteoporosis patients to avoid leakage in spine surgery. Osteoporosis is a common skeletal disorder of the spine and hip in the elderly population. Spine surgeons often encounter patients with osteoporotic spines that require spinal decompression and management with surgical instrumentation due to degenerative and traumatic spinal diseases ,2. PedicDifferent methods have been used to enhance the short- and long-term stability of implanted screws in the osteoporotic lumbar spine ,7. The iAlthough there have been many experimental screw-bone interaction studies, it is technically difficult to determine the region of effect (RoE) by observing the screw-bone interaction during pullout testing. Sources in the literature have reported that the pullout strength of the pedicle screw increased from 147% to 300% -12 when Based on the pullout experiments ,7,17 andSixteen conventional pedicle screws (CDH Ø 6.5 × 40 mm) and polyurethane foam ) were used. Two different types of polyurethane foam with different properties — ρ = 0.16 g/cc, porous cell size 0.5 to 2.0 mm, and Young’s modulus of 23 MPa; and ρ = 0.32 g/cc, porous cell size 0.5 to 1.0 mm, and Young’s modulus of 137.5 MPa, ,21, whicThe screw-foam samples were prepared according to the surgical procedure. First, guiding holes with diameters of 3.5 mm and depths of 50 mm were drilled into the polyurethane blocks, and these guiding holes were then tapped manually, using the taps provided by the manufacturer . Finally, the 6.5 × 40-mm pedicle screws with 13 threads were screwed into the the polyurethane block to the full dept. The guiding holes were 10 mm deeper than the desired screw insertion depth, to ensure that the tip of the screw was not pressed against the bottom of the guiding hole, thus avoiding the generation of undesired pretension on the foams block . SixteenFigure Because the screw-bone interface was periodically symmetric, and a linear correlation existed between the number of threads and pullout strength ,16, a quFor a reasonable representation of the actual matching geometry between the foam/bone and screw threads, a fine mesh (0.1 mm in average length) was adopted, while a relatively coarser mesh (0.3 mm in average length) was adopted for the other regions. For the region between the interfaced threads, significant mesh refinement was used to obtain better stress distribution around the cancellous bone tissue in this area ,25. The To simulate the contacts between the foam/bone and screws at their interfaces under pullout loading conditions, the surface-to-surface contact relationship was employed in the model. Contact pairs were defined between the foam/bone and screws, with the trailing edge of the screw chosen as the master surface and those elements of the foam/bone chosen as the slave surface.The material properties of the foams adopted from the literature , which wy-axis acting along the axis of the screw length, and the x- and z-axes pointing radially. The nodes in the circumferential surfaces of the foam were fixed in all degrees of freedom. The degrees of freedom in x- and z- direction translation were restricted, and a constant velocity of 0.01 mm/s, with displacement of 2.7 mm along the positive y-direction translation was prescribed to the pedicle screw [The validation of the FE screw-foam model was conducted by evaluating the predicted screw pullout strengths and the pullout force-displacement curves against the experimental data obtained from the current test and from Hashemi et al.’s study . By applle screw .Q, was extracted from all of the fully restrained nodes in the circumferential surfaces during the screw pullout procedure. The pullout force for a complete screw-foam model with N threads inserted into the cancellous bone, defined as F, was calculated based on equation (1):The maximum reaction force, defined as Fwith N = 13 to correlate with those in the current experiment and in the studies by Hashemi et al. .For the material sensitivity study, the foam material properties of the validated screw-foam model were changed appropriately to emulate those of the cancellous bone. In this study, the cancellous bone was assumed to be homogeneous and, isotropic with an elastic-perfect plastic material, similar to those described in Hayes et al.’s study . The matΔr beyond the outer pedicle screw radius, and the volume of the RoE for the complete screw, with a purchase length of L, defined as VA, was calculated from equation (2):During the screw pullout procedure, the reaction force at the screw head gradually increased to the maximum and then decreased, and the pullout force was defined as the maximum reaction force. The region of effect (RoE) was defined by the region surrounding the cancellous bone tissue with a von Mises stress value > 0.01 MPa at the maximum reaction force. Then, based on the surrounding region, the RoE was estimated to be a circular area with a radius of Where:r: Outer radius (mm) of pedicle screw;Δr: Radius (mm) of RoE;L: The purchase depth (mm) of pedicle screw into the cancellous bone of vertebral bodyΔr beyond the outer perimeter pedicle screw was also documented.At maximum pullout force, the stress distribution and magnitude in the cancellous bone along The comparisons between the current FE predicted and experimental screw pullout strengths and the load–displacement graphs against those obtained from Hashemi et al.’s study , are shoThe predicted pullout forces from the FE screw-bone models of different bone material properties are shown in Table Figure Δr) and volume of RoE (VA) for the stabilization of the pedicle screw in screw-bone fixation. Table Δr and VA in cancellous bone around the pedicle screws increased with the stiffness of the bone block. Δrs was 4.73, 5.06 and 5.4 mm in the simulated age group > 75 years old, 75 years old > age > 50 years old and age < 50 years old, respectively. The corresponding VA was 6.67, 7.35 and 8.07 ml, respectively.The results show that there existed a hypothetical effective region with radius of RoE  for screw stabilization. The FE computed results showed that the stress distribution pattern in cancellous bone around the pedicle screw was circular in shape. The stress magnitude was the highest in the region near the pedicle screw, and it decreased with the distance from the screw. This result supported the hypothesis that there existed a circular region around the pedicle screw, called the region of effect, which could play a pivotal role in screw stabilization.Δr ranged from 4.73 to 5.40 mm for standard 6.5 lumbar of specified thread profiles, and this range could vary depending on the screw type and thread profile (such as the thread shape factor and the inclination of the leading edge), which will be investigated in a further study.As shown in the results, the range of RoE and screw pullout forces were affected by the mechanical properties of cancellous bone. However, Morgan et al.  and Hou In this study, the predicted RoE is based on the stress contour during the pullout simulation. The region with higher stress (near the pedicle screw) plays an important role in stabilizing the pedicle screw. The region with lower stress (distance away from the pedicle screw) carries less of a load in stabilizing the pedicle screw. We adopted the region with a von Mises stress value < 0.01 MPa to be ineffective in the stabilization of the pedicle screw. Based on the material properties of the cancellous bone, with minimum yield stress of the cancellous bone in the age > 75 years old group of 0.615 MPa, and the value of 0.01 MPa less than 2% of the yield stress, the region with von Mises stress value < 0.01 MPa could therefore play a minor role in pedicle screw stabilization.The development of any computational model requires a number of assumptions regarding the geometry, materials, and interactions between components. In the present study, the model was a quarter screw-foam/bone unit excluding the cortical bone. The commonly used standard of the 6.5 mm pedicle screw type in clinical application for fixation in osteoporotic patients was used in the modeling. Elastic-perfect plastic material properties were adopted for cancellous bone tissue in the current study, based on the Hayes et al.’s study , and theIn conclusion, the RoE was calculated based on an experimentally validated FE model in the present study. The results showed that there existed a circular region of effect around the pedicle screw during the pullout simulation. The RoE was sensitive to the material properties of the cancellous bone. The results suggested that the RoE could play a significantly role in pedicle screw augmentation, and the proper amount of injection cement for augmentation could be estimated in the treatment of osteoporosis patients for spine surgery.The authors declare that they have no competing interests.SL, WQ and YZ have made substantial contributions to conception and design, and acquisition of data, and analysis and interpretation of data; ZXW have been involved in drafting the manuscript and revising it critically for important intellectual content; YBY have given final approval of the version to be published; and WL agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.SL: Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, China;WQ: Surgery Department of 520th Hospital of PLA, Mian yang, China;YZ: Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, China;ZXW: Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, China;YBY: Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, China;WL: Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, China.Shuai Liu, Wei Qi and Yang Zhang are the co-first authors.


