Staphylococcus aureus) is Gram positive and the second type (Escherichia coli) is Gram negative (by using agar well diffusion method). Finally, it was found that compounds show different activity of inhibition on growth of the bacteria.New binuclear Co(II) and Co(II) complexes of ONO tridentate heterocyclic Schiff base derived from 4-aminoantipyrine with salicylaldehyde have been synthesized and characterized on the bases of elemental analysis, UV-Vis., FT-IR, and also by aid of molar conductivity measurements, magnetic measurements, and melting points. It has been found that the Schiff bases with Cu(II) or Co(II) ion forming binuclear complexes on (1 : 1) “metal : ligand” stoichiometry. The molar conductance measurements of the complexes in DMSO correspond to be nonelectrolytic nature for all prepared complexes. Distorted octahedral environment is suggested for metal complexes. A theoretical treatment of the formation of complexes in the gas phase was studied, and this was done by using the HyperChem-6 program for the molecular mechanics and semi-empirical calculations. The free ligand and its complexes have been tested for their antibacterial activities against two types of human pathogenic bacteria: the first type ( Amino heterocyclic compounds containing two or more potential donor centers play an important role in the study of competitive reactivity of an bidentate ligand system . HeterocPhenazone ligand can form mononuclear and binuclear complexes –5. Transition metal complexes containing a salicylaldehyde are commonly found in biological media and play important roles in processes such as catalysis of drug interaction with biomolecules . PhenazoIn this paper we are reporting the synthesis of the binuclear Co(II) and Cu(II) complexes of some heterocyclic Schiff base ligands  containiA Fisher-100 infrared spectrophotometer was used to record the IR spectra as KBr and CsI disc, and UV/VIS spectra were measured by a HITACHI U-2000 spectrophotometer. Determination of all metals percentage by atomic absorption spectrophotometry on AA-680G (Shimadzu). Electrical conductance was measured on conductivity CDC304 (Jenway4070) melting points determined by an electric heated block apparatus  and were uncorrected. Room temperature magnetic susceptibility measurements were carried out on a B.M 6 BRUKER type magnets, balance. Diamagnetic correction was done using pascal constants.All the chemicals and solvents used for the synthesis were reagent grade and procured from . Metal salts were purchased from E. Merck and used as received. All solvents were dried and purified before used.The ligands were prepared by condensation of 4-aminoantipyrine with salicylaldehyde in ethanol. This preparation was performed as cited in the literature .The general structures of ligands obtained from chemical analysis and spectral methods are given in 2·4H2O (0.20 g)] in 15 mL ethanol was added dropwise with continuous stirring. The mixture was stirred further for 2-3 h. at 80°C. The precipitated solid was then filtered off, washed with diethylether, followed by cold ethanol, and dried under vacuum. The same method was applied for the preparation of [CoCl2·6H2O] complexes by using the corresponding (L1 or L2) working in the same conditions with their respective molar ratio.1.00 mm of the ligands were dissolved in 30 mL of ethanol, and solution of 1.00 mm of the metal salts [CuClThe physical properties of prepared complexes are listed in Escherichia coli that are gram negative (−ve) and Staphylococcus aureus that are gram positive (+ve) to be cultivated and as control for the disc sensitivity test [ The biological activity of the ligands and their metal complexes were studied against two selected types of bacteria which included ity test . This me−1 cm2 mole−1−3 M) at room temperature. Polydentate complexes were obtained from 1 : 1 molar ratio reactions with metal ions and L1 and L2 ligands. The ligands L1 and L2 on reaction with Cu(II) and Co(II) salt yields complexes are corresponding to the formulas [Cu2(L1)2H2O], [Co2(L1)2H2O], [Cu2(L2)2(H2O)4], and [Co2(L2)2(H2O)4].The Schiff bases ligands are soluble in common organic solvents. But its metal complexes are generally soluble in DMF and DMSO. The elemental analytical data of the complexes reveal that the compounds have “metal : ligand” an ion stoichiometry ratio of 1 : 1; the analytical data and other spectral analysis are in good agreement with the proposed stoichiometry of the complexes. The colors, yields, melting points, IR, and electronic absorption spectral data of all the compounds are presented in −1 for L1, 1720, 1625, and 1290 cm−1 for L2 which may be ascribed to the stretching vibrations of C=O groups, C=N (imine) and C–O (phenolic) groups, respectively [−1 due to the stretching mode of the C=N group in the spectrum of the free ligands shows a remarkable negative shift with splitting in the 1590–1610 cm−1 region in all the complexes spectra suggesting that the coordinating azomethine nitrogen atoms of the Schiff bases are involved in the complexes formation [The most important infrared spectral bands of the investigated metal complexes in the present article are summarized in ectively . The ban−1 is shifted to lower frequency (10–30 cm−1). It is suggested that the oxygen atom of this phenolic (C–O) group is bridged to the metal ions. An additional band at 1160 cm−1 suggests that water molecules are coordinated to metal ion [−1. Other band of M–O and M–N bands appear respectively at 535–515, 460–470 cm−1In the spectra of all binuclear complexes, the phenolic band at 1260–1270 cmetal ion . This ban-π* transitions of the azomethine group. During the formation of the complexes, these bands are shifted to lower wavelength, suggesting that the nitrogen atom of the azomethine group is coordinated to the metal ion. The values in the 325–245 nm range are attributed to the π-π* transition of the aromatic rings. In the spectra of the complexes, these bands are shifted slightly to lower wavelength.The electronic spectra were recorded in DMSO. In the spectrum of the ligand, the bands in the 380–340 nm range are assigned to the +2) On the basis of the magnetic moment measurements, the Cu(II) complexes at room temperature probably have a binuclear structure with phenolic oxygen bridges. The magnetic moment lies in the range 0.9–1.69 B.M  ions [−1.The electronic spectra of the copper complexes II) ions . The spe2B1g → 2A1g  (dx2 − y2 → dz2),2B1g → 2B2g  (dx2 − y2 → dzy), and 2B → 2Eg  , and these transitions suggest D4h symmetry. The energy level sequence will depend on the amount of tetragonal distortion due to ligand field and Jahn Tellar distortion effect.These bands may be considered to the following three spin allowed  transitiAt room temperature the magnetic moment measurements of Co(II) complexes at 4.90–5.04 B.M correspond to three unpaired electrons, −1; these bands may be assigned to the following transitions: 4T1g  (F) → 4T2g  (ν1), 4T1g → 4A2g  (ν2), and 4T1g(F) → 4T2g  (P)  (ν3), respectively [The electronic spectra of all the Co(II) complexes display absorption at 10370, 14380, 18720, and 356960 cmectively . It is dectively , 21, 22.According to the results obtained from  measurements for the prepared complexes, the proposed molecular structure of the complexes has an octahedral structure as shown in The ball and cylinders and some of selected structural parameters (bond length and angles) of the optimized geometries are shown in As shown in this figure, there is no obvious trend for the variation of these parameters. The values of the bond length and angles of the optimized geometries are quite similar to the experimental results of the corresponding compounds.Staphylococcus aureus (gram positive) and Escherichia coli (gram negative) bacteria at 25°C. The disc of whatman no. 4 filter paper having the diameter 6.00 mm was soaked in the solution of compounds in DMSO (1.0 mg cm−1). After drying it was placed on nutrient agar plates. The inhibition area wasobserved after 48 hr. DMSO used as control Staphylococcus aureus, which is known as a resistant to most commercial antibiotic. The Schiff base ligands had more antibacterial activity than other ligands used, and this effect may be due to the presence of –ph, –OH and –N=C– groups which are electron releasing. The antibacterial results evidently showed that the activity of the ligand compounds became more pronounced when coordination to the metal ions.The ligand and its transition metal complexes were evaluated against different species of bacteria , 24. The


Microtus rossiaemeridionalis population.Here, we present the morphotypic variety of the m1 and M3 teeth diagnostics for the recently formed isolated population of the sibling vole in Far Eastern Russia. In the Far Eastern population, the prevalence of the individuals with m1 with a complicated crown of the forward unpaired loop of the paraconid is characteristic. Namely, m1 in these individuals shows well-expressed sixth exterior and fifth interior salient angles. The structure of the M3 morphotypes is also unique in the sibling voles in Far Eastern Russia. The dominant morphotypes were typica (47 %) and simplex (45 %), whereas the abundance of the duplicata morphotype was 0.08 %. The frequencies of various m1 and M3 morphotypes found in casually introduced sibling voles in the Far East are not typical of any previously studied  The original area of distribution of the sibling vole is located between 30–60° E and 60–40° N. Isolated sibling vole populations are found in the south of Russia, in Krasnoyarskij Kraj, Khakassia, and Irkutsk Oblast. In 2009–2010, a new isolated population of this species was discovered in the south of the Russian Far East in Khabarovskij Kraj in the vicinity of Sovetskaya Gavan City   and 64 right and left upper molars (M3) belonging to 33 individuals caught in Khabarovsky Krai, in urbanized biotopes in the vicinities of Sovetskaya Gavan City and two nearby settlements  (Nadachowski simplex (s), typica (t), duplicata (d), and variabilis (v). The simplex morphotype is defined by interior and exterior tooth sides with three salient angles each. The typica morphotype is defined by four salient angles at the interior edge and three salient angles at the exterior edge. The duplicata morphotype is defined by four salient angles at each edge. The variabilis morphotype is defined by four to five salient angles at the interior edge and five to three or four salient angles at the exterior edge.In this study, we focused primarily on qualitative traits corresponding to the development of additional salient angles on the lingual or/and on buccal side of a tooth. For m1, we used the classification proposed by Markova et al. , includiMicrotus rossiaemeridionalis found in the Far East do not surpass the variability limits of the species. The following averaged measurements characterize the Far Eastern individuals. All measurements were made in millimeters. The body length is 97.6 ± 9.2 (n = 18), the tail length is 35.2 ± 3.5 (n = 16), the hind foot length is 15.4 ± 1.4 (n = 18), and the ear length is 11.2 ± 1.0 (n = 18). The condylobasal length is 23.3 ± 1.5 (n = 17), the length of the upper teeth is 5.6 ± 0.4 (n = 16), the length of m1 is 2.65 ± 0.10 (n = 16), and the length of M3 is 1.78 ± 0.09 (n = 16). All individuals studied have six plantar calluses.Basic morphological and craniological characteristics show that the M. rossiaemeridionalis, morphotype I of m1 and typica of M3 are predominant in all samples  and typica and duplicata .M. rossiaemeridionalis populations, including ten populations from Europe (338 m1/329 M3), two populations from Middle Urals (180 m1/180 M3), three populations from Southern Urals (62 m1/44 M3), and one population from Siberia (30 m1/30 M3)  and simplex (45 %), whereas the abundance of the duplicata morphotype was 8 % Fig. . A similM. rossiaemeridionalis easily enters man-made structures and reaches high population numbers in urbanized areas (Tikhonov et al. M. rossiaemeridionalis have been collected near the tidal zone in the vicinity of the port facilities in the settlement of Lososina.M. rossiaemeridionalis. Morphotype analyses of m1 and M3 exist only for series of selected areas of the entire range of this species (Markova et al. It is still not possible to infer the area of origin of the introduced populations of


