Lignin is a complex phenolic structural component of the secondary cell walls of all vascular plants. It is an irreversible end point of a major metabolic pathway in plant secondary metabolism. Lignin is fundamental to the adaptation of plants to land, the evolution of vascular transport and the resistance of plants to pests and pathogens. Lignin is a major barrier to the utilization of biomass for energy, for papermaking, and for forage digestibility due to the interaction of lignin with cellulosics in the plant cell secondary wall. Populus trichocarpa , and the systems approach including advanced quantitative methods of genomics, proteomics, metabolomics, biochemistry and structural chemistry, to provide a comprehensive analysis of the regulation of lignin biosynthesis. A perturbation strategy is used to systematically knock down the expression of all pathway and regulatory genes known to be involved in lignin biosynthesis during wood formation, and the effects on lignin biosynthesis  analyzed using advanced genomic methods available. This information forms the foundation of statistics-based mechanistic modeling and lignin quantity/structural predictions for a quantitative model of lignin biosynthesis. Details of systems data generation, data analyses and model development will be presented. Substantial data have been generated for gene-specific (amiRNA & RNAi) transgenic P. trichocarpa, enzyme kinetics, protein regulation and numerical modeling, and stable-isotope-dilution based absolute quantitation of proteins and of metabolites. These results will be discussed. Our long term goal is a predictive model of lignin biosynthesis and quantity/structure for greater understanding of the plant response to environmental stress and for more precise strategies to improve plant productivity and the production of energy, biomaterials and food.The past research on lignin biosynthesis is substantial, creating one of a few opportunities in higher plants to integrate genomics, proteomics, biochemistry, chemistry and modeling to develop a comprehensive understanding of biosynthesis and structure of a major component of morphology and adaptation. We are conducting a systems biology study on regulation of lignin biosynthesis in wood formation. We seek to build models to quantitatively illustrate how the entire pathway is organized and regulated and to reveal regulatory and metabolic flux control mechanisms, leading to lignin quantity and structures. We use the model woody plant,


This paper describes the analysis of a database of over 180,000 patient records, collected from over 23,000 patients, by the hearing aid clinic at James Cook University Hospital in Middlesbrough, UK. These records consist of audiograms (graphs of the faintest sounds audible to the patient at six different pitches), categorical data  and brief free text notes made by the technicians. This data is mined to determine which factors contribute to the decision to fit a BTE (worn behind the ear) hearing aid as opposed to an ITE (worn in the ear) hearing aid.2) test, which can also be used in the model. The final model can act a decision support tool to help decide whether an individual patient should be offered a BTE or an ITE hearing aid.From PCA  four main audiogram types are determined, and are related to the type of hearing aid chosen. The effects of age, gender, diagnosis, masker, mould and individual audiogram frequencies are combined into a single model by means of logistic regression. Some significant keywords are also discovered in the free text fields by using the chi-squared (χThe final model was tested using 5-fold cross validation, and was able to replicate the decisions of audiologists whether to fit an ITE or a BTE hearing aid with precision in the range 0.79 to 0.87.A decision support system was produced to predict the type of hearing aid which should be prescribed, with an explanation facility explaining how that decision was arrived at. This system should prove useful in providing a "second opinion" for audiologists. This research looks for factors influencing the choice between two common hearing aid types: BTE (worn behind the ear) or ITE (worn in the ear). This choice is typically made by audiology technicians working in out-patient clinics, on the basis of audiogram results and consultation with the patient. In many cases, the choice is clear cut, but at other times the technicians might benefit from a second opinion given by an automatic system with an explanation of how that second opinion was arrived at. The production of such a decision support system is the main goal of this paper. Our data set is unusual in that ITE hearing aids are not generally available on the British National Health Service in England, as they are more expensive than BTE hearing aids. However, both types of aid are prescribed at James Cook University Hospital in Middlesbrough, UK. The data, collected between 1992 and 2001, consists of the following types of records:• Audiograms , e.g., 40, 35, 35, 35, 85, 70, 15, 20, 20, 30, 55, where the first six values are AC (air conduction) and the last five are for BC (bone conduction). AC is measured by placing headphones over the ears, and determines the overall level of hearing. BC is measured by placing the sound source tightly on the mastoid bone behind the ear, and measures the level of hearing of the inner part of the ear. A constraint on the data is that BC must always be the same or better than AC. The difference between the AC and the BC is called the air-bone gap, and measures the hearing ability of the middle and outer parts of the ear.• Categorical data , e.g., M, TINNITUS, BE18.• Brief free text notes made by the technicians, e.g., IMPS. TAKEN FOR BINAURAL AIDS., where IMPS is an abbreviation for "impressions", and BINAURAL means "worn in both ears".This section describes how a Principal Component Analysis (PCA) was performed on the set of 11,462 audiograms where all AC and BC thresholds for the right ear were recorded, to determine the main audiogram types found among hearing aid users. The rows of our input matrix were the individual audiograms for the right ear, while the 11 columns were for six air conduction and five bone conduction thresholds. Although the patients were originally tested at 11 frequencies, the principle of PCA is that certain frequencies tend to vary together, and thus can be grouped into a smaller number of underlying variables called principal components (PC). Each PC has a set of coefficients in the range -1 to +1, corresponding to the degree of influence of each of the original thresholds on that PC, given in Table A method was devised for converting PCA scores to typical audiogram types, which is an approximation only. It assumes that the PCA scores are directly related to audiogram thresholds, whereas in reality they measure different things: PCA measures the importance of a threshold in distinguishing audiograms, while audiograms measure the degree of hearing loss at the same frequency. The range of PCA scores is -1 to +1, while the range of audiogram thresholds is 0 to 120 dB. If we assume that the relationship is linear, then for each frequency a PCA value of -1 corresponds to an audiogram threshold of 0, and a PCA value of 0 = an audiogram threshold of 60, and a PCA value 1 = an audiogram threshold of 120. The formula relating the two was:Each individual patient audiogram was classified into one of the main audiogram types identified, according to least Euclidean distance. A chi-squared test was then performed to determine whether there was any association between the audiogram class of each patient and the type of hearing aid worn. This test was done on the set of 7,437 records where all AC and BC thresholds were available for the right ear, and either a BTE or an ITE aid was specified. In the final logistic regression model, rather than simply using the identified broad audiogram types, each individual hearing threshold was used.In the previous section, it was shown that the choice of hearing aid type was related to the shape of the audiogram. This section describes how the simple chi-squared test was used to discover which of the category data fields were significantly associated with the choice of hearing aid type, and also to discover free-text keywords which were significantly associated with either BTE or ITE hearing aids.p < 0.001. This data showed, with 99.9% confidence that these free text words were not randomly distributed, but some text words are associated with hearing aid type. To find the association between individual free-text words and hearing aid type, the quantity 2 / E (O - E)was examined to rank the