Heat shock protein 20 (HSP20) has cardioprotective qualities, which are triggered by PKA phosphorylation. PKD1 is also a binding partner for HSP20, and this prompted us to investigate whether the chaperone was a substrate for PKD1. We delineate the PKD1 binding sites on HSP20 and show for the first time HSP20 is a substrate for PKD1. Phosphorylation of HSP20 by PKD1 is diminished by pharmacological or siRNA reduction of PKD1 activity and is enhanced following PKD1 activation. Our results suggest that both PKA and PKD1 can both phosphorylate HSP20 on serine 16 but that PKA is the most dominant. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd. HDAChistone deacetylaseHSP20heat shock protein 20ISOisoprenalinePKAcAMP‐dependent protein kinase APKD1protein kinase D1SDS–PAGEsodium dodecyl sulfate–polyacrylamide gel electrophoresisHeat shock protein 20 (HSP20), also referred to as P20 or HSPB6, is ubiquitously expressed in a number of tissues with the highest levels in the heart.Recently, we have identified protein kinase D1 (PKD1) as a novel partner for HSP20 using high‐density ProtoArray analysis.3(CH2)16COOH), making them cell permeable. PKD1 activator bryostatin 1 and inhibitor Go6976 were purchased from Calbiochem. For cell studies, peptides/compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma) and used at a final concentration of DMSO ≤ 0·1% (v v−1).Anti‐HSP20 antibody (#07‐490) was purchased from Millipore. Anti‐HSP20‐phospho‐Ser16 (#ab58522) and α‐tubulin (#ab‐18251) were purchased from Abcam. Anti‐PKD1 (#2052) and anti‐phospho‐PKD  antibodies were purchased from Cell Signaling. Horseradish peroxidase‐conjugated goat anti‐rabbit and anti‐mouse secondary antibodies, and PKA inhibitor KT5720 were purchased from Sigma. Anti‐RACK1 antibody (#sc‐17754) was purchased from Santa Cruz. PKD1–HSP20 disruptor peptide (disr pept) (GRDVAIKIIDKLRFPTKQESQLRNE) and control peptide (cont pept) (GAAVAIKIIAKLRFPTKQESQLRNE) were synthesised by GenScript and included an N‐terminal stearoyl group  and transferred onto nitrocellulose membranes for Western blotting. Immunoreactive proteins were detected using appropriate antibodies and visualised by enhanced chemiluminescence detection (Pierce). Quantification of the band intensity was accomplished by densitometry using Quantity One 1‐D software (Bio‐Rad).−1 or without active PKD1 protein (Abcam) in PKD phosphorylation buffer  supplemented with 100 μM adenosine‐5′‐triphosphate (ATP) for 1 h at 30 °C with agitation. The phosphorylated protein was then subjected to SDS–PAGE and immunoblotted with an anti‐phospho‐HSP20 (Ser 16) antibody.A range of purified His‐HSP20 concentration (0·5–2 µg) was incubated with 1·0 g ml2, 0·5 mM CaCl2, 1 mM DTT, 0·2 mg ml−1 BSA, 1 mM ATP) supplemented with 10 µg of active PKD1 (Abcam) and 5 μCi (0·185 MBq) of [γ‐32P] ATP EasyTides  for 30 min at 37 °C with gentle agitation. After the phosphorylation reaction, the membrane was washed extensively with 1 M NaCl followed by 5% H3PO4 and finally ethanol. The membrane was air dried and placed in intensifying screen cassette  for a week before developing using the Kodak® X‐Omat Model 2000 processor.Peptide arrays corresponding to the entire sequence of HSP20  were constructed as published previouslyt‐test or one‐way ANOVA tests followed by post hoc Tukey's tests. Values were considered significant if p < 0·05. Where representative immunoblots were shown, similar data were obtained n ≥ 3 times.Values are presented as mean ± SEM from at least three independent experiments. Statistical significances between groups were determined by the use of Student's 10WLRRXSAPLP20), we sought to evaluate whether the heat shock protein could be phosphorylated by PKD1. We used two experimental approaches to examine the effect of PKD1 activity on the phosphorylation level of HSP20 at serine 16. Firstly, we utilised pharmacological inhibition/activation of the kinase. Cells were treated with activator bryostatin 1 (10 nM)p < 0·05) and diminished significantly (p < 0·01) after treatment with Go6976. Interestingly, the phosphorylation of HSP20 increased along with activation of PKD1 and converse inhibition of PKD1 decreased HSP20 phosphorylation , a site which is similar to the phospho‐motif of the AGC kinase subfamily .in vitro phosphorylation assay carried out on HSP20 peptide arrays using [γ‐32P‐ATP] revealed an optimal putative PKD1 phosphorylation site at Ser16 (Figure in vitro kinase assay was carried out using recombinant purified His‐HSP20 protein and PKD1 active protein and then probed with HSP20 phospho‐Ser16 antibody to validate the specificity of the phosphorylation site. Phospho‐bands were detected when active PKD1 was added. No appreciable immunoreactivity was evident when the assay mix was devoid of PKD1 (Figure In an attempt to map the binding site of PKD1 on full‐length HSP20, we used peptide array, a technique previously utilised by us to pinpoint the interaction sites between HSP20 and PDE4.in vitro and in vivo studies,As our data suggest a direct interaction between HSP20 and PKD1, we were keen to determine whether the disruption of PKD1–HSP20 interaction affects levels of HSP20 phosphorylation. Using a cell permeable peptide disruptor of the PKD1–HSP20 complex previously characterised in both In this study, we report for the first time that HSP20 is a substrate for PKD1. In identifying the binding sites and phosphorylation site for PKD1 on HSP20, we suggest that PKD1 binds directly to the heat shock protein to enable phosphorylation at a PKD consensus site, which contains serine 16. Indeed, the complex between these proteins has previously been reported.The results outlined earlier are of particular interest in light of previous research, which has described increased PKA phosphorylation of HSP20 as a cardioprotective mechanism following sustained β‐adrenergic stimulation.Y. Y. S. and G. S. B. conceived the study, designed the experiments, and wrote the manuscript. Y. Y. S. performed the experiments.