We propose that variabilities should be separately measured from the VO2 and VCO2 averages to understand technological differences among metabolic monitors when they estimate the REE. To prove this hypothesis the mixing chamber (MC) and the breath-by-breath (BbB) techniques measured the VO2 and VCO2 averages and their variabilities. Variances and power spectrum energies in the 0–0.5 Hertz band were measured to establish technique differences in steady and non-steady state. A hybrid calorimeter with both IC techniques studied a population of 15 volunteers that underwent the clino-orthostatic maneuver in order to produce the two physiological stages. The results showed that inter-individual VO2 and VCO2 variabilities measured as variances were negligible using the MC while variabilities measured as spectral energies using the BbB underwent 71 and 56% (p < 0.05), increase respectively. Additionally, the energy analysis showed an unexpected cyclic rhythm at 0.025 Hertz only during the orthostatic stage, which is new physiological information, not reported previusly. The VO2 and VCO2 inter-individual averages increased to 63 and 39% by the MC (p < 0.05) and 32 and 40% using the BbB (p < 0.1), respectively, without noticeable statistical differences among techniques. The conclusions are: (a) metabolic monitors should simultaneously include the MC and the BbB techniques to correctly interpret the steady or non-steady state variabilities effect in the REE estimation, (b) the MC is the appropriate technique to compute averages since it behaves as a low-pass filter that minimizes variances, (c) the BbB is the ideal technique to measure the variabilities since it can work as a high-pass filter to generate discrete time series able to accomplish spectral analysis, and (d) the new physiological information in the VO2 and VCO2 variabilities can help to understand why metabolic monitors with dissimilar IC techniques give different results in the REE estimation.The aim of this paper is to understand how to measure the VO Finally, the inter-individual VO2 and VCO2 averages were computed with the purpose to compare the techniques’ performance in the REE estimation.The objective of this work was to develop a pilot study where young healthy volunteer population was submitted to the physiological clino-orthostatic maneuver (COM) with the idea to generate steady/clinostatic and non-steady/orthostatic stages as a manner of an instrumental bench test  and vVCO2(f) with units in (ml/bth)2/Hz in the frequency range of 0–0.5 Hertz. Thus, individual spectrum functions were generated when each subject (N = 15) was submitted to the steady/clinostatic and then to the non-steady/orthostatic stages of the COM.The Figure vVO2(f) and vVCO2(f) showed that their main energy was concentrated in the low frequency (LF) and medium frequency (MF) bands, where the analysis bands were defined as LF = 0–0.04, MF = 0.04–0.15 and HF = 0.15–0.50 Hertz. Here, it is noteworthy that the band definition for the variabilities was analogous to how the heart rate variability is analysed with the purpose to facilitate their physiological interpretation (Task Force of the European Society of Cardiology vVO2(f) and vVCO2(f) have an unexpected cyclic rhythm energy with central frequency at 0.025 Hz that is generated only during the non-steady/orthostatic stage of the COM with an energy increment of 71% and 56%, respectively, when this is compared with the steady state energy.The averaged functions 2 increments by frequency bands. For instance, the total energy of vVO2(f) was computed as the integral below the function so that in orthostatic stage is approximately 50% higher in magnitude than the total energy of the vVCO2(f) for the same stage. A comparative analysis for the variability energies as a consequence of the COM application is shown in the Table The Figure SDVO2)2 and (SDVCO2)2 are compared against the total variabilities’ averaged energy obtained from the vVO2(f) and vVCO2(f) functions.The Table vVO2(f) and 56% for the vVCO2(f) (p < 0.05) as it is also seen in Figure The variance averages increment due to the COM were not statistically significant (p > 0.1), while the spectral total energy increment were 71% for 2 and VCO2 average comparison according to the model of the Figure VO2[n] and VCO2[n]) model was considered in order to compute separately the averages for the MC and BbB techniques. Then, the averages for the MC technique (VĊO2[n]MC) were incremented 63% and 39% (p < 0.05), while the averages for the BbB technique (VĊO2[n]) were only increased 32% and 40% (p < 0.1), when the COM was applied. The statistical differences analysis was based on the Welch t-test for unequal variances.The Table t-test for the inter-individual VO2 and VCO2 averages, after applying an ANOVA one-factor test for multiple measurements. However, it is worth to observe that the difference between the Although all computed averages in Table 2 and VCO2 variabilities in order to understand the causes of variations in the REE estimation. The Table 2 and VCO2 variabilities. The MC technique works as a low pass filter such that it suppresses the high frequencies of the variabilities generating more adequacies to measure the averages of the VO2 and VCO2. Thus, the analysis of averages in Table 2 and VCO2 averages  has been accepted as the reference instrument when new metabolic monitors are compared against its performance, mainly during IC studies in critical care patients. It is clear that the canopy in the Delta Track II performs as an open circuit MC technique with the capability to reject high frequency variabilities. Deltra Track’s hardware can be modelled as a time average filter so that the VOvVO2(f) and 53% for the vVCO2(f) when subjects are submitted to the orthostatic stage. These results suggest, the BbB technique is more suitable for monitoring and controlling the subjects’ physiological condition. This is an improvement over only using the traditional concept of CV. In addition, the cyclical rhythms found at 0.025 Hz in Figure vVO2(f) and vVCO2(f) in frequency bands similarly to the way that heart rate variability (HRV) is processed; after which, one second step would be to correlate the energy found in LF and MF with the LF energy of the HRV in order to discard whether or not the rhythmicity is due to sympathetic neural control or not  The MC technique is the appropriate method to estimate the VO2 and VCO2 averages, whereas the BbB technique is the most suitable procedure to provide physiological information to determine how stable or unstable an IC study is. (c) The cyclical rhythm in the BbB technique cannot be interpreted as instrumental noise since the O2 and CO2 transducers are performing independent measurements in open circuit calorimeters that allow to compute the vVO2(f) and vVCO2(f) separately. (d) The discrete gas exchange modeling of the Figure 2 and VCO2 variability measurements can be considered as an input–output paradigm assessment in order to search for the metabolic monitors’ performance.New and advanced IC metabolic monitors designs should consider the following issues: (a) the VO2 and VCO2 variabilities are defined at the output as SDVO2 and SDVCO2 and vVO2[n] and vVCO2[n], respectively. Likewise, the averages are defined as VĊO2[n]MC for the MC technique and VĊO2[n] for the BbB method.The Figure VO2[n] and VCO2[n]) in which the variabilities are implicitly included before they are separately measured. The argument n = 1,2,3… stands for a discrete time series that represents the breath by breath gas exchange during an IC study. The model in Figure This model for the MC and BbB techniques considers a discrete gas exchange at the input of the mouth (VO2(t) and VCO2(t)) is sampled by the lung’s mechanical ventilation. Then a discrete gas exchange VO2[n] and VCO2[n] is generated when the breath by breath instant flow f(t) works as a sampling function as in Equations ..VO2(t) af (t) is the instant expired flow (L/sec). VT is the tidal volume (ml) without BTPS  to STPD  volumetric corrections in order to preserve the simplicity of the model, FIO2(t) – FEO2(t) is the inspired-expired oxygen fraction difference and the FECO2(t) is the expired CO2 gas fraction. All gas fractions are in atmospheric percentages (%).Where: f(t) × VO2(t) and f(t) × VCO2(t) generate the continuous sampling for the O2 and CO2 uptake during each expired breath with time duration D1, D2, …Dn. Hence, individual and different breath-by-breath sample volumes are produced as VO2[Dn] and VCO2[Dn]. These volumes are computed as in Equations (The products quations  and 4)..f(t) × Vquations .34g (t – Dn) are continuous gate functions with the same time duration D1, D2,..Dn that allow synchronization to integrate the products between the instant flow and the instant gas fractions as it is seen in Figures f(t) × VO2(t) and f(t) × VCO2(t) are needed in order to match with the f(t) peak amplitude the VO2(t) and VCO2(t) values so that Equations   g  and the time gas fraction signals (FEO2(t) and FECO2(t)). This time lag depends on the sensors’ time response and the delay produced by the tubing length which utilizes a flow of 150 ml/min to sample the sensors. The hybrid calorimeter with the open pneumatic circuit is sketched in Figure VO2[Dn] and VCO2[Dn] value is placed in a time series using the sequence δ[n] with mathematical proprieties that allow the generation of the BbB discrete time series according to Equations  as it is seen in Figure Dn is extended as a dumb variable  just to be interpreted as an index to generate the BbB discrete time series VO2[n] and VCO2[n]. The Figure νVO2[n] and νVCO2[n] variabilities are computed. The average values (VĊO2[n]) are calculated from the discrete gas exchange as in Equations ..δ  and νVCO2[n] is shown in the Figure VO2[n] and VCO2[n]. Then, one sample per second was used to resample the reformatted discrete time series in order to obtain a frequency domain analysis in the range of 0.0 to 0.5 Hz. The processing window was selected to capture at least 15 minutes of data so that a Welch power spectrum estimator allowed a maximum resolution of 0.005 Hertz using 50% of data overlapping. The frequency band analysis was defined in three main regions: low frequencies (LF = 0–0.04 Hz), medium frequencies (MF = 0.05–0.15 Hz) and high frequencies (HF = 0.16–0.5 Hz). These band divisions are similar to the heart rate variability analysis with special emphasis in the LF and MF bands since the HF band is assumed to be related with instant flow’s frequency (respiratory frequency) activity as it is seen in the example of the Figure An example of the power spectrum analysis of the VO2[n] and VCO2[n], having the model in Figure VĊO2[n]MC are computed using the criteria of 30 averages to smooth enough the gas exchange. Thus, each one of the 30 averages is formed with M breaths to obtain one average sample during a total of 15 minutes per each IC study.The measurement of the averages and variabilities using the MC technique requires modeling the effect of the mixing chamber upon the discrete gas exchange The measurement of the variabilities in the MC technique was computed as in Equations  and 14)14). EvenA specific open-circuit hybrid indirect calorimeter (MGM-3) was designed and manufactured for the purpose of this work which was based in the design of Westenskow et al. . The MC 2, 10% CO2, complemented with N2. Additionally, two more gas certified mixture cylinders  were used to adjust the transducer offsets and gains for the case of doing IC studies in ambulatory patients. The MC technique was implemented by displaying values of VE (expired volume minute in L · min-1), RF (respiratory frequency in breaths · min-1), VO2 (ml · min-1), VCO2 (ml · min-1), VT  and RQ (respiratory quotient VO2/VCO2) every 20 seconds. These readings were automatically corrected and displayed at STPD conditions after measuring volumes and fractions at BTPS conditions . The VO2 was computed using the Haldane correction.The MGM-3 calibration and quality control unit was a microprocessor based design and was calibrated every 5 minutes using a reference gas cylinder with a certified mixture of 21% O-2. All subjects gave signed informed consent to be studied in the morning after 8 hours of fasting. Subjects were asked to perform the active clino-orthostatic maneuver (COM). First, a 5 minute period of relaxing was used before he/she lied down on a couch and was then submitted to the COM while connected to the MGM-3 calorimeter. Two 30 minutes periods were used to implement the measurement protocol: 15 minutes for the MC technique and 15 min for the BbB technique in each COM position. All of the measurements were made in the same room maintaining constant temperature and data collection by the same expert team in all cases.A population of 15 young normal volunteer subjects without a history of any chronic disease was studied. The ages ranged from 18 to 30 years with a body mass index (BMI) average of 24.2 ± 3.8 Kg · mVĊO2[n]MC were compared against the BbB averages SDVO2)2 and (SDVCO2)2 were compared against total spectral energy averages. The statistics analysis was parametric since the variables were considered to be Gaussians, once they were tested by the Kolmogorov–Smirnov test. Then, two-tailed paired t-tests (Welch version) were used as appropriate for unequal variances. In all cases, the null hypothesis was rejected when p ≤ 0.1 since this experiment was considered to be a pilot study.Comparative statistical paired data analysis was applied intra-groups. The MC averages