keywords according to importance. Some of the keywords in Table To discover those free-text keywords which were significantly associated with either BTE or ITE hearing aids, first a large contingency table was created where the rows stood for hearing aid type, while each column stood for a candidate keyword (one of 664 distinct words found to have occurred at least once in the free-text fields of the entire record set). The observed value in each cell was the number of times that word had been found in the free-text fields of patients with that type of hearing aid. This table Table  had an oHaving determined that audiogram frequencies, gender, presence of tinnitus, use of tinnitus masker, age, mould and certain keywords were all associated with the decision between fitting a BTE or an ITE aid, all these factors were combined into a single logistic regression model. The input to the model was the matrix of patient data, where columns corresponded to attributes and rows corresponded to individual patient records. The output was a formula  in the f0 is a constant, and b1 to bk are called the coefficients of the model. The values x1 to xk are all either 1 or 0, depending on whether a given attribute in the patient's record is present or absent. The overall value L is greater than 0 if it is more likely that the patient should be given a BTE aid, while it is less than 0 if it is more likely that the patient should be given an ITE aid.In our case p is the probability that the patient should be fitted with an ITE aid, while (1- p) is the probability that the patient should be given a BTE aid. bBefore performing the logistic regression, the actual set of records was divided into two parts, one containing 80 percent 5,736) of the records and the other containing the remaining 20 percent 1,433) of these records. The 80 percent subset was used as the training set for model construction, and the remaining records for testing the model. The sampling method was to extract every fifth record for testing. The logistic regression was performed on the records which had all fields filled for the right ear: AC (air conduction) and BC (bone conduction) thresholds, gender, age and text keywords , of which 128 also had non-null entries for diagnosis, 98 had non-null entries for masker, and 3983 had non-null entries for mould. This data was converted into discrete numeric values as inputs to the model, as follows: For AC thresholds below the first quartile (40 db) a value of 0; for thresholds between the first and second quartile (55 dB) a value of 1; for thresholds between the second and third quartile (75 dB) a value of 2; and for thresholds above the third quartile a value of 3. The same method was used to assign values for BC thresholds and age, except in that the quartile thresholds were 25, 40 and 55 dB and 60, 70 and 78 years respectively. The values for diagnoses were 0 for no tinnitus diagnosis and 1 for tinnitus. Finally, for gender, 0 was assigned for male and 1 for female. Regression coefficients and associated p values were found for all the model variables and those variables with p values more than 0.05 for the constant were discarded. Thus, BC4000 (bone conduction at 4000 Hz), age, diagnosis and masker were not considered in the final model as the p values of their constants was more than 0.05, as shown in Tables 33 of the36 of theDue to data sparseness, it was not possible to incorporate all the keywords discovered by an analysis of the free text into a single model. However, a few significant keywords  were all negative and approximately equal. This suggests that the main source of variation between the patients was simply the overall degree of hearing loss. The coefficients of the second PC (PC2) were negative for frequencies at or below 1000 Hz, but positive for higher frequencies, for both air and bone conduction, and thus differentiated patients according to whether they have a predominantly high frequency or low frequency hearing loss. The coefficients of the third PC (PC3) were positive for air conduction at all frequencies, but negative for bone conduction, showing a contrast between patients with and without an air-bone gap. The fourth component (PC4) was similar to the third, but corresponded to an air-bone gap at low frequencies. No clear patterns were seen for the fifth or subsequent principal components. The first four PCs corresponded to audiogram types frequently encountered in audiology clinics. The percentage of the overall variability in the data explained by the first four principal components respectively was 59.5, 13.4, 9.7, and 5.2, giving a total of 87.8%.2 , calculated as the sum of the cells in Table 2 / E values which make the greatest contribution to the overall χ2 value are those in the PCA1 and PCA3 columns. Thus, flat hearing loss (PCA1) audiograms without air-bone were associated with ITE aids and flat audiograms with additional air-bone gaps (PCA3) were associated with BTE aids. This result is in accordance with Stephens [The thresholds corresponding to the first four PCs by using formula (1) are given in Table Stephens  which prO) are not enclosed in brackets, Expected frequencies (E) are in  and the quantity 2 / E (O - E)is in . The overall chi-squared value  was 33.68, which for one degree of freedom is significant at p < 0.001. Males tended more to use ITE hearing aids and females tended more to use BTE hearing aids. For the relationship between hearing aid type and a diagnosis of tinnitus (ringing in the ear), the overall chi-squared value was 31.75, again significant at p < 0.001 for one degree of freedom. Patients with tinnitus tended more to wear ITE hearing aids. The relationship between the wearing of a tinnitus masker (a soothing sound source designed to drown out tinnitus) and hearing aid type, among patients diagnosed with tinnitus, had the overall chi-squared value of 17.16, which for one degree of freedom, was also significant at p < 0.001. The data for the cross-tabulation of hearing aid type and age produced the overall chi-squared value of 10.53, which for one degree of freedom, showed significance at p < 0.001. Mould type was also cross-tabulated with hearing aid type and the overall chi-squared value was 9844.18, which for 30 degrees of freedom was significant at p < 0.001. Thus all the category data types were significantly associated with hearing aid type. All the data in the patient records was used without considering confounding effects, where for example it might have been the choice of hearing aid type affecting the choice of mould, rather than vice versa. It is believed that this may have been the case, since many mould types never occurred in conjunction with one or the other hearing aid type.The contingency table showing the relationship between gender and hearing aid type is shown in Table The set of free-text keywords which tended to occur significantly more and less often  in records where the patient wore either BTE or ITE aids are shown in Table In Table As shown in Table To show how the model works, a sample record from the test set is taken, as shown in Table Testing of these logistic regression models showed that overall there was 81.64% agreement between the predictions of our model and the actual hearing aid chosen by the audiologist (as given in the "type" field) as shown in Table In Table The theoretical upper bound of classifier performance is the inter-annotator agreement , in our Although this LR model did not find age as a significant factor, Meredith and Stephens  have fouThe associations between hearing aid type and audiogram type were confirmed by both the PCA/chi-squared and LR experiments described in this paper, and also by the authors' previous work on associations between words found in the database and hearing aid type, and the previous findings by audiologists . These aIt is planned to validate these results by obtaining feedback from a professional audiologist, and by using an approach (Bayesian networks) which constructs model with interaction between variables. A major advantage of both Naïve Bayes and Logistic Regression is that they enable an explanation facility to be incorporated into any decision support tool, since it is easy to read back and see exactly which variables contributed exactly how much to the final decision of whether to fit a BTE aid or an ITE aid.The authors declare that they have no competing interests.MNA conducted the statistical analyses and drafted the manuscript together with MPO. MPO was project supervisor. Both authors contributed to the design and conception of this work. Both the authors have read and approved the final version of the manuscript.