Cα and the Cα of the other sites. According to a mechanistic stress model proposed recently, rates are determined by packing because mutating packed sites stresses and destabilizes the protein’s active conformation. While WCN is a measure of Cα packing, mutations replace side chains. Here, we consider whether a site’s evolutionary divergence is constrained by main-chain packing or side-chain packing. To address this issue, we extended the stress theory to model side chains explicitly. The theory predicts that rates should depend solely on side-chain contact density. We tested this prediction on a data set of structurally and functionally diverse monomeric enzymes. We compared side-chain contact density with main-chain contact density measures and with relative solvent accessibility (RSA). We found that side-chain contact density is the best predictor of rate variation among sites (it explains 39.2% of the variation). Moreover, the independent contribution of main-chain contact density measures and RSA are negligible. Thus, as predicted by the stress theory, site-specific evolutionary rates are determined by side-chain packing.In protein evolution, due to functional and biophysical constraints, the rates of amino acid substitution differ from site to site. Among the best predictors of site-specific rates are solvent accessibility and packing density. The packing density measure that best correlates with rates is the weighted contact number (WCN), the sum of inverse square distances between a site’s  Why do some protein sites evolve more slowly than others? Protein evolution is driven by random mutations and shaped by natural selection . Mutatiospecific properties account for site-dependent rates of evolution? The most studied predictors are structural site-specific properties  , predicts evolutionary rates at least as well as RSA (We can reformulate the question opening the previous paragraph: What operties . For yeaty (RSA) . Howeverl as RSA .The relationship between WCN and substitution rates can be understood in terms of a mechanistic stress model of protein evolution . Given aCα to the Cα of other sites: it is a measure of Cα packing density. Therefore, previous substitution rate vs. WCN studies were based on main chain (Cα) packing  (A site’s WCN is the sum of inverse square distances from its  packing . Howeverde 1CA2) . The envde 1CA2)  differs de 1CA2) . When ThTo address this issue, we extended the stress model to consider main and side chains explicitly and we theoretically derived that substitution rates depend only on side-chain of packing. We tested the theory on a data set of monomeric enzymes. In agreement with predictions, site-specific substitution rates correlate better with side-chain packing than with main-chain packing measures and RSA. Moroever, partialing out the effect of side-chain packing, the independent contributions of main-chain packing and RSA are negligible.In this section, we show that the mechanistic stress model of protein evolution predicts that the substitution rate of a protein site is determined by the packing density of its side chain. This prediction and its empirical assessment are the point of this paper.FC is the concentration of folded protein and ρ(ractive) its probability of adopting the active conformation. Assuming that Cmut/Cwt is equal to the ratio of partition functions, from basic statistical physics it follows that: β represents the selection pressure and The stress model was proposed by βδV∗ ≪ 1 (weak selection), from i, iK, is proportional to (minus) the change in stability of the active conformation averaged over mutations at i, 〈δV∗〉i. This is the basic equation of the stress theory.Assuming that predict whether evolutionary rates will be determined by main chain packing or side chain packing. To address this issue, here we represent each residue using two nodes: a main-chain node α, placed at the residue’s Cα, and a side-chain node ρ, placed at the geometric center of the residue’s side chain (Gly’s are represented using only one node at Cα). The energy function is: rinjn is the distance between nodes in and jn (n is α or ρ), kinjn is the force constant of the spring connecting these nodes, and dinjn the equilibrium spring length.In i will replace iρ, affecting only the parameters of the energy function related to this node. We emphasize: while the mutation may well induce global structural changes involving the backbone and other side chains, the only parameters that will change are those of the mutated side chain. Following i by adding random perturbations to the lengths of the springs connected to iρ: diρjρ → diρjρ + δiρjρ and diαjρ → diαjρ + δiαjρ, to find, using i we find: A mutation at site ρ of the site considered (subscript) and nodes α and ρ of the other sites (superscript). Therefore, the stress model, combined with a two-nodes-per-site pfANM energy function, predicts that site-specific rates will depend on the contact density of the side chain To finish, we assume, as in the parameter-free Anisotropic Network Model (pfANM) of independent effect of By analogy with diαjα is the distance between the the alpha carbons of sites i and j. For the sake of clarity, wherever it is convenient we will use the notation Cα of a site (subscript) and the Cα of the other sites (superscript) are considered. Therefore, Cα–Cα interactions).To assess the prediction of the previous section, we also consider the following structural properties. First, the Weighted Contact Number WCN, which was introduced by ρ rather than Cα to define: ρ–ρ interactions.Second, by analogy with Finally, we also consider the Relative Solvent Accessibility, RSA, which is the most studied structural determinant of evolutionary rates. The RSA of a residue is obtained by dividing its area accessible to the solvent (SA) by the maximum SA for the given amino acid type .To test our theory, we used the data set of relative rates were obtained by dividing site-specific rates by their average over all sites of the protein. We denote the empirical rates by KR4S.We used the empirical site-specific rates of evolution of x, we quantified its predictive power using the squared Pearson correlation coefficient R2. According to the theoretical predictions, independent contribution of each of the other structural descriptors by partialing out the effect of unique contribution of predictor x. Also, it is the amount by which the explained variation of KR4S (R2) would increase when going from the single-variable linear fit p-values were obtained by averaging protein correlations and semipartial correlations for 10,000 bootstrapped replicas of the dataset of 209 proteins.For each protein of the dataset, we used the pdb structure to calculate the five site-dependent structural properties defined above: p-values were calculated using cor.test. Semipartial correlation coefficients and p-values were calculated using spcor.test. For bootstrapping with used boot with default options.For statistical analysis we used R . CorrelaKR4S were obtained from multiple sequence alignments using Rate4Site. We compared KR4S with ρ − ρ weighted contact number), α − α weighted contact number), and RSA (relative solvent accessibility). For each protein, we calculated correlation coefficients between KR4S and each structural property and semipartial correlations to measure independent contributions. Protein-by-protein results . As we mentioned in the Introduction, main chain environments and side-chain environments are different . Accordied rates . The twot better  and are t better : the R2 R2 values of 0.392 ± 0.008 and 0.319 ± 0.008 for R2 = 0.073 ± 0.003 is significantly positive . Therefore, We repeated the previous assessment for each of the 209 enzymes of the data set . Empiricsole predictor of substitution rates. Any correlation between rates and unique contribution of R2 would increase when going from the one variable model p < 10−3, bootstrapping), but very small: The stress model predicts ρ that represents the side chain of a site with all other nodes, ρ and α of the network. ρ − ρ contacts. R2 = 0.0024 ± 0.007 is significant , but very small (just 0.24% of explained variation). Thus, ad hoc, theoretically derived.Currently, WCN  = 0.314 ± 0.007. The difference is ΔR2 = 0.078 ± 0.003, which is statistically significant . Thus, not only does −3, bootstrapping). Despite statistical significance, the unique contribution of WCN is just 0.2%, which is negligible. Thus, We found that The most studied structural predictor of site-dependent evolutionary rates is the relative solvent accessibility RSA . TherefoR2 = 0.327 ± 0.007. The difference is ΔR2 = 0.065 ± 0.004, which is statistically significant . Thus, p < 10−3, bootstrapping), but very small. Therefore, According to protein-by-protein results, We used the the mechanistic stress model to predict theoretically that site-specific rates of evolution depend solely on the side-chain contact density To finish, we note that the structural properties studied do not explain all of the variation of substitution rates among sites. The best predictor, 10.7717/peerj.911/supp-1Table S1Correlation coefficients for all proteins.Click here for additional data file.


Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis. Understanding the mechanism of tachyzoite invasion is important for the discovery of new drug targets and may serve as a model for the study of other apicomplexan parasites. We previously showed that Plasmodium falciparum expresses a homolog of human calcium calmodulin-dependent protein kinase (CaMK) that is important for host cell invasion. In this study, to identify novel targets for the treatment of Toxoplasma gondii infection (another apicomplexan parasite), we sought to identify a CaMK-like protein in the T. gondii genome and to characterize its role in the life-cycle of this parasite.T. gondii by using purified proteins with various concentrations of calcium, calmodulin antagonists, or T. gondii glideosome proteins. Indirect immunofluorescence microscopy was performed to detect the localization of this protein kinase by using the antibodies against this protein and organellar maker proteins of T. gondii.An in vitro kinase assay was performed to assess the phosphorylation activities of a novel CaMK-like protein in T. gondii, T. gondii CaMK-related kinase (TgCaMKrk), which exhibits calmodulin-independent autophosphorylation and substrate phosphorylation activity. However, calmodulin antagonists had no effect on its kinase activity. In T. gondii-infected cells, TgCaMKrk localized to the apical ends of extracellular and intracellular tachyzoites. TgCaMKrk phosphorylated TgGAP45 for phosphorylation in vitro.We identified a novel CaMK homolog in T. gondii motility and infection, the interaction between parasite protein kinases and glideosomes, and drug targets for protozoan diseases.Our data improve our understanding of  Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis  1–866) was amplified by RT-PCR using parasite mRNA as the template and the following primers: forward, 5'-GCG CKrk Fig. . To gene2+ or calmodulin upon translation, DMSO (negative control), the calmodulin antagonist W-7 , or calmidazolium  was added to the SUB-AMIX solution.Protein expression using a wheat germ cell-free protein synthesis system (CellFree Sciences) was achieved as described previously , 28. BriEscherichia coli XL-1 blue transformed with plasmids harboring MBP-TgGAP45, MBP-TgACT1, MBP-TgMLC1, or MBP-TgGAP50 was performed as described elsewhere ATP. After incubation, the samples were washed three times with TNE buffer , and the phosphorylated proteins were separated by 10 % SDS-PAGE. The gels were stained with Coomassie Brilliant Blue (CBB), dried, and exposed to X-ray film ATP by phosphorylation  that physically associates with GST-TgCaMKrk, or that it was non-specifically selected for by the glutathione-Sepharose beads. To exclude these possibilities, we constructed a mutant that lacked intrinsic kinase activity but retained its overall structure. We used site-directed mutagenesis to replace Lyssee Fig. . Lys218 activity , and becactivity . The aution Fig. , Lane 2,ion Fig. , Lane 3.2+ and/or calmodulin dependent, as was shown for PfPK2 [2+. The kinase activity of TgCaMKrk was the same, regardless of the presence of Ca2+. Next, we assessed the effect of Ca2+ and calmodulin on the phosphorylation activity of TgCaMKrk. We found that TgCaMKrk activity was not affected by the presence of 10 μM Ca2+ or 10 μM calmodulin, while the autophosphorylation of PfPK2 depended on the calcium concentration.To determine whether the kinase activity of TgCaMKrk is Caor PfPK2 , we perfP. falciparum [2+ nor calmodulin is required for the kinase activity of TgCaMKrk. These results are consistent with the fact that the putative amino acid sequence of TgCaMKrk has a conserved catalytic domain but lacks the regulatory domain, which comprises the autoinhibitory and calmodulin-binding domains in CaMK-like proteins . The catalytic domain of TgCaMKrk shares 88 % similarity with that of PfPK2; however, TgCaMKrk has no regulatory domain. The regulatory domain of PfPK2 consists of autoinhibitory and calmodulin-binding domains. The lack of such a domain in TgCaMKrk is reflected by the fact that calmodulin antagonists  did not inhibit TgCaMKrk autophosphorylation or the histone phosphorylation activity of TgCaMKrk. Some regulation of TgCaMKrk must occur, however, because continuous phosphorylation by this protein would presumably affect the parasite’s life-cycle. A BLAST search via ToxoDB (http://toxodb.org/toxo/) using the putative amino acid sequence of TgCaMKrk did not reveal a domain homologous to the regulatory domain of PfPK2. Therefore, it is likely that another parasite protein regulates the kinase function of TgCaMKrk.This is the first report to describe the identification and characterization of a CaMK-like protein in P. falciparum indicated that PfPK2 is also expressed in the parasite’s inner membrane [Our IFA data showed that TgCaMKrk localizes to the apical ends of extracellular tachyzoites. The localization patterns of TgCaMKrk in extracellular tachyzoites were more posterior to those of GAP45. Antibodies against TgGAP45 stained the inner membrane complex, but not the plasma membrane . Previoumembrane . These f163 and Ser167. Mutation of these residues to Glu prevented association of the MyoA-MLC1-GAP45 complex with GAP50, but did not have any obvious effects on motility or host cell invasion, suggesting that Ser163 and Ser167 phosphorylation controls the final step in assembly of the myosin XIV motor complex [TgCaMKrk specifically phosphorylated TgGAP45 in the glideosome in vitro. The residues of TgGAP45 phosphorylated by TgCaMKrk were most likely Ser complex .163 and Ser167 of TgGAP45 are not conserved in TgGAP70. Further analysis of TgCaMKrk in extracellular tachyzoites is needed to clarify its substrates.TgGAP70 localized to the apical cap of extracellular tachyzoites, whereas TgGAP45 localized to the periphery . When weT. gondii kinases play important and diverse roles in the parasite’s life-cycle [T. gondii tachyzoites and could be associated with the glideosome. Future studies should focus on the regulatory mechanism for this protein kinase and its overall impact on the life-cycle of T. gondii.fe-cycle . InhibitT. gondii-infected cells and localizes to the apical ends of extracellular and intracellular tachyzoites. TgCaMKrk phosphorylates TgGAP45 in vitro. Our data further our understanding of T. gondii motility and infection, the interaction between parasite protein kinases and glideosomes, and drug targets for protozoan diseases.In this study, we identified TgCaMKrk, which exhibits autophosphorylation and histone phosphorylation activity. TgCaMKrk is expressed in


There are several synergistic methods available. However, there is a vast discrepancy in the interpretation of the synergistic results. Also, these synergistic methods do not assess the influence the tested components , have upon one another, when more than two components are combined.Heteropyxis natalensis, Melaleuca alternifolia, Mentha piperita and the green tea extract known as TEAVIGO™. The synergistic combination was tested against the oral pathogens, Streptococcus mutans, Prevotella intermedia and Candida albicans. Inhibition data obtained from the checkerboard method, in the form of binary code, was used to compute a logistic response model with statistically significant results (p < 0.05). This information was used to construct a novel predictive inhibition model.A modified checkerboard method was used to evaluate the synergistic potential of Based on the predictive inhibition model for each microorganism, the oral pathogens tested were successfully inhibited (at 100% probability) with their respective synergistic combinations. The predictive inhibition model also provided information on the influence that different components have upon one another, and on the overall probability of inhibition.Using the logistic response model negates the need to ‘calculate’ synergism as the results are statistically significant. In successfully determining the influence multiple components have upon one another and their effect on microbial inhibition, a novel predictive model was established. This ability to screen multiple components may have far reaching effects in ethnopharmacology, agriculture and pharmaceuticals. Synergistic interaction between components i.e. drugs, plant and natural extracts can enhance their efficacy and bioactivity against a target. Furthermore, synergy reduces toxicity, lowers the dosage and decreases adverse side effects, as well as combating antimicrobial resistance,2. SeverThere is also limited information available with regards to assessing the influence multiple components have upon one another in combination. Three-component combinations have been proven successful in enhancing bioactivity-8. HowevHeteropyxis natalensis was combined with the essential oils of Melaleuca alternifolia and Mentha piperita as well as the green tea extract known as TEAVIGO™. Combinations of these were used against the oral pathogens, Streptococcus mutans, Prevotella intermedia and Candida albicans[An indigenous South African plant,  albicans.H. natalensis was collected. The plant parts were collected from the University of Pretoria’s experimental farm during January, 2013. A voucher