Interaction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1α (ck1α) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV.Serine 232 of NS5A is known to be phosphorylated by human ck1α. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1α has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1α has been identified from the model and these are found to be conserved well in the ck1 family. ck1α – substrate peptide complex has also been used to understand the structural basis of association between ck1α and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available.Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified.The substrate interacting residues in ck1α have been identified using the structural model of kinase - substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy. A small molecule targeted to specifically bind to the suggested part of NS5A to prevent its interaction with ck1α could be very useful.The crystal structure of p53 transactivation domain containsModeling of p53 transactivation domain [PDB id: 1YCR] bound to ck1α showed the analogy of substrate recognition and interaction with ck1α. The substrate interacting residues arginine 214, glycine 251 and lysine 260 makes ionic interactions with the substrate constraint residue glutamic acid 17 in the crystal structure of the transactivation domain of p53 tumour suppressor was useda19 was NS5A could have independently evolved the binding features of human PKR which is a mechanism commonly observed in viruses to evade the host system. This asHowever, the 3-D structure of NS5A is unlikely to be similar to the tertiary structure of PKR and also a previous study showed tA portion of NS5A ISDR that could mimic the binding of a PKR protomer to other PKR protomer has been shown in the FigureThe conservation of the predicted PKR interacting residues from all NS5A-1b sequences (genotype 1b −331 sequences) was found to be above 90%. Even though Val 296 is similar to the dimer interface residue in the alignment, it was conserved below 80% and this position is highly variable with substitutions like I, P and M. So this position is not predicted as a PKR interacting residue.It was further considered that the predicted PKR binding residues from genotype 1b would be different to those equivalent residues in genotype 2a and 3a which are unable to bind and inhibit PKR efficiently. Multiple sequence alignment  predicted to be PKR interacting residues in NS5A 1b by the method of aligning NS5A 1b and PKR, has also been picked by evolutionary trace analysis. This clearly shows the importance of these residues in binding to PKR.These observations again emphasizes that both ISDR as well as V3 region are involved in PKR inhibition which are primarily genotype specific as already shown by experimental studies,29,30,40The finding from evolutionary trace analysis is consistent with the experiments done previously showing the importance of NS5A ISDR and V3 region to bind to PKR.V3 region in NS5A was found to be crucial for providing specificity to binding/ poor binding efficiency to PKR in the case of genotype 1b and genotype 2a/3a respectively,40. The It should be noted that not all the variable positions within NS5A 1b sequences may be completely responsible for their interaction with PKR. Moreover NS5A has binding partners other than PKR and neutStructural modeling has provided insights about the structural basis of interaction and recognition of NS5A peptide containing the phosphorylation motif with ck1α. This shows the viral mimicry of the substrate recognition motif for human ck1α. Mutation of Arg 214 and Lys 260 which are predicted to interact with phosphorylated Ser in ck1α could serve as an interesting validation of the model.Analogy of kinase recognition and substrate interaction is shown by considering another substrate of ck1α which is p53 tumour suppressor transactivation domain whose crNS5A phosphorylation by ck1α serves as a regulatory switch between viral replication and RNA translation/packaging. On the It is interesting to speculate that HCV infection could trigger phosphorylation of p53 by ck1α as a host response which gets counteracted by the HCV NS5A sequestering the p53 to prevent apoptosis,45.Thus, identification of residues in human ck1α which interacts with NS5A and p53 in this work could provide further insights to understand the interplay between ck1α and its two substrates (NS5A and p53) which could influence the outcome of the viral infection.Two different methods have been used to narrow down the range in NS5A which might interact with PKR. Critical residues responsible for interaction of NS5A 1b with PKR and residues unable to bind and inhibit PKR efficiently in NS5A 2a/3a have been predicted for the first time. The functional significance and nature of interaction of ISDR and V3 region in NS5A with PKR which was previously identified experimentally,29,30,40Even though the clinical response to interferon therapy has been studied for other HCV genotypes like 4, 5 and 6, the molRecent work on evolution of PKR showed that each of its 3 domains evolve rapidly to evade the viruses. Few residues within the dimerisation region of PKR have been shown to undergo positive selection. This coThe information of interface residues in NS5A 1b and PKR would be very helpful to design inhibitors that would prevent this interaction to enable the HCV genotype 1 infected patients respond well to the interferon therapy like the other genotype infected patients of HCV.Schizosaccharomyces pombe using COMPOSER[Human ck1α sequence [NCBI: NP_001020276.1] was retrieved from NCBI database. It was searched for similar sequences with known crystal structure in the protein databank by using BLAST program. The homCOMPOSER,49, whicCOMPOSER,51. An iNS5A peptide S(P)SAS has been modeled on to the structural model of ck1α which is re-generated based on the crystal structure of cAMP dependent kinase [PDB id: 1ATP]. This isA model of p53 transactivation domain for which the crystal structure is available containing the phosphoacceptor serine bound to ck1α model was generated as discussed in the previous section. It was made sure that the structure of p53 transactivation domain does not have any short contact with the kinase and makes favourable interactions with the residues of the kinase.http://www.kinase.com/kinbase) was carried out using CLUSTALW[Multiple sequence alignment of the members of casein kinase 1 family . The refIn this study, trace residues have been defined as those residues which show a distinct sequence pattern in a particular position of both the multiple sequence alignment which is compared. Trace residues are picked up if complete/ high conservation (mostly >90% and very few residues showing >80%) / conservative substitution of two different residues or a residue and a gap from both multiple sequence alignment are being compared.Seven representative sequences each from genotype 1b, 2a & 3a were chosen for the evolutionary trace analysis,32. The The authors declared that they have no competing interests.GS carried out the analysis. GS, YD and NT carried out the protein structure modeling. NS and SD conceived the study. All authors read and approved the final manuscript.Multiple sequence alignment of members among casein kinase 1 family. The additional file provides multiple sequence alignment and the details of substrate interacting residues in human ck1α conserved within casein kinase 1 family. Arginine 214, lysine 260 is absolutely conserved and conservatively substituted respectively. Glycine 251 is not conserved because the main chain nitrogen is involved in its interaction with the substrate constraint residue – phosphoserine 229. These substrate interacting residues are highlighted in blue. The aspartic acid 140 of human ck1α probably avoids the proximity of substrate constraint acidic residue phosphoserine 229 of NS5A. Aspartic acid is conservatively substituted among the ck1 family and is highlighted in yellow.Click here for filePrediction of PKR interacting residues in NS5A 1b. (1) Alignment of PKR dimerisation region with ISDR (Interferon sensitivity determining region) of NS5A. (a) Dimer interface residues are colored in magenta in PKR sequence. The residues in NS5A 1b, 3a equivalent to the dimer interface residues of PKR are colored in cyan and yellow respectively. (b) Dimer interface residues are colored in magenta in PKR sequence. The residues in NS5A 1b, 2a equivalent to the dimer interface residues of PKR are colored in cyan and green respectively. (2) Trace residues are identified when NS5A from genotype 1b which interacts and inhibits PKR is compared with NS5A 3a and NS5A 2a which are unable to bind and inhibit PKR efficiently in few representative sequences. All the trace residues highlighted are highly conserved (> 80%) in the entire dataset. (a) Trace residues picked when MSA of genotype 1b Vs 3a is compared. (b) Trace residues picked when MSA of genotype 1b Vs 2a is compared. (c) Trace residues picked when MSA of genotype 1b Vs 3a and 2a are compared.Click here for file


Hydrogen generation amount increases but hydrogen generation rate decreases with La content increasing. There is an optimized composition that Al-15 wt% La-5 wt% NiCl2/NaBH4 mixture  has 126 mL g−1 min−1 maximum hydrogen generation rate and 1764 mL g−1 hydrogen generation amount within 60 min. The efficiency is 88%. Combined with NiCl2, La has great effect on NaBH4 hydrolysis but has little effect on Al hydrolysis. Increasing La content is helpful to decrease the particle size of Al-La-NiCl2 in the milling process, which induces that the hydrolysis byproduct Ni2B is highly distributed into Al(OH)3 and the catalytic reactivity of Ni2B/Al(OH)3 is increased therefore. But hydrolysis byproduct La(OH)3 deposits on Al surface and leads to some side effect. The Al-La-NiCl2/NaBH4 mixture has good stability in low temperature and its hydrolytic performance can be improved with increasing global temperature. Therefore, the mixture has good safety and can be applied as on board hydrogen generation material.The effect of La on Al/NaBH Many achievements showed that NaBH4- and Al-based hydrogen generation system attached to PEMFC can provide 2 W–10 kW powers [4 and Al have many disadvantages. For example, NaBH4 is an expensive raw material and alumina layer on Al surface reduces Al reactivity in water. The low solubility of NaBH4 and NaBO2 reduce hydrogen generation density and catalyst reactivity and durability [Hydrogen is a nonpolluting fuel and a clean energy which can be consumed and transformed to electricity in proton exchange membrane fuel cell (PEMFC). The development of PEMFC in vehicles and portable electronics requires large amounts of hydrogen storage at moderate temperature with high efficiency and low cost . On boarW powers , 4. Neverability . So the 4 or NaBH4/Al mixture in little water amount presents a good solution to overcome the disadvantages of traditional NaBH4- and Al-based hydrogen generation system. It has high hydrogen generation density and fast hydrolytic kinetic as exothermal reaction proceeds in limited water amount. Liu et al. [4 and Ru-based catalyst has 7.3 wt% hydrogen generation density at 298 K, meeting the required target of USA department of energy (6.5 wt.