An accurate understanding of the electrical interaction between retinal prostheses and retinal tissue is important to design effective devices. Previous studies have used modelling approaches to simulate electric fields generated by epiretinal prostheses in saline and to simulate retinal ganglion cell (RGC) activation using passive or/and active biophysical models of the retina. These models have limited scope for studying an implanted human retinal prosthesis as they often do not account for real geometry and composition of the prosthesis-retina interface. This interface consists of real dimensions and location of stimulation and ground electrodes that are separated by the retinal tissue and surrounded by physiological fluids.et al. in their Investigative Ophthalmology and Visual Science (IOVS) paper published in 2008 using the Argus I epiretinal implants. Finite element method (FEM) based computations were used to estimate threshold currents based on a threshold criterion employing a passive electric model of the retina.In this study, we combined the prosthesis-retina interface elements into a framework to evaluate the geometrical factors affecting stimulation thresholds for epiretinal prostheses used in clinical human trials, as described by Balthasar Threshold currents and impedances were estimated for different electrode-retina distances. The profiles and the values for thresholds and impedances obtained from our simulation framework are within the range of measured values in the only elaborate published clinical trial until now using Argus I epiretinal implants. An estimation of resolution for the electrodes used in these trials was provided. Our results reiterate the importance of close proximity between electrodes and retina for safe and efficient retinal stimulation.The validation of our simulation framework being relevant for epiretinal prosthesis research is derived from the good agreement of the computed trends and values of the current study with measurements demonstrated in existing clinical trials on humans (Argus I). The proposed simulation framework could be used to generate the relationship between threshold and impedance for any electrode geometry and consequently be an effective tool for design engineers, surgeons and electrophysiologists. These dTwo major electrical parameters responsible for affecting the efficiency of retinal prostheses  are: (i)An electrode-retina distance contributes to the varying current spread from the electrodes causing changes in the stimulation area in the retina and therefore affects the resolution of the prosthesis. In vitro electrophysiological data and analytical calculations suggest that the threshold currents rise rapidly with increasing distance of the electrodes from the retinal surface ,15.Electrode geometry has an effect on the current required for RGC activation. In vitro experiments  have estPresently, proximity of the retina to the electrodes is verified by two different techniques after implantation of retinal prostheses. Optical coherence tomography (OCT) is one of the methods that reveal only proximity of the edges of the device to the retina for a non-transparent retinal implant. The other technique, known as impedance analysis  uses theDiscretisation methods such as Finite Element Method (FEM) can be employed to compute electric fields within the retina for different electrode geometries and electrode-retina distances in either epiretinal or subretinal schemes. For real geometries, it is cumbersome to analytically calculate and predict the effect of these factors on retinal stimulation. To resolve such a complex electrostatic problem, FEM can be used in a simulation framework. For a successful simulation, the framework should include anatomically correct retina model describing electrical characteristics of the retinal layers  with dueet al. [For our studies, a simulation framework was built integrating the prosthesis-retina interface elements involved in an epiretinal prosthesis closely resembling the one used in the framework of the only and most comprehensive published human trials until now using Argus I epiretinal implants by de Balthasar et al. . Followiet al.  that inc17 Ω·m [5 Ω·m, a standard value for bulk platinum. The ground electrode is placed on the photoreceptor  side and axially shifted by 15 mm away from the stimulation disc electrode. The ground was defined as a 100 mm diameter platinum disc electrode.Elements of the framework are: physiological fluid, stimulation and return (ground) electrodes, and retina. Physiological medium encompasses the implant until the photoreceptor layer of the retina and defined to have a resistivity of 2 Ω·m. The retinal pigment epithelium (RPE) is represented by a highly resistive block above the retina, which is set to a resistivity of 500 Ω·m. The stimulation electrode is positioned on the RGC layer side of the retina in the form of a planar disc embedded in an insulated substrate and located at the geometrical centre of the entire model geometry. The insulation (flexible sheet of the implant) was defined with a resistivity of 1 × 1017 Ω·m  correspoin vivo [The retina was modelled as an inhomogeneous element with a piecewise linear change in resistivity, from 35 Ω·m at the photoreceptor layer to 2 Ω·m in the RGC layer. These values were extrapolated from measurements carried out in macaques, in vivo . Even thin vivo . The selin vivo , the impin vivo  due to tin vivo . Consideg) and electrode disc diameter (d). The retinal resistivity model is positioned according to the electrode-retina distance, between 0 μm and 1500 μm. The diameter of disc electrodes was defined to be 260 μm and 520 μm to mimic the electrode geometry used in the human trials [GL h- depth at which ganglion cells are assumed to be located, and Ret h- depth where the retina ends and the RPE starts. GL hwas defined to be 20 μm outwards from the epiretinal side, i.e., (g+20) μm from the surface of the implant. Figure Two geometrical factors affecting the activation thresholds were included in the simulation model as variable parameters, electrode-retina distance  -29, stimet al.  employinet al. . They caet al.  that weret al. [-4 ms [GL hlayer.An RGC activation threshold criterion can be extracted from one of the multiple strength-duration curves computed by Boinagrov et al.  using a . [-4 ms   was performed on the stimulation and the ground electrodes to ensure current conservation. The data extracted from the simulations were post-processed to generate the required plots. The current delivered by the electrode was computed by a boundary integration of the normal component of current density over the ground electrode. Impedance is computed as the ratio between the applied voltage stimulus and the resulting current seen at the electrode taking into consideration the retina with or without an electrode-retina gap.Comsol MultiphysicsThe time-varying bio-electric fields, currents and voltages in a biological medium can be examined in the conventional quasi-static limit . In our GL his a sufficient condition to activate the RGCs. The FEM simulations used a monopolar stimulation scheme for which the return electrode is located in the far field. Simulations were performed with a potential difference applied between the stimulation and ground electrodes. The area of activation for a 400 μm electrode is graphically illustrated in Figure Based on an earlier explanation, we emphasise that in our simulation framework, injecting a current that will produce an electric field of 1 kV/m at et al. [The effectiveness of this computational study can be evaluated by directly comparing clinical and electrophysiological results with outcomes based on our simulation framework. One of the principal results for comparing our study are the only exhaustive measurements made during the clinical study conducted by de Balthasar et al.  on humanin vitro experiments conducted by Jensen et al. [A computed threshold current is plotted as a function of