specimen was prepared and identified at the H.G.W.J. Schwelcherdt Herbarium (PRU), University of Pretoria. Melaleuca alternifolia essential oil , Mentha piperita essential oil , and TEAVIGO™ (Chempure (Pty) Ltd, Silverton, South Africa), were purchased for the present investigation.Aerial plant parts, comprising of leaves and twigs of The plant material was air dried at room temperature (25°C), and ground to a fine powder using a standard food processor. The powdered material was extracted with ethanol  Ltd Wadeville, South Africa) under pressure (100 bar) and regulated temperature of 50°C in a BUCHI Speed Extractor, E-916 . The solvent was evaporated at low boiling point in a Genevac, EZ-2 plus , after which the extract was subjected to antimicrobial tests.Prevotella intermedia (ATCC 25611), Streptococcus mutans (ATCC 25175) and Candida albicans (ATCC 10231). The bacteria were grown on Casein-peptone Soymeal-peptone (CASO) Agar  Ltd Wadeville, South Africa) enriched with 1% sucrose  Ltd Wadeville, South Africa) under anaerobic conditions in an anaerobic jar with Anaerocult® A  Ltd Wadeville, South Africa), at 37°C for 48 hours. Candida albicans was grown on Sabouraud Dextrose 4% Agar (SDA)  Ltd Wadeville, South Africa), at 37°C for 48 hours. Sub-culturing was done every second week. Inocula were prepared by suspending microbial test organisms in their respective broths until turbidity was compatible with McFarland Standard 1  Ltd Wadeville, South Africa)[P. intermedia was 40 × 107 (CFU/ml), S. mutans was 30 × 107 (CFU/ml) and C. albicans was 4 × 107 (CFU/ml) for each bioassay.The microorganisms used in this study included ans ATCC 231. The H. natalensis, M. alternifolia essential oil, M. piperita essential oil and TEAVIGO™, the minimum inhibitory concentration (MIC) of each component was determined first using the anti-microbial microtiter-plate method[H. natalensis was prepared in 20% dimethyl sulphoxide (DMSO)  Ltd); while TEAVIGO™ was dissolved in distilled water. The stock solutions were serially diluted in enriched CASO broth  Ltd) for the bacteria and Sabouraud Dextrose 4% broth  Ltd) for Candida; in the 96-well microtiter-plate adding 100 μl of a McFarland Standard 1 inoculum of 48 hour old microorganisms grown at 37˚C. The final concentration of the extract and TEAVIGO™ ranged from 0.10–12.5 mg/ml and the positive control, 1.25% v/v chlorhexidine gluconate (CHX) , ranged from 4.77 × 10-6–0.31% v/v. The essential oils were dissolved in 10% Tween (80)  Ltd Wadeville, South Africa). The final concentration tested of the essential oils ranged from 1.6 × 10-5–1.25% v/v. The highest concentration of the solvents DMSO (5%) and Tween 80 (2%) were found to be non-toxic to the microorganisms tested. The inoculated plates were incubated at 37°C, under anaerobic and aerobic conditions respectively for 24 hours before adding 20 μl of the colour indicator PrestoBlue[To determine the effects of combinations of e method. A stockH. natalensis (50 μl) in horizontal orientation, and the second plate, was used to make five-fold serial dilutions of M. alternifolia in the vertical orientation. Both dilutions were made in enriched CASO broth for the selected bacteria, and Sabouraud Dextrose 4% broth for the yeast, C. albicans. For the two-fold dilutions, 50 μl of broth was pipetted per well for the first plate and 200 μl for the five-fold dilutions in the second plate. Using a multichannel pipette, 50 μl of M. alternifolia was transferred to the first plate, 50 μl of the respective broth was added and then 50 μl of bacterial suspension was added to each well and incubated for 24 h at 37°C; after which 20 μl of PrestoBlue was used to indicate bacterial growth[H. natalensis in combination ranged from 0.097 – 12.5 mg/ml, while the essential oils ranged from 1.6 × 10-5 – 1.25% v/v.The synergistic activity of the samples was determined using a modified checkerboard method. The basic design is a logarithmic design with the dosages halved at each step. Full 2-factor factorial designs were used for two factors at a time with equally spaced dosages for the other factors. The individual designs were compounded in such a way that all two-way interactions and some three-way interactions could be estimated. Two 96-well plates were prepared: the first one was used to make two-fold serial dilutions of l growth,13. The M. piperita was added at a sub-MIC value at a fixed concentration to all wells. The sub-MIC concentrations of M. piperita and TEAVIGO™ were determined on the basis of MIC values previously obtained.A third plate was prepared at the same time in the exact same manner as the first plate except that instead of 50 μl of additional broth; 50 μl of a third component, This process was repeated for all the combinations of the four components for each microorganism tested.H. natalensis and TEAVIGO™ ranged from 0.097 – 12.5 mg/ml, while the essential oils ranged from 1.6™ 10-5 – 1.25% v/v. CHX was again utilized as a positive control.The MIC’s of each component tested (as previously described) were also conducted at the same time acting as controls and a comparison. The concentration range of Once the plates were developed with PrestoBlue, each well was assigned either a 0 to indicate no inhibition or a 1 to indicate inhibition for the logistic response model. This information was used to construct a predictive inhibition model (IBM© SPSS© version 22) for each microorganism where the antimicrobial ability of the different combinations was tested as described in the antimicrobial susceptibility testing.The checkerboard method was utilized as a screening tool for the reduction of MIC values. This method also provided numerous concentration variables for the components under investigation and their inhibition potential Figure . The resP. intermedia  both essential oils MIC’s were reduced from 1.17% v/v to 0.29% v/v overall. In plates G and H with H. natalensis and M. alternifolia and the addition of M. piperita in plate H there was a significant decrease in both components MIC’s. Even though the pattern of inhibition to no-inhibition was a little scattered, the overall reduction of the MIC of H. natalensis from 12.5 mg/ml to 1.56 mg/ml and for M. alternifolia from 1.17% v/v to 4.5 × 10-3% v/v was obtained. There is a significant increase in the antimicrobial activity of the components in combination when compared to the MIC values of the individual components.In determining the antimicrobial susceptibility of  Figure , the addC. albicans  to 0.07% v/v.S. mutans . The same effect was exhibited with H. natalensis, M. alternifolia and the addition of TEAVIGO™ in plates C and D. There was no apparent effect of TEAVIGO™ on the essential oils, M. piperita and M. alternifolia (plates E and F). There was a marked increase in the inhibition by M. alternifolia with the addition of M. piperita; from 1.17% v/v to 0.02% v/v (plates G and H). Melaleuca alternifolia on its own inhibited S. mutans at 0.29%. In combination with H. natalensis and M. piperita this inhibitory concentration was reduced to 0.02% v/v.In determining the inhibitory effect of three components on  Figure , there wThe logistic response model was usedThe response variable is Y as follows:Y = 0 means no responseY = 1 means inhibitionX =  is the combination of dosages with X1 representing H. natalensis, X2 - M. alternifolia, X3 - M. piperita and X4 - TEAVIGO™X) = the probability of inhibition given the dosage combinationp(X) is the odds of obtaining inhibitionO(X)}The log(odds) is LN{O(The logistic regression model is a linear model (linear in terms of the regression coefficients) that links the log (odds) to the dosages and to interaction terms between the dosages.The function is estimated by means of maximum likelihood. In this case Table , the estThe estimated odds of inhibition is thenThe estimated probability of inhibition is thenPrevotella intermedia }, obtained from the logistic regression model, combines the data of the four components in the predictive inhibition model. This enabled the probability of inhibition to be calculated utilizing all four components. The predictive inhibition model also provided information on the influence, the different components tested, had upon one another and on the probability of inhibition .Based on the predictive inhibition model where 1 indicates the probability for 100% inhibition; a Table , C. albis Table  and S. ms Table  were sucThere is a reduction in the MIC values of the individual components, when used in combination for each of the microorganisms tested Table . And theM. piperita, H. natalensis and TEAVIGO™, against P. intermedia, C. albicans and S. mutans all had similar outcomes, resulting in an increased H. natalensis activity against these microorganisms  resulted in an increase in the activity of H. natalensis against S. mutans and both H. natalensis and M. alternifolia against C. albicans. However, this combination had no apparent effect on P. intermedia. The reverse situation occurred for the combination of M. piperita, M. alternifolia and TEAVIGO™ (plates E and F), where an increase in antimicrobial activity was noted for M. piperita and M. alternifolia on P. intermedia but there were no