%). Sodium borohydride/nanoaluminum could supply stable hydrogen generation with about 7 wt.% yield when their mass ratio is 1 : 1 [4/NaOH mixture had controllable hydrogen generation performance via regulating the amount and rate of CoCl2 solution. In such case, water acts as an oxidizer for both aluminum and metal borohydride, thus as a source of hydrogen. There also exists an interaction of Al/NaBH4 hydrolysis which improves their hydrolytic kinetics. However, there is a great shortcoming that nanoaluminum particle or alkaline solution has to be used to start the hydrolytic reaction. It is necessary to find an environmental-friendly metal which can improve aluminum reactivity in neutral aqueous solution at moderate temperature. Metal lanthanum is a good candidate. It is a good hydrogen generation material and its hydrolysis byproduct presents alkaline, which accelerates the hydrolysis kinetic of magnesium and aluminum [3 from depositing densely on the surface of composites and stimulated Al hydrolysis.Hydrogen generation from solid-stage NaBHu et al.  found thaluminum . Luo et aluminum  found th2/NaBH4 mixture and reduce the alkali corrosion problems via the lanthanum additive (La). The relative hydrolysis mechanism has been discussed to gain the mechanistic understanding of the effect of La.We herein report a simple but effective method that can control hydrogen generation performance of micro-Al-NiClμm; Angang Group Aluminum Powder Co., Ltd., China), La powder (99.0% purity), NiCl2 (99.0% purity), and NaBH4  were used as starting materials. The composites of the Al-15 wt.% La-5 wt.% NiCl2 mixture  were weighed and mixed in an argon-filled glove box. The total weight of the mixture was 2 g, and ball milling was performed by a QM-ISP3 planetary ball miller under 0.2–0.3 MPa argon atmosphere. Ball-to-powder mass ratio corresponded to 30 : 1 at a milling time of 15 h and a rotation speed of 400 r/min.Elemental aluminum powder  mixture shows 68 mL g−1 min−1maximum hydrogen generation rate and yields 1293 mL g−1within 60 min. Upon increasing NiCl2 content to 15 wt%, hydrogen generation maximum rate and amount of the mixture include 122 mL g−1 min−1 and 1362 mL g−1, respectively. Reaction 3 particle [NiCl2B deposits on Al surface and acts as a cathode of a microgalvanic cell (Al-Ni2B), which stimulates the electrochemical corrosion of Al according to  was fixed except Al and La contents. The La content was fixed at 0, 5, 10, 15, and 20 wt%. We examined the impact of La content on hydrogen generation performance of the mixture. The results are shown in −1 within 60 min at 15 wt% La content. The efficiency is 82%. Further increasing La content deteriorates hydrogen generation performance. It can be explained from the microstructure change in 2 with different La content. The peaks of La, Al, and AlNi alloy are identified, reflected that La and Al have not formed alloy compound in the milling process. However, broadened lines are observed with increasing La content. Combined with the Scherrer equation (Dhkl = kλ/βcosθhkl), the crystal size can be roughly calculated. Its value decreases from 238 Å to 190 Å with La content increasing from 5 to 15 wt%, but further increases to 197 Å with La content further increasing to 20 wt%. In addition, La metal has high reactivity and reacts with water to produce some hydrogen at 298 K.−1 min−1 with La content increasing from 5 to 10, 15, and 20 wt%, respectively. It is known that hydrolysis byproduct La(OH)3 has low solubility in water and deposits on Al surface. So the contact area of Al and H2O is decreased and hydrogen generation rate is reduced therefore. However, the hydrogen generation rate is conversely proportional to La content, which can be obviously obtained in 2/NaBH4 mixture has low reactivity in low temperature. The results in −1 with about 30% efficiency at 303 K. In practical application, the mixture seldom reacts with water and has good stability at 298 K. But with increasing global temperature, hydrogen generation performance can be improved. In order to further understand the effect of La on hydrogen generation performance of Al/NaBH4 mixture, the relationship of activation energy and La content was analyzed. Using the Arrhenius equation (Al-La-NiClk) on the temperature (T) and the activation energy (Ea). The values of k can be determined from the maximum hydrogen generation rate at different temperatures. Using the linear relationship of Ink ~ 1/T in 4 mixture with different La content can be calculated. It can be found that activation energy is increased with increasing La content in 2/NaBH4 with different weight ratios. Al-15 wt% La-5 wt% NiCl2 has low hydrogen generation rate and amount, but the hydrogen generation performance is increased with decreasing weight ratios of Al-15 wt.% La-5 wt.% NiCl2/NaBH4, especially that Al-15 wt.% La-5 wt.% NiCl2/NaBH4 with weight ratio of 1 : 3 has best hydrogen generation performance. Its maximum hydrogen generation rate and amount are up to 126 mL g−1 min−1 and 1764 mL g−1 within 60 min. The efficiency reaches 88%. La and NiCl2 have little effect on Al hydrolysis, but seriously affect NaBH4 hydrolysis. The hydrolysis byproducts of NaBH4 hydrolysis stimulate Al hydrolysis in some degree, according to reactions  has 126 mL g−1 min−1 maximum hydrogen generation rate and 1764 mL g−1 hydrogen generation amount within 60 min. The addition of La has great effect on NaBH4 hydrolysis but little effect on Al hydrolysis at 323 K. Increasing La content is helpful to decrease the particle size of A-La-NiCl2 in the milling process, which induces that the hydrolysis byproduct Ni2B is highly distributed into Al(OH)3 and the catalytic reactivity of Ni2B/Al(OH)3 is increased therefore. Meanwhile, there exists a synergistic effect of hydrolysis byproduct NaBO2 and Ni2B to simulate Al hydrolysis. The Al-La-NiCl2/NaBH4 mixture has good stability in low temperature and its hydrolytic performance can be improved with increasing global temperature. So the mixture has good safety and can be applied as on board hydrogen generation material.La additive has greatly affects hydrogen generation performance of Al/NaBH


A(γ) is defined as In our publication , a confuA(γ) is an additive relationship matrix that satisfies the usual recursions but has the peculiar feature that base animals in the pedigree are related (with relationship coefficient γ) and inbreed (with inbreeding coefficient γ/2) as mentioned in the main text of [Instead it should have said that it is defined as I apologise for any inconvenience.


Transcription and replication of the influenza A virus RNA genome are mediated by the viral RNA polymerase from a promoter consisting of the partially base-paired 3′ and 5′ termini of viral genome segments. Here we show that transcription and replication can be uncoupled by mutation of an unpaired adenosine in the 5′ strand of the promoter. This residue is important for transcription but not replication by being essential for the cap-binding activity of the RNA polymerase. As leotides  and F.To address the question of whether the unpaired nature of 5′10A or the chemical properties of the residue are important for supporting viral transcription, we generated (i) a 3′ uridine insertion opposite 5′ A10 to enable its base pairing (3′iU10), (ii) a 5′dA10 and 3′i10U double mutation as a control for 3′iU10, and (iii) a 5′A10U substitution to control for the chemical properties of position 10 . As showin vivo, we transfected 293T cells with plasmids expressing PB1, PB2, tandem affinity purification (TAP)-tagged PA , RNAs consisting of about 22 to 27 nucleotides (nt) of the 5′ end of vRNA, as cofactors (In the absence of an influenza A virus RdRp structure, we can currently only speculate about the exact mode of action by which 5′A10 controls capped RNA binding by the RdRp without interfering with the ability of the RdRp to replicate RNAs. Deletion of 5′10A may result in an altered vRNA promoter structure, in which, for instance, base pair formation between 5′9C and 3′9G can efficiently compete with the formation of the 5′ and 3′ intrastrand base pairs, as proposed by the corkscrew model. Indeed, the formation of these 5′ intrastrand base pairs was previously shown to be critical for endonuclease activity of the RdRp , 13, sugtructure . In addiofactors . Our dat


Unlike previous extended peptide networks, the imidazole side chain of the histidine residue is deprotonated to afford Zn–imidazolate chains, with bonding similar to the zeolitic imidazolate framework (ZIF) family of porous materials. ZnCar exhibits permanent microporosity with a surface area of 448 m2 g−1, and its pores are 1D channels with 5 Å openings and a characteristic chiral shape. This compound is chemically stable in organic solvents and water. Single-crystal X-ray diffraction (XRD) showed that the ZnCar framework adapts to MeOH and H2O guests because of the torsional flexibility of the main His-β-Ala chain, while retaining the rigidity conferred by the Zn–imidazolate chains. The conformation adopted by carnosine is driven by the H bonds formed both to other dipeptides and to the guests, permitting the observed structural transformations.The peptide-based porous 3D framework, ZnCar, has been synthesized from Zn Metal–organic frameworks (MOFs) are crystalline porous materials composed of inorganic nodes, either single ions or clusters of ions, bridged by organic linkers through metal–ligand coordination bonds. Recently2 and CH4 adsorption for microporous materials.4⋅10 H2O is the only other known compound of histidine where metal–imidazolate bonding is employed.[Crystals of ZnCar⋅DMF in the shape of rectangular prisms and 50 to 100 μm in length  were obtained from the reaction of zinc nitrate with carnosine in DMF/Hith Zn2+ . The Zn ith Zn2+ , and eacole ring . The che1 and its 3D structure is directed by the carnosine conformation  at room temperature.The structure and the composition of ZnCar⋅DMF, obtained from single-crystal X-ray diffraction data are in good agreement with the characterization results of the bulk sample. The powder XRD pattern perfectly matches the simulated pattern from the single-crystal structure and is indexed to the same unit cell . CHN analysis results are very close to theoretical values (Table S2) and the DMF content, calculated as 20 wt % from the single-crystal data, was verified by thermogravimetric analysis . The solid-state 3 (by ≈5 %) between 388 and 394 K as DMF is removed from the pores . The relaxation of the ZnCar framework from the DMF-solvated state at 388 K . In contrast, the three torsional angles responsible for the orientation of the imidazolate ring and the carboxylate group on histidine each changed by less than 2°, indicating the rigidity of the bc planes shown in The ZnCar⋅DMF framework is structurally flexible and displays permanent porosity after the removal of DMF, as demonstrated by the variable-temperature single-crystal X-ray diffraction data. While the crystallinity of the material is retained over the whole temperature range that was studied, the unit-cell volume decreases sharply from 762.82 to 726.78 Åat 388 K  a to theat 388 K  b result−5 mbar) at 130 °C for 5 hours. The structure of the evacuated sample, ZnCar, was confirmed by powder XRD and the complete removal of DMF was verified by TGA . The porous properties of desolvated ZnCar were investigated with the CO2 adsorption–desorption isotherm  obtained at 195 K up to 1 bar. ZnCar is purely microporous, as the isotherm shape corresponds to that of type I according to the IUPAC classification. The BET surface area is calculated as 448 m2 g−1 using the relative pressure range, 0.01<P/Po<0.1. The total pore volume, estimated at 0.19 cm3 g−1, is slightly smaller than the solvent-accessible volume of the desolvated ZnCar structure of 0.21 cm3 g−1. The isotherm shows no gate opening, but a low pressure hysteresis loop associated with the strong adsorption of CO2.The bulk desolvation of ZnCar⋅DMF was achieved by evacuation on a high-vacuum line (102 and Zn(Gly-Thr)2 frameworks, which are stable