electrode-retina distances for the two electrode sizes: 260 μm and 520 μm are presented in Figure n et al. . At largn et al. .2 [2 was chosen for our study considering a real geometry of the electrode and a pulse width of 0.975 ms. Currents corresponding to this charge limit are 190.6 μA for 260 μm and 762.4 μA for 520 μm electrodes. It can be observed that the current injection limit can be reached at an electrode-retina distance of about 270 μm for 260 μm diameter electrodes and nearly 600 μm for 520 μm diameter electrodes. Close proximity of RGCs to the electrodes is thus a critical issue for safe and chronic retinal stimulation.Safe stimulation is critical for a chronic usage of a retinal prosthesis. Platinum electrodes have a charge density limit ranging between 0.05  and 0.492  per stim2 . The ran2 . A theorin vitro experiments conducted by Jensen [et al. [A range of disc electrodes with diameters ranging between 10 μm and 1500 μm were used to simulate the relationship between the stimulation threshold and electrode-retina distances - 0 μm (in contact with retina), 10 μm and 100 μm. In retina contact condition presented in Figure y Jensen  and the y Jensen  groups i [et al.  as the sComputed threshold currents for 260 μm and 520 μm electrodes differ only slightly at electrode-retina distances from ~200 μm onwards. This similarity between threshold currents for the two electrodes was also observed in Argus I clinical trials . It is i2) is plotted in Figure Electrode-retina distance influences the threshold current values for various electrode sizes having a pronounced effect on safe stimulation of the retina. A trend line between threshold current limits for different disc electrodes based on the electrochemical limit for platinum  have been correlated with changes in the tissue resistivity surrounding the electrode  and elecOur framework computed the trend between impedance and electrode-retina distance and is shown in Figure GLh) from the stimulation electrode. During actual experiments, to ensure stimulation, there is a tendency to use stimulation currents 10-20% above the pre-determined minimum threshold current. In our study, we define lateral extent as the horizontal distance (measured from the electrode axis) covered at GL hcorresponding to a threshold electric field of 1 kV/m caused by a 20% excess on the threshold current. An illustration of the assumption and the lateral extent definition is presented in Figure In our study, we have computed threshold currents for activation of a single RGC located at the cell activation depth (Simulations on the effect of geometrical factors, viz. electrode size and electrode distance to the retinal surface affecting impedance and threshold values is an indication of the importance of proximity between the electrode array and the retina for a successful retinal implant. Resolution of the implant can be estimated for different electrode-retina distances considering the computed lateral extents of stimulation. Electrode breakdown and tissue encapsulation effects, in spite of being extremely important in the viability of neural prostheses, have not been observed to be dominant in implanted human retinal prostheses studied until now. This could be due to the fact that they have not been studied long enough to evaluate their performance under long term exposure to retinal milieus. Based on the threshold and impedance data collected during clinical epiretinal trials conducted in human subjects until now ,41, the An integrated simulation framework computing electric fields in the electrode-retina interface could help in understanding the effective operation of a retinal implant. Knowledge of current densities in the retinal tissue can resolve significant questions which include: design of implantable electrode arrays, a proper location for the implant to be placed, optimal electrode geometry and ground placement, efficiency of different shapes and sizes of electrodes, optimal inter-electrode spacing, maximum amount of current injected safely for a given configuration, efficiency of current injection and current circulation in a tissue for a particular scenario.Gregoire Cosendai is affiliated with Second Sight Medical Products. The other authors declare that they have no proprietary, financial, professional, or other personal competing interests of any nature or kind.HK and PR designed the model and simulations. HK performed all computations and simulations. HK and WH wrote the manuscript together. WH along with HK was involved in systematic organisation and representation of results. GC and AB supplied critically important intellectual content in revising the manuscript. All authors read and approved the final manuscript.


We aimed to determine whether the clinical characteristics and electrodiagnostic classification of nerve injury, and response to treatment differed in patients diagnosed with chronic inflammatory demyelinating polyneuropathy (CIDP) with and without diabetes.CIDP patients with diabetes (CIDP+DM) (n = 67) and without diabetes (CIDP-DM) (n = 67) underwent clinical examination and nerve conduction studies (NCS). CIDP-DM patients were selected using age and gender matching with the existing CIDP+DM cohort. Patients treated with immunotherapies were classified as responders (R) (n = 46) or non-responders (NR) (n = 54) based on clinical response to treatment. The groups were compared using analysis of variance, contingency tables and Kruskal-Wallis analyses.CIDP+DM subjects had more severe neuropathy based on higher lower limb vibration potential thresholds (VPT)(p = 0.004), higher Toronto Clinical Neuropathy Score (TCNS) (p = 0.0009), more proximal weakness (p = 0.03), more gait abnormality (p = 0.03) and more abnormal NCS. CIDP+DM subjects had more abnormal sural NCS with lower sural sensory nerve action potential amplitudes  and slower sural nerve conduction velocities . CIDP-DM subjects were more likely to receive immune therapies , despite no significant differences in treatment responder rates (p = 0.71). Patients who responded to therapy had shorter duration of CIDP than non-responders .The clinical phenotype and electrophysiological profile of CIDP patients differs according to the presence or absence of diabetes. Despite CIDP+DM patients having more severe clinical and electrophysiological neuropathy, they are less likely to receive disease-modifying/specific therapy, yet have similar response rates to treatment as those without diabetes. Specifically, the duration of neuropathy - not diabetes status - was associated with treatment response. Chronic inflammatory demyelinating polyneuropathy (CIDP) is an immune-mediated inflammatory disorder of the peripheral nervous system. Classically, CIDP results in demyelination of peripheral nerves, as indicated by a significant reduction of motor conduction velocity and prolonged distal motor latencies We therefore aimed to determine whether the clinical characteristics, electrodiagnostic classification of nerve injury and treatment response differed in diabetes and non-diabetes patients diagnosed with CIDP; and whether these characteristics could be useful to differentiate the two entities.CIDP subjects attending the Neuromuscular clinic for management of their immune-mediated polyneuropathy between 1997 and 2013 were evaluated for this study. Our current study involved the extraction of demographic data, clinical history, physical examination, laboratory test results, and electrophysiologic data from previously coded charts of CIDP patients. The Research Ethics Board of the University Health Network approved the current study protocol, based on chart review and collection of de-identified data.All subjects were ≥18 years of age and had a diagnosis of CIDP. All subjects with a confirmed diagnosis of either type 1 or type 2 diabetes mellitus (DM) and CIDP were included in this study (n = 67). The duration of diabetes mellitus was taken from the time when the patients first developed symptoms or from the time