noticeable effects on C. albicans and S. mutans. Both Gram-positive and Gram-negative bacteria's cell walls consist of peptidoglycan. Peptidoglycan is comprised of N-acytyl-muramic acid and N-acetyl-glucosamine cross linked by peptide side chains and cross-bridges; however, peptidoglycan is thicker in Gram-positive bacteria. Gram-negative bacteria also possess a periplasmic space which lies between the outer membrane and the cytoplamic membrane. It is within this space that some Gram-negative bacteria produce the lactamase enzyme that can resist drugs such as penicillin[M. piperita, M. alternifolia and TEAVIGO™ may target the transenvelope efflux pump in P. intermedia which does not occur in S. mutans or the eukaryotic C. albicans[M. piperita, M. alternifolia and H. natalensis (plates G and H) all resulted in an increase in M. alternifolia antimicrobial activity but only on P. intermedia was the activity of H. natalensis also increased. Overall it would seem that TEAVIGO™ increases the antimicrobial inhibitory activity of H. natalensis; while M. piperita has a similar effect on its essential oil counterpart M. alternifolia.The synergistic combination of the components had different effects on each of the microorganisms tested. This may indicate the possible mechanism of action of these components. The combinations of nicillin. The com albicans. The comp < 0.05) from the logistic response model. This has reduced the need to calculate the fractional inhibitory concentration (FIC) or equivalent values.The predictive inhibition model provides information of the influence the different components tested have upon one another and on the probability of inhibition. This ‘determination of influence’ goes beyond the classification of synergism, indifference and antagonism. A probability of inhibition value was assigned to the concentration of each individual component and in various combinations of two to four. The concentrations of each component can then be adjusted to obtain a 100% probability of inhibition. The predictive inhibition model is also based on statistically significant results  relating to the content of the manuscript. No financial benefits have been received by the authors.CJHS conceived the study, carried out the experimentation, and drafted the manuscript. FES conducted the statistical analysis and edited the manuscript. FSB and NL supervised the project and edited the manuscript. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/190/prepub


Equations to estimate GFR have not been well validated in the elderly and may misclassify persons with chronic kidney disease (CKD). We measured GFR and compared the performance of the Modification of Diet in Renal Disease (MDRD), the Chronic Kidney Disease-Epidemiology Collaboration (CKD-Epi) and the Berlin Initiative Study (BIS) equations based on creatinine and/or cystatin C in octogenarians and nonagenarians.Using cross-sectional analysis we assessed 95 very elderly persons (mean 85 years) living in the community. GFR was measured by iohexol (mGFR) and compared with estimates using six equations: MDRD, CKD-Epi_creatinine, CKD-Epi_cystatin, CKD-Epi_creatinine-cystatin, BIS_creatinine and BIS_creatinine-cystatin.2. Bias was smaller with the CKD-Epi_creatinine-cystatin and the CKD-Epi_creatinine equations (-4.0 and 1.7 ml/min/1.73 m2). Accuracy (percentage of estimates within 30% of mGFR) was greater with the CKD-Epi_creatinine-cystatin, BIS_creatinine-cystatin and BIS_creatinine equations . Among the creatinine-based equations, the BIS_creatinine had the greatest accuracy at mGFR < 60 ml/min/1.73 m2 and the CKD-Epi_creatinine was superior at higher GFRs . The CKD-Epi_creatinine-cystatin, BIS_creatinine-cystatin and CKD-Epi_cystatin equations yielded the greatest areas under the receiver operating characteristic curve at GFR threshold = 60 ml/min/1.73 m2 . In participants classified based on the BIS_creatinine, CKD-Epi_cystatin, or BIS_creatinine-cystatin equations, the CKD-Epi_creatinine-cystatin equation tended to improve CKD classification .Mean mGFR was 55  ml/min/1.73 m2, the BIS_cr equation seems to be the best alternative.GFR-estimating equations CKD-Epi_creatinine-cystatin and BIS_creatinine-cystatin showed better accuracy than other equations using creatinine or cystatin C alone in very elderly persons. The CKD-Epi_creatinine-cystatin equation appears to be advantageous in CKD classification. If cystatin C is not available, both the BIS_cr equation and the CKD-Epi_cr equation could be used, although at mGFR < 60 ml/min/1.73 m Chronic kidney disease (CKD) is a major public health problem and its prevalence is rising, particularly in the elderly, reflecting mainly the worldwide increased life expectancy of the population ,2. The mCystatin C is considered to be a potential alternative to serum creatinine for estimating GFR ,6. EvideThe aims of this study were to measure GFR, to compare the performance of some frequently used or recently described GFR estimating equations based on creatinine and cystatin C, alone or combined, and test their usefulness in the classification of CKD in individuals 80 years of age or older.This is a cross sectional study of community-dwelling independent elderly aged 80 years or older recruited in the southern region of the city of São Paulo, Brazil. The elderly were identified through regional surveys among those individuals living in the neighborhood of the Federal University of São Paulo campus from November 2010 through December 2011. During this period, 294 elderly were identified and after interview and physical examination 200 were enrolled in a longitudinal epidemiologic study at the Department of Geriatrics. All clinically stable individuals and independent in activities of daily living were acceptable to participate in the study. The exclusion criteria were: to be institutionalized, unable or unwilling to give consent, to have an acute infectious disease, a moderate or severe cognitive impairment, heart failure (NYHA class III or IV), known cirrhosis, previously received dialysis, unstable chronic pulmonary disease, previous immunosuppressive therapy within 6 mo., previous chemotherapy for cancer, known HIV infection, and previously reported allergic reaction to iodine. From August 2011 to May 2012, of the 294 initial individuals, 94 were excluded after checking the selection criteria. The purpose and the procedures for this study were explained by telephone to the remaining 200 and, of these, 97 returned to the clinics for the renal function studies scheduled within a week of the date of interview. There were no sociodemographic or clinical characteristics significantly different between the participants (n = 97) and the remaining 103 individuals enrolled in the epidemiologic Geriatric study that did not undergo renal function tests. The study protocol was approved by the Research Ethics Committee of the Federal University of São Paulo and all participants gave their written informed consent.After the blood sample was collected from each participant, the material was immediately processed and analyzed in the same day and always in the same laboratory at the university Hospital do Rim. Serum creatinine was measured using a modified kinetic Jaffé colorimetric method on an autoanalyzer , which was calibrated to isotope dilution mass spectrometry (IDMS) using a standard reference material (914a) traceable to the National Institutes of Standards and Technology (NIST). Total analytical variation (CV%) of the colorimetric method was 1.4-3.0%. The intra-assay coefficient variation (CV) for creatinine was 1.9%.Plasma cystatin C levels were determined by an automated particle-enhanced immunoturbidimetric method  using a 2 body surface area [Five mL of iohexol  were administered intravenously in an antecubital vein and blood samples were obtained at 2, 3, 4 and 5 hours after infusion. Iohexol clearance (referred to as ‘measured GFR’ (mGFR)) was calculated based on the measured decline in plasma concentrations of iohexol, using the slope intercept method (one-compartment model) . Plasma ace area .1. The Modification Diet in Renal Disease (MDRD) equation .2. The Chronic Kidney Disease Epidemiology creatinine (CKD-Epi_cr) equation .3. The Chronic Kidney Disease Epidemiology cystatin C (CKD-Epi_cys) equation .4. The Chronic Kidney Disease Epidemiology creatinine-cystatin C (CKD-Epi_cr-cys) equation .5. The Berlin Initiative Study creatinine (BIS_cr) equation .6. The Berlin Initiative Study creatinine-cystatin C (BIS_cr-cys) equation .2. The significance of the differences among equations was determined by the paired Student t test, and McNemar’s test for differences in proportions.Values are expressed as mean ± SD or median and interquartile range. Bias, precision and accuracy were measured to determine the performance of each equation . Bias wa2 for CKD [2 [The diagnostic accuracy of eGFR equations was evaluated using receiver operating characteristics (ROC) plots. The GFR determined with iohexol clearance was used as the gold standard and the cutoff value was set at 60 ml/min/1.73 m for CKD . Analyser CKD [2 .2 on the basis of one equation versus the other. Analyses were performed with the use of SPSS