only in organic solvents, is attributed to the Zn–imidazolate bonding that is the basis of the chemical stability of ZIFs.The chemical stability of ZnCar⋅DMF was examined by soaking fresh material in water under stirring for 3 days (Table S4). ICP analysis of the supernatant aqueous solution every 24 hours showed very low Zn leaching, corresponding to 1.7 wt % of the Zn added to water in the form of ZnCar⋅DMF. The stability of ZnCar⋅DMF in water, unlike Zn(Gly-Ala)2O and MeOH. The resolvated crystals resulted in two new structures, ZnCar⋅MeOH and ZnCar⋅H2O. The ZnCar framework proved to be adaptable to the presence of guest molecules, as the peptide conformation changed in both cases without breaking the 4:4 connectivity motif between Zn and carnosine. ZnCar⋅MeOH adopts the monoclinic P21 space group with slightly altered unit-cell parameters, but without significant changes in the pore shape compared to the desolvated structure , which form different sets of H bonds. Thus, two types of pores are formed, a wide and a narrow one  were significantly changed compared to those of all the other structures . In the narrow pore, there are two distinct H2O molecules that form three and two H bonds, respectively .The desolvated ZnCar structure is obtained after H2O removal  and the experimentally measured pore volume, 0.21 cm3 g−1 , equals to the estimated value from the ZnCar single-crystal structure. H2O vapor adsorption isotherms on ZnCar exhibit broad hysteresis loops and a maximum uptake of 15 wt % , and were cycled three times without change. The exhibited reversible resolvation–desolvation of the ZnCar framework with H2O proves that the properties of ZnCar remain unaffected after its exposure to water. Unlike ZnCar⋅MeOH and ZnCar⋅DMF, the DFT-calculated energy minimization of the empty ZnCar⋅H2O structure did not converge to that of the desolvated material, but relaxed to a different stable state that was 53 kJ mol−1 higher in energy (Table S6). This means that the desolvated ZnCar and ZnCar⋅H2O structures are separated by an energy barrier, and the calculated energy increase for the framework is counterbalanced experimentally by the formation of the water network that is rich in H bonds. The framework transformations, driven by the different guests, are facilitated mainly by the torsional flexibility of the peptidic chain. The 2D layers constructed from the Zn–imidazolate chains constitute the rigid part of the framework, as indicated by small changes in the Zn-im-Zn angle, the Zn–Zn distance, and the distance between the chains (Table S7).The structure of the water-resolvated ZnCar⋅Hrrow one  d, and t H bonds  a, threeructures  a–c. Carructures  b, one w2 and CH4 adsorption isotherms of ZnCar were collected at 283, 293, and 303 K . The gravimetric uptakes were found to be around 19 wt % for CO2 and 5 wt % for CH4 at 15 bars. The isotherms are of type I, and in each case 80 % of total uptake was adsorbed at 4 bars, showing the entirely microporous character of this material. The ZnCar MOF exhibits strong binding affinity for both gases, as shown by the isosteric heat of adsorption Qst . For CO2, the Qst value at zero coverage is 49 kJ mol−1, which is amongst the highest ever reported for MOFs. Cu-BTTri-mmen exhibits a Qst value of 96 kJ mol−1,[Qst at zero coverage ranges from 25–50 kJ mol−1.[2 carbon atom . The molecular dynamics (MD) simulations of CO2 diffusion along the pores have also shown this configuration to be the most favorable for adsorption, and showed that the CO2 molecules diffuse by hopping between the cavities in the pore structure through smaller channels that connect the pores . This is a moderate value for MOFs, lower than that of Mg2(dobdc), which is 60,[The selectivity of adsorption between COch is 60, in accor2 and CH4.The β-dipeptide carnosine is a special MOF linker that offers chemical stability, structural diversity and chirality, and permanent porosity in the ZnCar⋅DMF compound. ZnCar⋅DMF is an analogue of ZIFs where the involvement of the imidazole ring of the histidine side chain in Zn–imidazolate bonding both affords the chemical stability of the framework in water and produces a 3D structure, in contrast to previously studied peptide-based MOFs. The main His-β-Ala chain connects the rigid imidazolate-based layers, retaining its flexibility and conferring structural adaptability on the framework in the presence of different guest molecules. The ZnCar framework exhibits permanent microporosity and strong adsorption affinity for CO


The purpose of this study was to determine the α/β ratio for normal liver with hepatitis by analyzing the toxicity data from patients with unresectable hepatocellular carcinoma treated with helical tomotherapy.Between March 2006 and February 2012, 98 patients were eligible for this study. 66 patients received 45–50 Gy in 4.5-5 Gy fractions (Group A) and 32 patients received 36–60 Gy in 2.5-3 Gy fractions (Group B). Radiation-induced hepatic toxicity was defined as an increase of at least 2 points in the Child-Pugh score within 4 months of completing helical tomotherapy. We attempted to find the statistically significant parameters in the 2 groups using α/β ratios of 2, 4, 6, 8, or 10, and compared the estimated probability curves of each significant parameter. We hypothesized that the α/β ratio associated with the best matches for the curves between the 2 groups would be equivalent to the α/β ratio for the normal liver.BED30 for α/β ratio = 2 and VBED25 for α/β ratio = 4, Group B: VBED25 for α/β ratio = 2 and VBED20 for α/β ratio = 4). When using an α/β ratio of 6, 8, or 10, VBED20 was found to be a statistically significant parameter in both groups. Comparison of the estimated probability curve of each significant parameter between the groups revealed that an α/β ratio of 8 resulted in the best matches.When using an α/β ratio of 2 or 4, different parameters were found to be statistically significant in a multivariate analysis (Group A: VWe suggest that the α/β ratio of the normal liver with hepatitis is 8. We hope that previously reported parameters and their values can be effectively used in different fractionation schemes by calculating the biologically effective dose using an α/β ratio of 8. Radiation-induced hepatic toxicity (RIHT) is a serious dose-limiting toxicity in patients with hepatocellular carcinoma (HCC) receiving radiotherapy (RT) because there is currently no effective treatment for RIHT, which can ultimately cause liver failure . SeveralWhen treating patients with different fraction size compared to those of previous studies, the expected tumor response and hepatic toxicity could be estimated by calculating the biologically effective dose (BED) according to the linear-quadratic model. An α/β ratio of 10 is commonly used to calculate the BED delivered to the tumor, but the BED for the normal liver could not be calculated as its α/β ratio was unknown. In previous studies on hepatic toxicity, an α/β ratio of 2–3 or 10 was used to analyze the toxicity data arising from different fraction sizes and total doses of radiation ,10.In addition, the majority of patients with HCC have hepatitis, unlike patients with metastatic liver tumors, especially in eastern countries. In patients with HCC, the tolerance of the normal liver to radiation was considerably reduced . DetermiIn this study, we attempt to determine the α/β ratio for the normal liver with hepatitis by analyzing the hepatic toxicity data from patients treated with 4.5-5 Gy per fraction (Group A) and 2.5-3 Gy per fraction (Group B), respectively, and comparing the 2 groups.The inclusion criteria for this study were as follows: 1) unresectable locally advanced HCC, 2) prior treatment with hypofractionated helical tomotherapy with a curative aim, 3) a follow-up period of 4 months, 4) at least 2 laboratory studies within 4 months of completing helical tomotherapy, 5) at least 2 radiologic study within 4 months of completing helical tomotherapy, and 6) no intrahepatic disease progression within 4 months of completing helical tomotherapy.Between March 2006 and February 2012, a total of 98 patients were found to meet these inclusion criteria. All of the patients received hypofractionated RT using TomoTherapy Hi-Art  at Seoul St. Mary’s Hospital and Incheon St. Mary’s Hospital, the Catholic University of Korea. Of those, 66 patients received 45–50 Gy in 4.5-5 Gy fractions (Group A) and 32 patients received 36–60 Gy in 2.5-3 Gy fractions (Group B). The patients’ clinical and dosimetric data were retrospectively collected following Institutional Review Board approval . The patients’ characteristics are shown in Table The gross tumor volume (GTV) was defined as that which was enhanced in the arterial phase and diluted in the delayed phase of the computed tomography (CT) scan. The planning target volume (PTV) was generated by adding 5–15 mm to the GTV in 63 of the 98 patients, facilitating asymmetric margin expansion to reduce irradiation to the stomach, duodenum, and small intestine. In the remaining 35 of the 98 patients, 4-dimensional CT (4D-CT) was performed to generate the internal target volume to compensate for respiration-induced liver movement because of the installation of 4D-CT in March 2009 at Seoul St. Mary’s hospital and in March 2011 at Incheon St. Mary’s hospital. Organs at risks such as the total liver, non-target normal liver (NTNL), stomach, duodenum, intestine, kidney, and spinal cord were also contoured for evaluation of the irradiated dose. The NTNL volume was the total liver volume minus the PTV.The radiation dose was 45–50 Gy in 4.5-5 Gy fractions for Group A and 36–60 Gy in 2.5-3 Gy fractions for Group B, prescribed to 95% of the PTV. 4.5-5 Gy per fraction was used in smaller PTV and 2.5-3 Gy per fraction was used in larger PTV. Prior to actual beam delivery, megavoltage cone-beam CT was performed during every treatment session. The patients’ set-up and position were corrected using automated image registration, and the anatomical accuracy was always evaluated by a radiation oncologist.RIHT was defined as an increase of at least 2 points in the Child-Pugh score (CP score) within 4 months after the completion of helical tomotherapy. The CP score, which is calculated on the basis of the serum bilirubin and albumin levels, the prothrombin time (PT), and the presence and degree of ascites or encephalopathy, is used as an assessment of hepatic function and an increase in the CP score reflects a deterioration in hepatic function .Patients were evaluated weekly by a physician during the treatment and followed up every 1–2 months after the completion of treatment. At every visit, physical examinations and blood tests were performed to assess hepatic toxicity. The levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), serum albumin, total bilirubin and the PT were examined and we also checked the presence of ascites and hepatic encephalopathy.We attempted to identify the statistically significant parameters in the 2 groups using an α/β ratio of 2, 4, 6, 8, or 10, and compared the estimated probability curves of each significant parameter , more than a BED of 10 Gy (VBED10), more than a BED of 15 Gy (VBED15), more than a BED of 20 Gy (VBED20), more than a BED of 25 Gy (VBED25), more than a BED of 30 Gy (VBED30), more than a BED of 35 Gy (VBED35), more than a BED of 40 Gy (VBED40), more than a BED of 45 Gy (VBED45) and more than a BED of 50 Gy (VBED50).The dose-volumetric values were calculated on the basis of dose-volumetric histograms (DVH) and converted to the BED according to the linear quadratic model  in all patients. The dose-volumetric parameters for predicting RIHT were the percentage of the NTNL volume receiving more than a BED of 5 Gy (VUsing logistic regression models and receiver operating characteristic (ROC) curves, statistically significant parameters were identified in each group using an α/β ratio of 2, 4, 6, 8, or 10. The values of the area under the curve (AUC) were obtained for each α/β ratio group and each treatment group (Group A and Group B). We then compared the estimated probability curve of each significant parameter between Group A and Group B to determine the α/β ratio that resulted in the best match between the 2 curves.p value of <0.05 was considered to be significant.Pearson’s chi-square test and the independent t-test were used to compare the clinical characteristics between the 2 groups (Group A vs. Group B). Binary logistic analysis was used for univariate analysis of dose-volumetric parameters associated with RIHT. Multivariate analysis involved stepwise procedures containing all significant variables according to the univariate analysis, and the ROC curve was used to evaluate the significant dosimetric parameters. Statistical analysis was performed using SPSS ver. 12.0  and a 3 (mean ± standard deviation) and 382 ± 396 cm3, respectively, and the volume of the NTNL for Group A and Group B was 1301 ± 548 cm3 and 1184 ± 302 cm3, respectively.A total of 98 patients were enrolled in this study. 