that they were diagnosed as having diabetes by their physician, if asymptomatic. CIDP-DM patients were selected from the full CIDP cohort (n = 1950) and were matched based on sex and age to the CIDP+DM cohort, using 1∶1 matching. DM was diagnosed according to the American Association of Diabetes criteria based on one of four abnormalities: haemoglobin A1c, fasting plasma glucose, random elevated glucose with symptoms, or abnormal oral glucose tolerance test CIDP patients, with or without DM, were also classified as responders (R) (n = 46) or non-responders (n = NR) (54) based on clinical response to treatment assessed by a combination of patient and physician evaluations in the 100 patients who were treated with immunotherapies. R subjects were those who stabilized after declining progressively, or improved after treatment, and NR subjects either worsened or did not change after treatment based on their clinical evaluation.Subjects were evaluated for neuropathy by neurological examination, the 19-point Toronto Clinical Neuropathy Score (TCNS), vibration perception thresholds (VPT), and sural, peroneal and tibial NCS Sural, peroneal and tibial NCS were performed using surface stimulating and recording techniques according to the standards of the Canadian Society of Clinical Neurophysiology and the American Association of Neuromuscular and Electrodiagnostic Medicine Vibration perception thresholds were measured using the Neurothesiometer  using the method of limits. Three tests were performed on the dorsum of each first toe proximal to the nail bed and a mean of the 3 responses was calculated. A “null” test was randomly inserted into the test sequence to ensure that the patient was reporting vibrating sensation accurately.2-test, while differences in continuous variables were assessed using the ANOVA, or the Kruskal-Wallis test for non-parametric data. Given the multiple comparisons, for each set of analyses significance was adjusted with Bonferroni correction, using a baseline p-value of 0.05, divided by the number of comparisons done.Statistical analysis was performed using JMP . Demographic data were expressed as means ± standard deviation (SD) for normally distributed data, or median and interquartile range [IQR] for data not normally distributed. Differences in categorical variables were assessed using the χ1c (HbA1c) of 7.1±1.9%. CIDP+DM subjects had higher systolic blood pressures (p = 0.04), higher lower limb vibration potential thresholds (VPT) , and more abnormal sensory testing as indicated by Toronto Clinical Neuropathy Score (TCNS) (p = 0.0009) than CIDP-DM subjects. CIDP+DM subjects also had weaker hip flexors (p = 0.05) and weaker quadriceps (p = 0.03), and more abnormality of gait (p = 0.03) compared to CIDP-DM patients. CIDP+DM subjects had higher HbA1c values (7.7±2.0%) than CIDP-DM patients  as expected, and a higher prevalence of retinopathy, nephropathy and hypertension .The demographic data of the 134 CIDP subjects categorized with diabetes  or without diabetes  are shown in On NCS, CIDP+DM subjects had shorter peroneal distal motor latencies , shorter tibial distal motor latencies , lower sural sensory nerve action potential amplitudes  and slower sural conduction velocities  than CIDP-DM subjects. Details of immunomodulatory and immunsuppressive treatment are shown in Of the 134 CIDP subjects, with or without DM, 100 received treatment for CIDP and the clinical and electrodiagnostic features of the 46 NR and 54 R subjects are shown in We examined a matched cohort of CIDP patients with and without diabetes to compare their clinical characteristics, electrodiagnostic classification of nerve injury and response to treatment. This report is the largest cohort so examined. We observed that CIDP+DM patients had a different clinical profile when compared to patients with CIDP-DM. Specifically, CIDP+DM patients had more severe neuropathy and a similar treatment response rate compared with CIDP-DM patients as observed by Gorson Existing criteria for the diagnosis of CIDP are highly specific We have previously reported that diabetes patients demonstrate electrophysiological findings atypical for classic DSP, although insufficient to make a diagnosis of CIDP using existing criteria Limitations of the current study are that this is a retrospective review. Although outcomes are based on clinical evaluation by both the physician and patient, rigorous scales such as the inflammatory cause and treatment (INCAT) scale, the Rasch-built Overall Disability Scale (RODS) and the Overall Neuropathy Limitation Scale (ONLS) were not available for most patients when this study was done, although current practice has changedThe differentiation of CIDP in patients with and without diabetes is important due to the implications for therapy and prognosis. This study shows that CIDP+DM patients have more severe clinical and electrophysiological neuropathy than CIDP-DM patients, but that they do not receive the same treatment despite having similar treatment response rates. We know that severity of neuropathy does not prevent response to treatment as shown by the ICE study results


The objective of the paper is to evaluate the hearing preservation rate in patients with high frequency hearing loss, treated with Cochlear Nucleus Freedom Hybrid-L implant in the Otolaryngology Department, Poznan University of Medical Sciences in Poland. Study was designed as the retrospective analysis. Twenty-one patients were operated and implanted with Nucleus Freedom Hybrid-L implant. Pure tone thresholds were recorded prior to the surgery and at the time of speech processor switch-on. Patients were subdivided into two groups with respect to their PTA thresholds: group A—classic indications and group B—extended indications. Average PTA for three frequencies  were calculated for each patient pre- and postoperatively. In the group of 21 implanted patients in 17 cases we have observed preservation of hearing  with a mean value of 13.1 dB. In 4 out of 21 patients deafness on the implanted ear was noted. Our results clearly indicate that with standard procedure hearing preservation can be obtained in majority of patients. Hearing preservation was not achieved in 19 %, but owing to design of the electrode of the Cochlear Nucleus Hybrid-L that enables to work as CI platform alone, in patients who lost their hearing after surgery re-implantations were not required. This proves that EAS is a safe and reliable method to help patients with specific type of hearing loss. Selected group of patients with hearing loss at high frequency and still preserved hearing at low frequencies do not benefit from the classic hearing aid. Parallelly, due to residual hearing they do not meet the qualification criteria for classic cochlear implant. Nevertheless, ongoing development indicated that the hybrid system—concept of electro-acoustic stimulation  i.e., coPreliminary observations that proceeded research and development on the hybrid system were combined with observations on conservation of residual hearing in cochlear implants recipients. Results showed that 50 % of operated patients retained sufficient hearing for effective use of ipsilateral ITE hearing aid . Number Since then, a great progress has been made. New, less traumatic electrodes were introduced as well as the surgery improved from 1–1.2 mm cochleostomy to the round window approach. Atraumaticity i.e., preservation both hearing and vestibular function  become tNew Nucleus Freedom Hybrid-L system was introduced into the clinical practice in 2009. First experiences with new electrode were presented by Lenarz et al.  confirmin = 21) were included to the study. There were 12 females and 9 males, ranging in age from 16 to 77 years of age, with a mean of 49.5 years and median 52.5 years of age. Nucleus Freedom Hybrid-L device  was implanted in every patient by the same surgeon (WS) with the standardized surgical technique described previously [Study was designed as the retrospective analysis conducted