version 17  and MatLab version 5 , software.We assessed the use of the equations with best performances for the classification of CKD by means of the net reclassification index (NRI) statistic. We compared the proportion of participants who were reclassified as having a measured GFR that was less than 60 (or 45) ml/min/1.73 mOf the 97 elderly who underwent the renal function studies, two were excluded due to incorrect technical procedures for the iohexol determination, resulting in a sample of 95 participants for the analyses. The characteristics of the participants are shown in Table 2. Fifty-nine percent had mGFR <60 ml/min/1.73 m2 (Table 2 with the BIS_cr-cys equation and a lower percentage with the MDRD equation. Performance of the equations is summarized in Table Mean(±SD) iohexol clearance (mGFR) was 55 ± 15 ml/min/1.73 m2 (Table 2. The opposite was observed with mGFR ≥60 ml/min/1.73 m2. The best performances combining bias and precision (CRMSE) were obtained with both BIS equations and the CKD_cr-cys when GFR was <60 ml/min/1.73 m2; and with the CKD-Epi_cr-cys and the CKD-Epi_cr in the higher GFR subgroup. As for the accuracy within 30% the best performances were obtained with the BIS_cr-cys and the CKD-Epi_cr-cys equations at lower GFR levels; and the CKD-Epi_cr-cys and the CKD-Epi_cr at when GFR was ≥60 ml/min/1.73 m2 . The BIS_cr equation had a more stable performance in the two investigated GFR subgroups than the other creatinine-based equations and showed better accuracy at decreased mGFR levels.Similar trends were in general observed when these analyses were repeated according to mGFR < or ≥60 ml/min/1.73 m2) were the BIS_cr-cys, BIS_cr, and the CKD-Epi_cys; and the best specificity was achieved with the MDRD and the CKD-Epi_cr equations. The greatest area under the ROC curve was observed with the CKD-Epi_cr-cys and the BIS_cr-cys equations (0.88) followed by the CKD-Epi_cys (0.87) equation. Although the area under the curve of the CKD-Epi_cr-cys was greater compared with the MDRD and the CKD-Epi_cr equations, the differences were not significant . Considering the cutoff point of 45 ml/min/1.73 m2 the greatest area under the curve was again obtained with the CKD-Epi_cr-cys (0.81) equation.The performance of the equations in relation to the classification of the thresholds of mGFR was assessed by the area under the ROC curve Table . The equ2 on the basis of the other equations , BIS_cr  or the BIS_cr-cys  equations. Compared with the CKD-Epi_cr, reclassification with the combined CKD-Epi_cr-cys equation was not favorable and was more often incorrect in those with a mGFR ≥60 ml/min/1.73 m2 . In a similar analyses for reclassification based on a mGFR threshold of 45 ml/min/1.73 m2 the CKD-Epi_cr-cys equation tended to improve classification compared with all other equations; the NRI was statistically significant compared with the BIS_cr , marginally significant compared with the CKD-EPI_cr , and not significant versus the CKD-Epi_cys , and the BIS_cr-cys equation  . Some studies have reported that both the MDRD and, to a lesser extent, the CKD-Epi_cr equations overestimate GFR in individuals older than 70 years with CKD [2. The marker used for measuring clearance and the methodology of measuring creatinine were the same in our study and in the BIS and do not seem to explain these differences. One could speculate that residual differences in creatinine standardization  or differences in ethnicity, since the BIS participants were white Europeans, while our population is less homogeneous in terms of ethnicity, might play a role in the distinct findings. In fact, ethnicity influences creatinine-based GFR estimating equations. Flamant et al. [Among the creatinine based equations assessed, the MDRD equation overestimates mGFR and its use will decrease the diagnosis of CKD. It tended to be less accurate than the CKD-Epi_cr and the BIS_cr equations as previously reported ,9,15,20,with CKD . The BISt et al.  have sugt et al.  reportedSerum cystatin C appears to be less dependent upon muscle mass, less sensitive to metabolic and extra-renal factors than creatinine in the elderly and equations based on this marker seemed to be promising . In cont2 threshold. There was no significant difference in CKD classification comparing the equations using creatinine alone (CKD-Epi_cr and BIS_cr). The CKD-Epi_cr-cys combined equation tended to improve the reclassification of patients as having CKD compared with most of the others.The combined creatinine-cystatin C equations (CKD-Epi_cr-cys and BIS_cr-cys) had the greatest accuracy , although both (more markedly the BIS_cr-cys) underestimated GFR. As for the classification of CKD, the combined equations performed better than the others, followed by the CKD-Epi_cys. The ROC analyses revealed that the combined equations and the cystatin alone equation had the greatest areas under the curve at GFR =60 ml/min/1.73 mTo explain the tendency of best performance with the equations that combine creatinine and cystatin C it has been proposed that errors due to the non-GFR determinants of creatinine and cystatin C are independent and smaller in an equation that uses both markers than in an equation that uses only one marker. Possible reasons for the continued imprecision are the residual contribution of non-GFR determinants of each marker, as well as physiologic variation in GFR and error in measurement of GFR.Our study suggests that cystatin C should not replace creatinine in clinical practice; however, the combination of creatinine and cystatin C appears to provide more accurate GFR estimates in the very elderly. In adults, Inker et al.  showed tThe strengths of this study are the evaluation of mGFR using a precise method in a sample of very elderly persons, and the comparison and validation of several recently proposed equations to estimate GFR based on creatinine and cystatin C. None of the previous studies have specifically assessed the comparison of these new equations in this age group. The limitations of the study are: the relatively small sample of octogenarians and nonagenarians, owing to the difficulties in recruiting individuals in this age group for a lengthy procedure such as the GFR determination, resulted in a lower power to detect statistically significant differences; the number of participants and the recruitment methods used restrict the representativeness of the population as a whole; errors in the measured GFR may account for some imprecision and limiting interpretation of the analyses. The cystatin C assay was not calibrated against the international certified reference material ERM-DA471/IFCC prior the use in GFR estimating equations; this lack of standardization may have caused imprecision in the estimates. Therefore, these results should be interpreted with caution. Although the most reliable method to analyze plasma iohexol is with high performance liquid chromatography, previous studies have shown that measurements by capillary electrophoresis had an excellent correlation with those by high performance liquid chromatography . For rea2, the BIS_cr equation seems to be the best alternative.The combined CKD-Epi_cr-cys and BIS_cr-cys equations, although underestimating GFR, showed better accuracy than other equations using creatinine or cystatin C alone in very elderly persons. The CKD-Epi_cr-cys equation appears to be advantageous in CKD classification. If cystatin C is not available, both the BIS_cr equation and the CKD-Epi_cr equation could be used, although at mGFR < 60 ml/min/1.73 mCKD: Chronic kidney disease; GFR: Glomerular filtration rate; mGFR: Measured glomerular filtration rate; eGFR: Estimated glomerular filtration rate; MDRD: The modification diet in renal disease; CKD-Epi_cr: The Chronic kidney disease epidemiology creatinine; CKD-Epi_cys: The chronic kidney disease epidemiology cystatin C; CKD-Epi_cr-cys: The chronic kidney disease epidemiology creatinine-cystatin C; BIS_cr: The berlin initiative study creatinine; BIS_cr-cys: The berlin initiative study creatinine-cystatin C.The authors declare that they have no competing interest.MBL participated in the conception, design of the study, data collection, data analysis, data interpretation, and approved the manuscript. LQA participated in the conception, design of the study, data collection, data interpretation, and approved the manuscript. MTP participated in the data analysis, laboratory parameters analysis, data interpretation, and approved the manuscript. SKN participated in the data analysis, laboratory parameters analysis, data interpretation, and approved the manuscript. GMK participated in the conception of the study, data analysis, data interpretation, provided important intellectual content, critically revised the manuscript, and approved the manuscript. MSC participated in the conception, design of the study, data collection, data interpretation, provided important intellectual content, critically revised the manuscript, and approved the manuscript. RCS participated in the conception, design of the study, data analysis, data interpretation, provided important intellectual content, and has written the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/265/prepub2.Reclassification of the participants with the use of the CKD-Epi creatinine-cystatin C equation versus: the CKD-Epi creatinine, the CKD-Epi cystatin C, the BIS creatinine or the BIS creatinine-cystatin C for estimated GFR, according to the cut-off value of mGFR = 60 ml/min/1.73 mClick here for file2.Reclassification of the participants with the use of the CKD-Epi creatinine-cystatin C equation versus: the CKD-Epi creatinine, the CKD-Epi cystatin C, the BIS creatinine or the BIS creatinine-cystatin C for estimated GFR, according to the cut-off value of mGFR = 45 ml/min/1.73 mClick here for file