66 patients received a daily fraction size of 4.5-5 Gy (Group A) and 32 patients received a daily fraction size of 2.5-3 Gy (Group B). There was no statistically significant difference between the 2 groups in terms of gender, age, Eastern Cooperative Oncology Group performance status (ECOG PS), pretreatment CP class, American Joint Committee on Cancer (AJCC) stage, the level of alpha-fetoprotein (AFP), the presence or absence of hepatitis, liver cirrhosis and portal vein tumor thrombosis (PVTT), prior treatment and treatment after RT Table . The PTVBED30 for α/β ratio = 2 and VBED25 for α/β ratio = 4, Group B: VBED25 for α/β ratio = 2 and VBED20 for α/β ratio = 4). Therefore, we compared the same parameters in each group using an α/β ratio of 2 or 4 (VBED25 and VBED30 for α/β ratio = 2 and VBED20 and VBED25 for α/β ratio = 4). When an α/β ratio of 6, 8, or 10 was used, VBED20 was a statistically significant parameter in both groups. In addition, these parameters were verified using the ROC curve and the values of AUC. These results are shown in Figure p < 0.001).After the completion of helical tomotherapy, RIHT developed in 43 (43.9%) of 98 patients: 28 patients (42.4%) in Group A and 15 patients (46.9%) in Group B. The results of multivariate analysis evaluating the association between dose-volumetric parameters and RIHT in each treatment group using an α/β ratio of 2, 4, 6, 8, or 10, are summarized in Table Next, we compared the estimated probability curve of each significant parameter between the 2 groups to determine the α/β ratio for normal liver. According to the data in Figure RT has not been generally used for the treatment of HCC because of the low tolerable dose of radiation for whole liver, which is insufficient to achieve tumor control -16. HoweRIHT is a significant dose-limiting toxicity for RT in patients with HCC. Radiation-induced liver disease (RILD) is a well-established concept of hepatic toxicity , and preet al. used an α/β ratio of 2 for grade 3 or worse CTCAE hepatic toxicity [et al. used an α/β ratio of 2 or 2.5 for cases of classic RILD [et al. used an α/β ratio of 10 to calculate the BED for cases of grade 2 or worse CTCAE hepatic toxicity [et al. did not calculate the BED for classic RILD as there was no clear α/β ratio for the normal liver [There is some uncertainty when using different fraction sizes for treatment. Although an α/β ratio of 10 could be used to calculate the BED delivered to the tumor, the BED delivered to the normal liver could not be calculated as the α/β ratio for the normal liver was unknown. In spite of this uncertainty, various ranges of α/β ratio were used with respect to various different criteria of hepatic toxicity in previous studies. To calculate the BED delivered to the normal liver, Cheng sic RILD . Kim et al liver ,5.For these reasons, it is important to determine the α/β ratio for the normal liver. Here, we have attempted to determine the α/β ratio for the normal liver indirectly on the basis of the toxicity data from 2 patient groups treated with different fraction sizes.et al. also used an increase of at least 2 points in the CP score to evaluate the deterioration of hepatic function in patients who were treated with lamivudine [We defined RIHT as an increase of at least 2 points in the CP score. In our previous study, the progression of CP class was analyzed as a useful radiation dose-limiting toxicity, whereas the elevation of hepatic enzymes according to the CTCAE scale was not appropriate in these circumstance . Liaw etmivudine . The CP et al. and Liang et al., it occurred within 4 months after the completion of RT [et al. and Cheng et al. also reported that non-classic RILD hepatic toxicities occurred within 3 or 4 months [We analyzed the hepatic toxicities that occurred within 4 months of treatment. Classic RILD typically occurs 4–8 weeks after the completion of RT, although it has been reported to occur as early as 2 weeks and as late as 7 months . Accordion of RT ,5. Kim e4 months . TherefoIn this study, 4.5-5 Gy per fraction was used in Group A and 2.5-3 Gy per fraction was used in Group B; The difference in fraction size between the 2 groups was thus 2 Gy. Although the fact that the same fraction size was not used for treatment was considered a weak point, the maximal difference in groups was only 0.5 Gy and it did not affect our analysis because the dose-volumetric values were converted to the BED according to the linear quadratic model  in all patients.BED20, VBED25 and VBED30, which were a lower level of dose-volumetric parameters than those of previous studies. This result reflects that it is due to the characteristic of the planning and delivery method of helical tomotherapy. Finally, all patients in this study had hepatitis . However, the analysis results were still reliable because the AUC values of statistically significant parameters were more than 0.859 and 0.914, respectively.We suggest that the α/β ratio for the normal liver with hepatitis is 8. We hope that previous reported parameters and their values can be effectively used in different fractionation schemes by calculating the BED using this α/β ratio, and we hope for further confirmation in larger-scale studies.The authors declare that they have no competing interests.SHS, HL, HSJ, BOC and CSK performed all CT evaluations, target contouring, data collection and interpretation of the data. YNK performed the treatment planning and conducted all planning evaluations. SHS, HSJ, JWJ, and SKY took care of the patients. SHS, HSJ, JWJ, SKY and CSK participated in the study design. SHS performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.


Amyloid beta (aβ) protein assembles into larger protein aggregates during the pathogenesis of Alzheimer’s disease (AD) and there is increasing evidence that soluble aβ oligomers are a critical pathologic species. Diagnostic evaluations rely on the measurement of increased tau and decreased aβ42 in the cerebrospinal fluid (CSF) from AD patients and evidence for oligomeric aβ in patient CSF is conflicting. In this study, we have adapted a monoclonal single antibody sandwich ELISA assay to a Luminex platform and found that this assay can detect oligomerized aβ42 and sAPPα fragments. We evaluated oligomeric aβ reactivity in 20 patients with AD relative to 19 age matched controls and compared these values with a commercially available Alzbio3 kit that detects tau, phosphorylated tau and aβ42 on the same diagnostic platform. We found that CSF samples of patients with AD had elevated aβ oligomers compared to control subjects (p < 0.05) and the ratio of aβ oligomers to aβ42 was also significantly elevated (p < 0.0001). Further research to develop high sensitivity analytical platforms and rigorous methods of developing stable assay standards will be needed before the analysis of oligomeric aβ becomes a routine diagnostic assay for the evaluation of late onset AD patients. Alzheimer’s disease (AD) is the most common dementia in the elderly, affecting nearly half of people in the United States over the age of 85. Pathological hallmarks that are important for the neuropathologic diagnosis of AD include the assessment of amyloid beta (aβ) deposits, neurofibrillary tangles and neuritic plaques ,2. Due tThe amyloid precursor protein is sequentially processed by β and γ-secretase complexes resulting in two major aβ species terminating at positions Val40 (aβ40) or Ala42 (aβ42). The aβ42 protein has two additional hydrophobic amino acids on its C terminus and is more prone to aggregation than aβ40 peptide –7. MonomMeasurements of aβ42, total tau and phosphorylated tau can be used to diagnose AD with sensitivity and specificities ranging from 81% to 90% when all three analytes are measured –22. The Previous studies have used a variety of reagents and techniques to assess the presence of aβ oligomers in CSF from AD patients. One study using a Ban50 single antibody sandwich ELISA to detect HMW oligomers documented an elevation of aβ oligomers in CSF that is associated with cognitive decline . AnotherStudy participants were recruited from the Clinical Core of the University of Washington (UW) Alzheimer’s Disease Research Center (ADRC). All participants underwent standard neuropsychological evaluation, including Mini-Mental State Examination , ClinicaThe study protocols were approved by the Institutional Review Board at the University of Washington. Subjects were recruited from University of Washington Alzheimer’s Disease Research Center and written informed consent was obtained from all participants at enrollment. Because AD patients belong to a potentially vulnerable group, a health care surrogate signed the consent forms in addition to the patient (unless the patient was not capable of doing so). Subjects also demonstrated assent by cooperating with and not objecting to any procedures. Participation in this research was strictly voluntary and potential subjects who did not take part in this project were not disadvantaged in any way. Participation in this research project had no impact on eligibility for clinical benefits or services. All aspects of this investigation were conducted according to the principles described in the Declaration of Helsinki.Oligomers used for assay standards and controls were prepared from monomeric aβ42 according to previously described protocols [Size exclusion chromatography was performed on aβ oligomer preparations using an AKTA purifier 10 with two Superdex 75 10/300 GL columns connected in tandem . Molecular weight calibration was performed using gel filtration calibration standards  at a flow rate of 1 mL/min. After collection, the fractioned samples were promptly subjected to Alzbio3 or aβ oligomer assay and positive fractions were pooled, aliquoted and stored in a -80°C freezer.2O and transferred to a solution containing 100mM monobasic sodium phosphate, pH 6.2. The magnetic beads were activated by adding 10 microliters of 50 mg/mL Sulfo-NHS followed by 10 microliters of 50 mg/mL EDC (diluted in dH 20) and mixed gently by vortex. The microspheres were incubated for 20 minutes in the dark at room temperature with vortexing at 10 minute intervals. The microspheres were washed twice in 250 microliters of 50 mM MES, pH 5.0 with vortex and sonication and separated into three fractions with 5, 25 or 125 micrograms of Ban50 antibody  to optimize binding conditions. In order to couple the capture antibodies to the microspheres, antibodies were incubated in 500 microliters of 50mM MES, pH 5.0 overnight at 4 degrees on a nutator. The following day the antibody-coupled microspheres were washed in PBS –TBN solution  solution and incubated for 30 minutes at room temperature on a nutator. Two additional washes were performed in 1 mL of PBS-TBN and Ban50-coupled microsphere yield was determined manually by counting on a hemocytometer. To prepare the detection antibody, Ban50-Fab was biotinylated using EZ-Link Sulfo-NHS-Biotin  according to the manufacturer’s directions.Conjugated Magplex microspheres  were prepared according to the directions of the manufacturer. In brief, stock microspheres were resuspended in 100 microliters of dHCSF samples were thawed on ice and reagents from the Inno-Bia Alzbio3 kit  were allowed to come to room temperature. Microspheres were vortexed, sonicated and diluted in the buffer provided by the kit manufacturer. The conjugate 1 working solution was prepared by diluting the provided biotinylated secondary antibodies. After prewashing the plate, 100 microliters of diluted bead mix were transferred to each well. The plate was aspirated using a vacuum manifold. All