at the Department of Otolaryngology and Oncological Laryngology, Poznan University of Medical Science. Twenty-one patients with high frequency hearing loss , minimum 5 years of stable hearing loss, insufficient gain form hearing aid (30 % of speech understanding). Impressions for acoustic components were taken between 7th and 10th day after surgery. Speech processor switch-on was done in 5–8 weeks, depending on acoustic component delivery. Up to date, all the patients use electric as well as acoustic stimulation—none of the patient use only electric stimulation. Twelve patients use hearing aids contralaterally.n = 13) and second with extended criteria according to Lenarz et al.  with residual hearing loss [Studied cohort was divided into two subgroups with respect to preoperative PTA recordings. First one with classic indication for hybrid-L implant  were calculated. Acoustic thresholds >110 dB or without recordings were considered as hearing loss and for statistic evaluation marked as 120 dB. The difference between mean values before and after surgery was studied and defined as follows: hearing preservation—mean value up to 10 dB; hearing impairment—mean value between 10 and 30 dB; hearing loss for patients with no thresholds recorded postoperatively.In the group of 21 implanted patients in 17 cases we have observed preservation of hearing . In 4 out of 21 patients deafness on the implanted ear was noted , although all these 4 patients reported significant difference in hearing after acoustic component activation.In 17 patients with preservation of hearing analysis of audiometric results revealed that within the A group (13pts.) in 9 cases average hearing thresholds were calculated for 3 evaluated frequencies. In 3 cases hearing threshold for 1,000 Hz was not detectable, comparing to preoperative values: 80, 110 and 100 dB. The pre–post difference of mean hearing threshold was 0–10 dB in 6 cases, 11–20 dB in 3 cases, and 21–30 dB in 3 cases. Within the B group (8pts.) in 4 cases hearing threshold for three examined frequencies was established. The pre–post difference of mean hearing threshold was 0–10 dB in 3 cases and 21–30 dB in 2 cases. In one patient from the B group hearing threshold for 1,000 Hz was not recorded pre- and postoperatively  implanted in patients with residual hearing in low frequencies were very successful –19. AlthInsertion of 16 mm Hybrid-L electrode has a standardized surgical procedure. This 16 mm with 22 contacts electrode covers approximately 270° of the basal turn of the cochlea, which is an equivalent of the former Nucleus 24 straight electrode. The stopper and the wing ascertains the comparable electrode position in the cochlea of the operated patients. Thus, variable such as insertion depth is not an issue in Cochlear Nucleus Freedom Hybrid-L implant. Data presented by Lenarz et al. [The overall hearing preservation rate in the current study was 80.95 % (17/21). Within the classic indication group (Group A) it was 92.3 % (12/13), but unfortunately, for patients with extended indications (group B) hearing preservation rate was lower—62.5 % (5/8). Nevertheless, it is still over 50 % above the level indicated in the literature. Looking at the potential cause of hearing loss in patients from our study, surgery variations was not an issue, because all cases were operated by the same surgeon. Thus, other reasons for the failure such as age of the patients (two were over 70 years), diabetes, vascular problems or inflammatory responses should be taken under consideration. These variables were not evaluated in the study.Our results clearly indicate that with standard procedure hearing preservation can be obtained in majority of patients. Implantations were successful in all cases with no major surgical problems. Hearing preservation was not achieved in 19.05 %, but owing to design of the electrode of the Cochlear Nucleus Hybrid-L that enables to work as CI platform alone, in patients who lost their hearing after surgery re-implantations were not required. This proves that EAS is a safe and reliable method to help patients with specific type of hearing loss.


Brown cotton fiber is the major raw material for colored cotton industry. Previous studies have showed that the brown pigments in cotton fiber belong to proanthocyanidins (PAs). To clarify the details of PA biosynthesis pathway in brown cotton fiber, gene expression profiles in developing brown and white fibers were compared via digital gene expression profiling and qRT-PCR. Compared to white cotton fiber, all steps from phenylalanine to PA monomers (flavan-3-ols) were significantly up-regulated in brown fiber. Liquid chromatography mass spectrometry analyses showed that most of free flavan-3-ols in brown fiber were in 2, 3-trans form , and the main units of polymeric PAs were trihydroxylated on B ring. Consistent with monomeric composition, the transcript levels of flavonoid 3′, 5′-hydroxylase and leucoanthocyanidin reductase in cotton fiber were much higher than their competing enzymes acting on the same substrates . Taken together, our data revealed a detailed PA biosynthesis pathway wholly activated in brown cotton fiber, and demonstrated that flavonoid 3′, 5′-hydroxylase and leucoanthocyanidin reductase represented the primary flow of PA biosynthesis in cotton fiber. Naturally colored cotton is an important raw material for ecological textiles. With naturally colored cotton, textile manufacturers can eliminate dyeing during processing, and significantly reduce processing costs, environmental pollutions and chemical residues in fabrics PAs, also known as condensed tannins, are widely distributed in plants with various functions such as pigments in seed coat and protectants against herbivores and microbes In attempt to clarify the details of PA biosynthesis pathway in brown cotton fiber, we performed a digital gene expression (DGE) analysis to compare the gene expression profiles in brown and white fibers. A total of 24 PA synthase genes were identified to be significantly up-regulated in brown fiber. Furthermore, we determined the chemical properties of PAs in brown fiber by using liquid chromatography-mass spectrometry (LC-MS) method. It was found that the majority of PA initiating units consisted of gallocatechin and the main extension units in brown fiber were trihydroxylated on B ring. These results demonstrated a detailed PA pathway involved in the brown pigmentation in cotton fiber, which may be essential to manipulate the biosynthesis of pigment and other flavonoids in cotton fiber.NlaIII restriction, the 3′ cDNA ends were separated by magnetic method and linked to an adaptor containing a MmeI recognition site. After MmeI digestion and ligation to the second adaptor, the tags were sequenced on an Illumina Genome Analyzer  using Solexa technology. The raw sequence data were deposited in NIH Sequence Read Archive under the accession number SRP033354. Sequenced tags were annotated by aligning with Gossypium hirsutum unigenes and singleton ESTs  http://www.phytozome.net/search.php) A recombination inbred line (RIL) population derived from the cross between white fiber cotton cultivar Yumian No.1 and brown fiber line T586 was constructed as described Quantitative RT-PCR (qRT-PCR) was employed to detect the expression levels of predominant PA synthase genes in each 10 brown and white fiber RILs. The investigated genes and corresponding primers were listed in 2, and extracted in 50 ml 80% aqueous acetone twice at 4°C for 2 h. Solutions were centrifuged at 10 000 rpm for 5 min. Supernatants were combined and acetone was evaporated under vacuum at 35°C. Two-microliter aliquots of the remaining aqueous solution were applied to a packed Supelco Discovery DPA-6S polyamide cartridge . After washing with 5 ml 30% methanol, PAs were eluted with 2 ml 90% N, N-dimethylformamide. Finally, eluates were evaporated to dry under vacuum, re-suspended in methanol, and subjected to LC-MS analyses.PAs were extracted from developing brown