A receptors in hippocampal neurons. shRNA-mediated knockdown of CDK5 and pharmacological inhibition of cyclin-dependent kinases reduced phosphorylated gephyrin clusters and postsynaptic γ2-containing GABAA receptors. Phosphorylation of S270 is antagonized by PP1/PP2a phosphatase and site-directed mutagenesis and in vitro phosphorylation experiments indicate that S270 is a putative CDK5 phosphorylation site of gephyrin. Our data suggest that CDK5 plays an essential role for the stability of gephyrin-dependent GABAA receptor clusters in hippocampal neurons.CDK5 has been implicated in neural functions including growth, neuronal migration, synaptic transmission and plasticity of excitatory chemical synapses. Here we report robust effects of CDK5 on phosphorylation of the postsynaptic scaffold protein gephyrin and clustering of inhibitory GABA We report that knockdown or inhibition of CDK5 in hippocampal neurons results in reduced phosphorylation of postsynaptic clusters of the scaffold protein gephyrin, detected with the phospho-specific antibody mAb7a This study was carried out in strict accordance with the European Communities Council Directive (86/609/EEC) to minimize animal pain or discomfort. The local animal care and use committee (Interfakultäre Biomedizinische Forschungseinrichtung (IBF) Heidelberg) gave approval for the study under the number T-81/10.BamHI and HindIII restriction sites and were cloned in the expression vector pCMVTAG 3B (Invitrogen) resulting in N-terminal fusion constructs with a myc-epitope (myc-CDK5). Sequences encoding for three shRNA directed against CDK5 and scrambled shRNA were cloned into pFSGW, a modified version of the lentiviral vector pFUGW DNA fragments encoding the complete coding sequence of rat CDK5 were amplified by PCR from embryonic brain cDNA creating flanking Site-directed mutagenesis of 6xHistidine-tagged gephyrin was performed using the Quickchange Lightning mutagenesis kit from Stratagene following the suppliers' instructions. Mutated sequences were verified by sequence analysis.Recombinant lentiviral particles were produced as described earlier 2 as described previously Primary cultures of rat hippocampal neurons were prepared from E19 embryos plated at a density of 60.000 cells/cmTreatment of cells was done as described earlier AR gamma2-subunit antibody (1∶10) , rabbit anti-vesicular inhibitory amino acid transporter (VIAAT) antibody (1∶2000) , rabbit anti-CDK5 antibody (1∶200) . Analysis of antibody labeling was performed using an Axiovert 200M microscope (Zeiss) equipped with a SPOT RT CCD camera . Images were collected with a 40× or a 63× Plan-Apochromat (1.4 NA) objective (Zeiss). For quantifications MetaMorph and Fiji software were used. For quantification the fluorescence intensities of single synaptic puncta, mAb7a and Ab-175 fluorescence images were taken with a Leica confocal TCS SP8 microscope using a 63× Plan-Apochromat (1.4 NA) objective. To evaluate the number of gephyrin or GABAA receptor clusters, one to three proximal dendritic regions with a length of 30 µm were chosen. For determining the number of synaptic gephyrin clusters VIAAT-fluorescence was used for region selection and consequently the respective postsynaptic immunofluorescence signals were analyzed. After setting the same threshold for these regions for all immunolabelings of one experiment, the quantification of gephyrin or γ2 puncta was determined by the software. Data analysis was done using Excel and Prism software.Immunocytochemistry was performed as described earlier N-terminaly 6xHistidine-tagged gephyrin wild type and 6xHistidine-tagged gephyrin-mutant proteins were expressed and purified as described elsewhere 32P-labeled gephyrin was done using the gephyrin-specific antibody mAb5 in vitro phosphorylation assays performed in HEPES buffer with 30 µCi [γ-32P] ATP (3000 Ci/mmol) and 10 µM unlabeled ATP, 0.1 U CDK5/p25 GST protein complexes  at 30°C for 30 min and further processed as described earlier Immunoprecipitation of 32P-phosphorylation assay as mean ± SD. Values of P<0.05 were considered as significant.Experimental data were evaluated of at least three independent experiments without knowledge of the experimental conditions used. Statistical analysis was performed using the GraphPad-prism IV software with non-parametric ANOVA followed by Tukey's or Bonferroni's multiple comparison tests, other data were analyzed with Student's t-test. Data are shown as mean ± standard error of the mean (SE) or in the analysis of the in vitro To investigate the functional role of CDK5 for the phosphorylation of gephyrin clusters at GABAergic synapses of cultured hippocampal neurons, we first established a knockdown of CDK5 with three different shRNA expressing lentiviruses. After having established that these sequences efficiently reduced heterologously expressed, myc-tagged CDK5 in HEK293T cells (data not shown), the reduction of CDK5 expression by these shRNAs in cultured hippocampal neurons was analyzed by immunofluorescence microscopy. As control, a scrambled shRNA (mismatch) was used.Cells infected by the CDK5-kd viruses - as indicated by GFP expression  - revealA receptors, the subcellular localization of GABAA receptor γ2 subunits was investigated. GABAA receptors harboring this subunit are known to colocalize with and to depend on gephyrin for synaptic localization in hippocampal neurons A receptor γ2-immunoreactive puncta along dendrites could be seen, whereas the immunoreactivity within the soma was preserved. The quantification shows that the number of GABAA receptor γ2 clusters is significantly reduced  was analyzed. Although a small reduction of the number of VIAAT puncta could be observed in kd2 infected cells  to 4.33±0.96 puncta/30 µm dendrite  dendrite  after thTo investigate the removal of gephyrin phosphorylation seen upon kinase inhibition we performed experiments inhibiting PP1/PP2a phosphatases known to be involved in the regulation of phosphorylation of gephyrin In order to study the level of mAb7a immunoreactivity and thus the phosphorylation of S270 more directly, we performed CDK activity recovery experiments. For this, we inhibited CDK5 with 5 µM SU9516 for 6 hrs, replaced the culture medium by conditioned medium from sister cultures to terminate CDK inhibition, thus allowing a putative increase of gephyrin phosphorylation for 1 or 2 hours. The incubation of cells was stopped by fixation and cells were double stained for gephyrin with Ab-175 and mAb7a. Next, relative protein levels of somatic and dendritic gephyrin clusters were estimated by quantification of Ab-175 immunofluorescence intensities in focal sections of stained cells by confocal microscopy. The same focus plane and cluster regions were also analyzed for relative fluorescence intensities of mAb7a immunofluorescence . The ratin vitro at serine and/or threonine residues by endogenous protein kinases S270A was subjected to in vitro kinase assays using CDK5/p25 and [γ-32P] ATP. Wild type gephyrin was labeled strongly with 32P after incubation with CDK5/p25  and dimerization of the carboxy-terminal domain (E-domain) These results are in agreement with the current model of gephyrin and receptor clustering. It was postulated that at postsynaptic membrane specializations gephyrin forms a regular hexagonal lattice by trimerization of the in vitro kinase assays using CDK5/p25 and [γ-32P] ATP. Consistent with earlier findings -/- mice revealed reduced glycine receptor cluster numbers To verify whether gephyrin serves as a CDK5 substrate, gephyrin and a gephyrin mutant were subjected to Other studies have also shown that gephyrin is phosphorylated on several serine and threonine residues, mostly in the central region (C-domain) A receptor subunits are regulated by phosphorylation of subunits 2+ calmodulin- dependent protein kinase II, resulting in a rapid insertion of GABAA receptors in the cell membrane A receptor γ subunits are phosphorylated at Y365 and Y367, reducing receptor internalization and increasing tonic inhibition A receptor cluster formation or stability independent of gephyrin phosphorylation.Posttranslational modification of receptor subunits is another mechanism to determine the formation of postsynaptic assemblies of ligand-gated ion channels and scaffold proteins. For example, protein kinase C was shown to regulate the glycine receptor (GlyR) diffusion properties and gephyrin interaction by phosphorylating residue S403 of the β-subunit of the GlyR A receptor clustering at inhibitory synapses.In spinal cord neurons, gephyrin clustering is regulated by GlyR activity