measurements were performed in duplicate with 25 µL of conjugate 1 and 75 µL of standards or samples. The plate was incubated overnight on a shaker at room temperature and protected from light.The following day, detection conjugate solution was prepared with the provided diluent. The plate was washed three times and incubated with detection conjugate for 1 hour at room temperature on an orbital shaker. The plate was washed an additional three times and reading solution was added to each well. The plate was incubated on the shaker for several minutes and read on a FlexMap3D platform .6 microspheres were vortexed and sonicated. A master mix containing Ban50 conjugated microspheres, biotinylated Ban50 detect conjugate and 1% BSA in PBS with protease and phosphatase inhibitors  was prepared and 25 microliters per well were dispensed into a low retention 96 well black plate assay plate . Samples and controls were added at 75 microliters per assay well. The plate was incubated overnight on a shaker at room temperature and protected from light with aluminum microplate sealing film.CSF samples and assay controls were thawed on ice and reagents were allowed to come to room temperature. Ban50 conjugates prepared at 125 micrograms of antibody per 1.25 x 10The following day, the plate was washed three times with PBS, 0.05% Tween and incubated at room temperature with 0.5 µg/mL of Lumigrade Streptavidin-R-Phycoerythrin  in PBS with 1% BSA for one hour on an orbital shaker. The plate was washed three times and 100 microliters of PBS was added to each well. The plate was incubated on the shaker for several minutes to resuspend the microspheres and read on a FlexMap3D platform . Assays were performed on de-identified coded samples. The investigator was blinded to diagnosis, genotype and demographic information during all of the manual steps of assay, and diagnostic groups were compared after the samples were read on the Luminex machine. Ban50 SAS oligomeric assays for AD and control samples were run on the same plate at the same time to minimize variability between assay runs and CSF was analyzed in duplicate wells.In vitro analysis of whether sAPPα interferes with aβ oligomer detection was performed at a range of sAPPα concentrations in the presence of 2% aβ oligomers from pooled SEC fractions. Immunodepletion experiments were performed by preclearing three pools of CSF from different AD patients with Protein A Sepharose  for one hour followed by an overnight incubation of the unbound CSF with 2.5 micrograms of 2B3  or IgG control antibody  on a nutator at 4 degrees. Immunoprecipitation was performed with Protein A Sepharose and unbound CSF was analyzed using the Ban50 assay and read on a FlexMap3D platform .Recombinant sAPPα and sAPPβ proteins  were oligomerized by incubating one microgram of protein in one milliliter of phenol red free F12 culture medium  at 4°C overnight. Monomeric sAPPα and sAPPβ were prepared by dissolving and evaporating 1 microgram of recombinant protein in HFIP three times before resuspending the protein in 1 milliliter of phenol red free F12 culture medium. Serial dilutions were made in PBS with 1% BSA and protease inhibitors  starting at 500 nanograms per milliliter. Continuous variables such as tau, phosphorylated tau, aβ42, oligomeric aβ, age at lumbar puncture, years of education, or cognitive testing scores were compared between AD and control groups using two-tailed Mann-Whitney tests or t-tests. Categorical variables, such as gender or ApoE genotype, were compared between AD and control groups using Fisher’s Exact Test. Receiver Operating Characteristic analysis was performed to evaluate test performance and determine appropriate cutoff values for oligomeric aβ assays. Linear regression and Pearson correlations were performed to examine the relation of cognitive tests  to oligomeric aβ. In experiments performed to assess crossreactivity of the BAN50 assay with sAPPα and sAPPβ, mixed between and within subjects ANOVA  was performed relating signal to monomeric and oligomeric sAPP fragments as well as to concentration. Significant main and interaction effects were followed up with post hoc simple main effects and polynomial contrasts across concentration levels. A Greenhouse-Geiser (GG) adjustment to the degrees of freedom (df) was used to adjust for correlated error in tests with df greater than one. In experiments evaluating the detection of oligomeric aβ in the presence of free soluble sAPPα at a range of physiologically relevant concentrations, a one-way repeated measures ANOVA was performed to assess the relation of concentration to signal. Immunodepletion experiments were analyzed using a t-test and confirmed using two nonparametric significance tests. Statistics and graphics were performed using SAS statistical software (Version 9.3), GraphPad Prism (Version 5.0), Origin (Version 8.0), and JMP Pro (Version 10) software.An age and gender matched cohort of patients seen at the University of Washington Alzheimer's Disease Research Center between 2004 and 2010 with a diagnosis of AD (n=20) or non-demented control (n=19) were assessed in this study. Demographic factors such as age, gender and years of formal education were not significantly different between the AD and control patient cohorts  analysis and found that the ratio of aβ oligomers/total aβ42 yields an Area Under the Curve (AUC) of 0.984 . A cutoff value of 0.503 corresponds to a sensitivity of 90 percent and a specificity of 94.7 percent using the oligomeric aβ / aβ42 ratio  between these parameters in the entire sample pooled . The MMSWe also examined whether there was any correlation between storage interval and the duration of time that samples were stored for any of the analytes measured in this study . We did When APP is cleaved by either the α- or β-secretase, larger fragments called sAPPα and sAPPβ are released extracellularly. We evaluated whether there was any cross-reactivity with sAPPα and SAPPβ fragments using the BAN50 SAS assay . Mixed bin vitro, oligomeric aβ was kept constant while monomeric sAPPα was assayed at a range of physiologically relevant concentrations. One-way repeated measures ANOVA showed no significant relation of concentration to BAN50 signal overall  or in its linear polynomial component . We also performed immunodepletion using pooled CSFs from AD patients comparing signal after incubating with either sAPPα specific antibody 2B3 or control IgG and did not find a significant change in Ban50 reactivity (t(4) = 1.49, p = 0.21, independent measures t test). In human CSF, depleting sAPPα does not diminish the amount of BAN50 signal detected, supporting the conclusion that oligomeric sAPPα is not likely to be a major biological confound in patient samples.In order to further examine the effect of free soluble sAPPα on the detection of aβ oligomers in the Ban50 assay in vitro experiments make it seem less likely that sAPPα oligomers account for the increased BAN50 SAS reactivity observed in AD CSF relative to normal controls, it is plausible that aβ42 or sAPPα species could influence the measurement of oligomeric species by interacting with capture or detect antibodies.The cross reactivity with sAPPα that we observe is consistent with prior studies indicating the Ban50 epitope is located at the N-terminus of aβ42 , which iPrevious studies investigating the presence of aβ oligomers in CSF from AD patients have employed a diverse range of techniques dedicated to investigating different species of aggregates. In designing this Luminex assay, we adapted a Ban50 single antibody sandwich ELISA that was previously shown to detect elevated HMW aβ oligomers in CSF . Interesin vitro [in vitro studies were performed using superphysiologic concentrations of aβ42 in a matrix that is compositionally different from CSF.Another consideration in experimental design is whether the dynamic equilibrium of aβ monomers and oligomers are affected during the assay protocol and whether monomeric aβ42 present in CSF might affect oligomeric aβ measurements. Luminex assays are typically incubated overnight at room temperature on an orbital shaker to maximize interaction of capture antibodies coated on the microspheres with analytes and detect antibodies in solution, conditions that are especially important for CSF assays that detect low abundance proteins . Howeverin vitro . It is uSeveral other assay designs have been utilized to assay oligomeric aβ in human CSF. One of the most promising assays is a nanoparticle based approach that was shown to yield excellent discrimination between AD and normal controls. This ultrasensitive assay uses biobarcode amplification to enhance signal, with estimated concentration medians for amyloid-β-derived diffusible ligands at 200 aM and 1.7 fM , howeverThe variability in results from the many prior studies on oligomeric aβ in CSF may be due to many factors. Most of the published studies use a relatively small number of clinical samples on a variety of diagnostic platforms with different antibody pairs. Some assays have a greater avidity for LMW or HMW aβ species and therefore may report differently than assays that detect all oligomeric forms. While we were able to confirm the prior study showing increased levels of aβ oligomers in patient samples , we founA major advance in the development of oligomeric aβ standards was recently reported by Kasai et al. who constructed a synthetic peptide connecting multiple BAN50 epitopes to a branching lysine core and was able to create a standard curve encompassing the physiological range of oligomeric aβ found in CSF . BecauseFigure S1A. Scatterplot of CDR and aβ oligomers for the entire sample pooled with linear regression in red. The correlation is not statistically significant  and is depicted with a solid red line. B. Scatterplot of Delayed Recall Score and aβ oligomers for the entire sample pooled with linear regression in red. The correlation is not statistically significant . C. Scatterplot of Immediate Recall Score and aβ oligomers for the entire sample pooled with linear regression in red. The correlation is not statistically significant .(TIF)Click here for additional data file.Figure S2A. Scatterplot of aβ oligomers relative to sample storage time with linear regression in red. The correlation is not statistically significant . B. Scatterplot of aβ42 relative to sample storage time with linear regression in red. The correlation is not statistically significant  C. Scatterplot of tau relative to sample storage time with linear regression in red. The correlation is not statistically significant . D. Scatterplot of tau 181P relative to sample storage time with linear regression in red. The correlation is not statistically significant .(TIF)Click here for additional data file.Figure S3A. Serial dilutions of monomeric or oligomeric sAPPα were assessed by BAN50 SAS assay. Mixed between- and within-subject ANOVA analysis followed by post hoc tests indicated that there was a significant increase in signal for oligomeric sAPPα , adjusted). No significant relation between concentration and assay signal was noted for monomeric sAPPα . Error bars represent standard deviations from three independent experiments. B. Serial dilutions of monomeric or oligomeric sAPPβ were assessed by BAN50 SAS assay. Mixed between- and within-subject ANOVA analysis followed by post hoc tests indicated that there was not a significant relationship between the assay signal and concentration of oligomeric sAPPβ , or monomeric sAPPβ . Error bars represent standard deviations from three independent experiments. C. Serial dilutions of monomeric sAPPα were added to samples containing constant levels of aβ oligomers were measured using the Ban50 assay to test whether soluble sAPP interferes with the measurement of oligomers in vitro. A one-way repeated measures ANOVA showed no significant relation of concentration to signal overall . Error bars represent standard deviations from three independent experiments. D. Immunodepletion of human CSF with either sAPPα specific antibody 2B3 or control IgG was performed to test whether Ban50 reactivity in patient samples is due to sAPPα. NS indicates difference between sAPPα immunodepleted and control groups were not significantly significant (t(4) = 1.49, p = 0.21).(TIF)Click here for additional data file.