cotton fibers and purified as described + ions were eluted at 8.64 and 14.12 min, while gallocatechin and epigallocatechin with [306+H]+ ions were detected at 3.92 and 6.62 min, respectively. Flavan-3-ol amounts were calculated by reference to the standard curves of authentic standards.To detect monomeric flavan-3-ols in the purified PAs, LC-MS analyses were performed on LC-MS 2010A system  with an Xtimate C18 column . Solvent A was water: formic acid , and solvent B was acetonitrile: formic acid . Around 5 µg PAs were injected and eluted with a gradient of solvent B to A at a flow rate of 0.2 ml/min. After 3-min isocratic elution in 90% solvent A and 10% solvent B, solvent B concentrations increased from 10% to 30% (v/v) in 12 min, followed 15-min washing with 100% of B and 3-min re-equilibration in 10% of B. Positive ions were monitored in a selected ion monitoring mode. Monomeric flavan-3-ols were identified according to the typical ions and retention times of authentic standards. Catechin and epicathechin with [290+H]+ at 14.81 min, [288+H]+ at 16.73 min, and [272+H]+ at 18.33 min, respectively. Anthocyanidins were quantified according to the areas of typical peaks by reference to the standard curves of corresponding standards.To elucidate the chemical properties of PA extension unit, purified PAs were hydrolyzed by acid butanol method G. hirsutum unigene or singleton EST . Gene expression levels in BCF and WCF bulks were compared according to the corresponding tag frequencies. A total of 2079 differentially expressed genes (FDR<0.001) were identified, among which 1165 genes were up-regulated (expression level ratio BCF/WCF>2) and 914 were down-regulated (BCF/WCF<0.5) in brown fibers  and/or pigment accumulation in cotton fiber may affect multiple aspects in fiber development other than fiber coloration.Gene ontology (GO) analysis suggested that the cellular components of vehicles (including membrane-bounded vesicle and cytoplasmic vesicle) and membrane were most significantly over-represented in differentially expressed genes (DEGs) between white and brown fibers . The mosIn higher plants, PAs are synthesized through the flavonoid pathway from phenylalanine to flavan-3-ols  [10], [1G. raimondii) were detected in brown and white fibers via qRT-PCR. Compared to white fiber, all of the 12 PA synthase genes were up-regulated in brown fiber  wholly activated PA biosynthesis pathway in cotton fiber.To verify the result of DGE analysis, expression levels of the predominant PA synthase genes identified for all steps from phenylalanine to flavan-3-ols  and 5 anDGE analysis also revealed that transcript levels of different flavonoid synthases varied dramatically in cotton fibers . In whitTranscriptional analysis revealed a PA biosynthesis pathway wholly activated in brown cotton fiber. To further clarify the details in this pathway, we employed LC-MS method to determine the monomeric composition of PAs in brown fiber. Four flavan-3-ols  were identified in the PAs from brown fiber by LC-MS analysis, with mol percentages of 85.4±1.4%, 3.0±0.2%, 10.8±1.2% and 0.8±0.1%, respectively . Among tThe anthocyanidin composition in the acid hydrolysate reflected the extension unit composition of corresponding PAs Taken together, transcriptome and biochemical analyses collectively demonstrated a detailed PA biosynthesis pathway wholly up-regulated in brown cotton fiber, in which F3′5′H and LAR represented the primary flow for PA biosynthesis .et al revealed much higher PAL activity in brown cotton fiber compared to white fiber Lc1) activated the whole PA biosynthesis pathway in cotton fiber. Furthermore, biochemical analyses demonstrated that the main PA units were trihydroxylated on B ring and most of free flavan-3-ols were in 2, 3-trans form. These results demonstrated that F3′5′H and LAR represented the major flow for PA biosynthesis in brown cotton fiber. By dissecting the details of PA biosynthesis pathway in brown cotton fiber, our results paved the way to manipulate the biosynthesis of pigment and other flavonoids in cotton fiber via biotechnology techniques.The exact chemical property of pigments is an important clue for exploitation of naturally colored cottons. Early extraction experiment suggested that pigments in naturally colored cotton belonged to flavonoids Medicago truncatula which consist mainly of epicatechin and/or catechin PAs are the predominant coloring compounds in seed coats, and may function as barrier to fungus infection of embryos In higher plant, PAs include a large number of oligomers or polymers of flavan-3-ols. In addition to various degrees of polymerization, difference in monomeric composition is a key factor influencing the complexity of PA components. There are two major branching points in the PA pathway which lead to different PA monomers . FirstlyFlavonoids may play roles in many aspects of plant growth and development Transcriptome analysis revealed that a whole PA pathway from phenylalanine to flavan-3-ol was activated in cotton fiber by the brown fiber gene. LC-MS analyses demonstrated that most of free favan-3-ols in brown cotton fiber were in 2, 3-trans form, and the main PA units were favan-3-ols trihydroxylated on B ring. The PA monomeric composition was consistent with the expression profiles of PA synthase genes, and suggested that F3′5′H and LAR represented the major flow of the PA biosynthesis pathway in brown cotton fiber.


Sildenafil citrate (Viagra), a phosphodiesterase 5 inhibitor (PDE5i), is a commonly prescribed drug for erectile dysfunction. Since the introduction of Viagra in 1997, several case reports have linked Viagra to sudden sensorineural hearing loss. However, these studies are not well controlled for confounding factors, such as age and noise-induced hearing loss and none of these reports are based on prospective double-blind studies. Further, animal studies report contradictory data. For example, one study (2008) reported hearing loss in rats after long-term and high-dose exposure to sildenafil citrate. The other study (2012) showed vardenafil, another formulation of PDE5i, to be protective against noise-induced hearing loss in mice and rats. Whether or not clinically relevant doses of sildenafil citrate cause hearing loss in normal subjects  is controversial. One possibility is that PDE5i exacerbates age-related susceptibility to hearing loss in adults. Therefore, we tested sildenafil citrate in C57BL/6J, a strain of mice that displays increased susceptibility to age-related hearing loss, and compared the results to those obtained from the FVB/N, a strain of mice with no predisposition to hearing loss. Six-week-old mice were injected with the maximum tolerated dose of sildenafil citrate (10 mg/kg/day) or saline for 30 days. Auditory brainstem responses (ABRs) were recorded pre- and post injection time points to assess hearing loss. Entry of sildenafil citrate in the mouse cochlea was confirmed by qRT-PCR analysis of a downstream target of the cGMP-PKG cascade. ABR data indicated no statistically significant difference in hearing between treated and untreated mice in both backgrounds. Results show that the maximum tolerated dose of sildenafil citrate administered daily for 4 weeks does not affect hearing in the mouse. Our study gives no indication that Viagra will negatively impact hearing and it emphasizes the need to revisit the issue of Viagra related ototoxicity in humans. Sildenafil citrate (Viagra), a phosphodiesterase 5 inhibitor (PDE5i), was originally synthesized for angina pectoris. It was patented in 1996, approved by the FDA in 1998, and then prescribed for erectile dysfunction and pulmonary hypertension. Within the first month of release, 300,000 prescriptions were written and more than 400 million dollars worth sold within its first quarter in the US One specific side effect that requires further investigation is sudden sensorineural hearing loss (SSHL). SSHL is characterized by a sudden or rapid loss of hearing, often causing distress in affected