Tendinopathies are a range of diseases characterised by degeneration and chronic tendon pain and represent a significant cause of morbidity. Relatively little is known about the underlying mechanisms; however onset is often associated with physical activity. A number of molecular changes have been documented in tendinopathy such as a decrease in overall collagen content, increased extracellular matrix turnover and protease activity. Metalloproteinases are involved in the homeostasis of the extracellular matrix and expression is regulated by mechanical strain. The aims of this study were to determine the effects of strain upon matrix turnover by measuring metalloproteinase and matrix gene expression and to elucidate the mechanism of action. Primary Human Achilles tenocytes were seeded in type I rat tail collagen gels in a Flexcell™ tissue train system and subjected to 5% cyclic uniaxial strain at 1 Hz for 48 h. TGFβ1 and TGFβRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFβ protein levels were determined using a cell based luciferase assay. We observed that mechanical strain regulated the mRNA levels of multiple protease and matrix genes anabolically, and this regulation mirrored that seen with TGFβ stimulation alone. We have also demonstrated that the inhibition of the TGFβ signalling pathway abrogated the strain induced changes in mRNA and that TGFβ activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFβ activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy. •Mechanical strain regulates multiple protease and matrix genes at the mRNA level.•Changes in mRNA level are analogous to those induced by TGFβ stimulation.•The inhibition of the TGFβ signalling pathway abrogated the strain-induced changes.•A SMAD activatory soluble factor is increased in activity in response to mechanical load. Activation has been reported to occur by a number of mechanisms: Cell surface integrins bind LAP via the RGD motif allowing protease enzymes to release active TGFβ In the canonical signalling pathway, active TGFβ binds cell surface TGFβ type I and type II receptors (TGFβRI and TGFβRII)—these receptors have serine/threonine kinase activity. Upon ligand binding, both type I and type II receptors form heterotetrameric complexes, TGFβRII phosphorylates TGFβRI, leading to phosphorylation and subsequent activation of SMAD proteins which translocate to the nucleus and cause transcriptional regulation of target genes  and 200 μl was pippetted into flexible bottomed collagen I coated flexcell tissue train plates, under 20% vacuum. The cell density was selected based on pilot experiments, which showed similar changes at low and high cell densities, but the response was greater at high cell density. Gels were allowed to polymerise for 1 h at 37 °C, at which point 3 ml serum free DMEM was added. After a further 72 h medium was replaced, appropriate treatments added ). Undetected samples were given a Ct value of 40. Relative expression levels in each gene of interest were analysed by normalising to endogenous control gene 18s (∆Ct [endogenous control gene Ct–gene of interest Ct]) and expressing the data as 22.4∆∆Ct. All data were normalised to the control, which is represented by the baseline value of 1. Data shown was normalised to TOP1, as GeNorme analysis demonstrated TOP1 to be the most stable housekeeping gene .The standard qRT-PCR programme was run using selected primer probe sets see  and the 2.5In order to test whether TGFβ protein levels were modified in response to mechanical loading, we measured total and active TGFβ in strain conditioned medium using a cell-based luciferase assay 2.6Data were presented as a mean ± Standard error (S.E.M.). Data were tested for normal distribution using the Shapiro–Wilk normality test. The Wilcoxon signed rank test and the students paired T test  were used to analyse qRT-PCR data and TGFβ luciferase data using SPSS as appropriate. p < 0.05 was chosen as the cut off for statistical significance.33.1In order to identify strain responsive genes we measured the expression of all 23 MMP, 4 TIMP and 18 ADAMTS mRNA and a selection of matrix genes using a Taqman Low Density Array . Of thesThere was a significant increase in the following genes with strain compared to non-strained controls at 24 and 48 h; ADAMTS2 (2.5 and 2 fold), ADAMTS4 (3.2 and 7.1 fold), ADAMTS16 (25.6 and 24.1 fold), MMP24 (5.2 and 35.2 fold) and TIMP3 (4.6 and 5.3 fold) (at 24 and 48 h respectively). MMP10 was increased with strain after 24 h (53.5 fold) and decreased after 48 h (4 fold). Thrombospondin-1 (8.6 fold), ADAMTS6 (3.6 fold) and ADAMTSS14 (3 fold) were increased and MMP11 was decreased (1.6 fold) after 24 h of strain. Lumican (1.4 fold), MMP3 (33.3 fold) and MMP17 (2 fold) were decreased and ADAMTS10 (2.6 fold) is increased after 48 h of strain.Using histological analysis we observed that cells became aligned to the axis of load and that there was no difference between strained and non-strained cultures (data not shown), similar to previous reported observations 3.2Using a real time PCR array, we confirmed that the majority of gene changes were similar with strain or TGFβ treatment; 12/14 ADAMTSs, 14/17 MMPs and 13/14 of matrix genes showed a similar response to strain and TGFβ at 24 and 48 h. Three out of the four TIMPs also showed similar responses to strain and TGFβ  . Additio3.5Following our observation that TGFβ signalling plays a role in the response to cyclical strain, we measured TGFβ1, TGFβ2 and TGFβ3 mRNA expression. TGFβ1 and TGFβ3 mRNA were significantly decreased with strain at 8 h (~ 1–2 fold) A and TGFStrain conditioned medium induced a significantly higher level of SMAD activity compared to conditioned medium from non-strained controls (~ 40 fold increase) B. Heat-a3.6MMPs  and serine proteases (thrombin and plasmin) have been reported to activate TGFβ by cleaving TGFβ from LAP TGFβ does not require physical separation from the LAP for activation; interaction of LAP with integrin and thrombospondin domains (RGD and LSKL motifs respectively) can result in a conformational change and consequent activation of TGFβ The M6P/IGF-II receptor has also been reported to be involved in the activation of TGFβ 4We are the first to study the effect of mechanical regulation on an array of protease genes in human tenocytes. We have chosen a strain of 5% as it is within the physiological range experienced by human tendons There have been a number of studies that have looked at the effects of mechanical strain on MMPs, mainly in rodent tendon in vivo, ex vivo or in vitro cell studies. Obvious limitations of these studies include the differences in species as rodents do not express MMP1; instead they only express MMP13, which is the nearest homologue No other published studies have looked into the mechanical regulation of MMP10, MMP24, the ADAMs, TIMPs 3 and 4 and such a large array of ADAMTS and matrix proteins at either the mRNA or protein level in human tendon. Our data suggests that generally there is a decrease in MMPs  and an increase in collagen at the mRNA level. We have also shown an increase in TIMP3 mRNA which is known to inhibit ADAM12, MMP1, MMP2, MMP3, ADAMTS4 and ADAMTS5 at the protein level Vogel showed stimulation of aggrecan with TGFβ, which is possibly similar to the current study Arnoczky's group has shown that mechanical strain is important in tendon ECM homeostasis in that MMPs are increased with the absence of tension Comparing the responses of metalloproteinase and matrix genes to strain and tendinopathy may shed light on the relationship between mechanical loading and tendinopathy development. MMP2, MMP3, MMP8, ADAMTS2, ADAMTS4, ADAM12 and collagen type I expression were regulated in a similar manner in tendinopathy compared to the current study TGFβ protein is increased in tendinopathy We have shown that the vast majority of changes reported in response to strain have shown a similar pattern of response to TGFβ. Some of the gene changes in response to TGFβ have previously been reported in other cell types. For example TGFβ is reported to regulate ADAM12 In some instances there is an additive effect when both strain and TGFβ are applied. ADAMTS5 is increased significantly with strain after 24 h (2.2 fold), in response to TGFβ there was a trend to increase, however with a combination of strain and TGFβ treatment ADAMTS5 was increased further than strain alone . ADAMTS5 stimulation with strain is not mediated via TGFβRI signalling therefore strain and TGFβ response rely on different mechanisms. We have also noted that the increase in elastin was reduced when strain and TGFβ were combined; elastin was increased 3.8 fold with strain, 27.5 fold with TGFβ and 11.2 fold with strain and TGFβ in combination. We have shown that elastin was regulated via TGFβRI however we have not fully characterised this response. In speculation, this may result from a negative feedback mechanism triggered when the levels of TGFβ reach a threshold; however more research is required to test this hypothesis.Limitations of the study include the sole use of tendon cells derived from tendinopathic tissue. However, we have studied a number of cell isolates derived from normal and ruptured tendon, which showed similar responses in terms of metalloproteinase regulation with strain (data not shown). This suggests that tendon cells derived from different disease phenotypes respond in a similar way to cells derived from normal tendon when cultured. In addition, as we seed the tenocytes into a simple collagen matrix which is lacking in many of the components of the ECM that occur in vivo, tenocytes may respond differently to mechanical loading than those in native ECM. Further study of TGFβ involvement in gene regulation with mechanical strain in tendon fascicles would confirm whether tenocytes respond in a similar manner in their native environment. Another limitation of this study is that we were unable to confirm that all inhibitors used to characterise mechanisms of TGFβ activation were effective although we were able to confirm the inhibitory ability of GM6001 after 48 h of culture, which was achieved using a quenched fluorescent substrate and active metalloproteinase (data not shown). Other inhibitors were used at concentrations more than or equal to those reported in similar studies (M6P and pefabloc Taken together our data suggest that activation of TGFβ and subsequent TGFβ signalling is part of the mechanotransduction response of tenocytes to moderately high levels of strain. TGFβ up-regulation at both the protein and mRNA level with mechanical strain has been previously reported in tenocytes 5We have shown that mechanical strain regulates multiple protease and matrix genes at the mRNA level and that changes in mRNA level are analogous to those induced by TGFβ stimulation. Furthermore, the inhibition of the TGFβ signalling pathway abrogated the strain-induced changes in mRNA level, demonstrating signalling via TGFβRI which mediates downstream phosphorylation of SMAD