patients. The incidence of SSHL is approximately 4,000 cases per year with an unknown etiology but can be attributed to multiple underlying causes, such as viral infections or ototoxic drugs et al presented a case report of SSHL, verified by audiometric data, in a 44-year-old man after he ingested sildenafil citrate at 50 mg/day for 15 days for erectile dysfunction et al enrolled 18 patients with erectile dysfunction who had been using a PDE5i in a prospective clinical study et al and Khan et al in which they found 88% (N = 17) and 66.7% (N = 18) of their patients, respectively, to have hearing loss in the same time frame. However, it is unclear whether baseline audiometric data was taken into account for each of the reported cases in Maddox et al and Khan et alet al also indicates that 4 of the cases reported by Maddox et al were in fact previously diagnosed with otologic disease, indicating that the reported data might be confounded by other factors such as pre-existing co-morbidities. In particular, the target population (older adults) for PDE5i prescriptions are at an increased risk for age-related hearing loss (presbycusis). Therefore, more studies are necessary to determine the relationship between PDE5i and SSHL.In 2007, Mukherjee et al showed that in both a mice and rat model vardenafil protects against noise-induced hearing loss (NIHL) in vivo that vardenafil activated the cGMP-Prkg1 signaling cascade in outer hair cells, satellite cells, and spiral ganglion nuclei, shielding the sensory epithelia in the cochlea from damage through the function of poly-ADP ribose polymerase (PARP). As a negative control, chronic administration of vardenafil (21 days) did not affect hearing function in rats as verified by audiometric testing. The data from Jaumann et al et al showed that a daily dose of sildenafil citrate in rats at 10 MPK for 135 days increased 4 kHz tone bursts (TB) thresholds 15 days into the treatment, 8 kHz TB thresholds at 135 days, and click thresholds as early as 100 days et al recently reported elevated caspase 3 immunoreactivity in 30% of the sildenafil citrate treated rats, suggesting apoptotic activation from 45 days of sildenafil citrate treatment at 1.5 MPK Rodents have served as excellent models in hearing research but three separate studies have found conflicting findings in the mouse or rat model with regard to PDE5i and hearing loss. Jaumann In light of these contradictory results, and with the ubiquitous use of sildenafil, this study attempts to clarify hearing function in chronic administration of sildenafil, which we define as a daily dose of sildenafil injected IP for 30 days.www.jax.org). For physiological data: The FVB/N mice were male and female (N = 4 treated with sildenafil and N = 4 treated with vehicle) at 6 weeks of age. The C57BL/6J mice were female and male (N = 7 treated with sildenafil and N = 9 treated with vehicle) at 6 weeks of age. For qRT-PCR analysis: 4-week-old C57BL/6J (N = 3 treated with sildenafil and N = 3 treated with vehicle) were both male and female. Ear punch was used to identify the animals.Mice from two inbred genetic backgrounds were used in this study: C57BL/6J, which is genetically predisposed to age-related hearing loss (a model for presbycusis), and FVB/N, which is not predisposed to acquire hearing loss and considered to be a ‘good hearing’ strain , USA. The protocol was approved by the Institutional Animal Care and Use Committee at CWRU (Protocol Number: 2012-0068).2 basis For the hearing experiments, we administered sildenafil citrate (Sigma Aldrich) dissolved in 0.9% NaCl solution at 2 mg/ml stored at 4° degrees Celsius for up to one week. This solution was administered daily intraperitoneal (I.P.) at 10 mg per kg of body weight (MPK) for 30 days. 10 MPK was chosen since it was a previously studied concentration ABR recordings were conducted as previously described 5′-AGAGAAGCCACAGCTGGGTA-3′ and 5′-CATCCACCTCGTCACCTTTT-3′), and GAPDH (5′-ATGTGTCCGTCGTGGATCTGAC-3′ and 5′-AGACAACCTGGTCCTCAGTGTAG-3′). The amount of cDNA template in each reaction was 10 ng of input RNA. Assays were performed in triplicate and data analysis was performed via Excel. The ΔΔCt method was employed to calculate fold changes. ΔCt represents normalized gene expression level . ΔCt for vehicle and sildenafil samples were averaged and then used to calculate ΔΔCt. CV, or coefficient of variation, is calculated using the formula STDEV/average.Mice were euthanized and the cochlea were dissected and immediately stored at −80°C In the laboratory mouse, hearing reaches adult levels of sensitivity around thirty days after birth and ABR threshold for normal (untreated) mice is typically 30±5 dB SPL We carried out longitudinal evaluation of hearing during treatment: Our results indicate that, within the mouse model, chronic, systemic (IP) administration (30 days) of sildenafil citrate at a concentration of 10 MPK does not affect hearing sensitivity in C57BL/6J  or FVB/NThe PDE5i mechanism of action has been well described in the literature Inability for activated sildenafil citrate to enter the cochlea could explain lack of hearing loss in the treated groups. In order to verify whether administered sildenafil citrate crossed the blood-cochlear barrier in an active form, we performed a quantitative real-time RT-PCR to quantify the level of PARP mRNA as a marker for penetrance and activity of sildenafil citrate. Twenty-four hours after an injection with sildenafil citrate at 10 MPK or equivalent vehicle into wild-type C57BL/6J mice, we found a 1.8 fold increase in PARP mRNA expression in the cochlea following sildenafil treatment . The co-Our study found that the chronic administration of PDE5i was not ototoxic at any time point for up to 30 days in the mouse model. One novel approach that our study uses is treating and testing two genetic backgrounds of mice. To test the possibility that PDE5i associated hearing loss is linked to age-related susceptibility to hearing loss we tested sildenafil citrate in C57BL/6J, a strain of mice that displays increased susceptibility to age-related and noise-induced hearing loss et al reported that vardenafil protects against noise-induced hearing loss (NIHL) in rodent models. Jaumann et al show that one of the key downstream mechanisms of protection against noise-induced hearing loss (NIHL) is PARP activation via the cGMP-PKG . The study demonstrated the presence of PDE5 and Prkg1a and Prkg1b within the cochlear hair cells, spiral ganglion neurons, and satellite cells in the rats and mice. In their Prkg1 knockout mice, one hour of acoustic trauma caused a significant increase in hearing thresholds, suggesting that Prkg1 is an important mediator in hearing loss due to NIHL. The study found that vardenafil treated mice and rats showed significant resistance to NIHL compared to untreated mice and saw an increase in PARP IHC stained cells in the vardenafil treated mice and rats et al suggest revisiting PDE5i related otoxoticity in humans.Jaumann et al findings; their study found statistically significant findings at 105 and 135 days of chronic 10 MPK injections in rats, with the earliest changes in hearing occurring fifteen days after the initial injection Two important factors that need further delineation are: 1) the temporal relationship between PDE5i ingestion and onset of symptoms and 2) dosing. The clinical studies previously described determined that PDE5i consumption caused SSHL based on the presentation of symptoms relative to the time of drug ingestion. Specifically, they found that the majority of the SSHL diagnosed was approximately 24 hours after ingestion. This suggests that the mechanism of ototoxicity is rapid in onset with an acute sequelae. Therefore, we would expect a rather acute process to present similarly in the mouse model. However, this is counter to the Hong 2 basis in humans Separately, the clinical reports do not report PDE5i dosing regimens. Dosing of sildenafil citrate for erectile dysfunction (ED) usually begins at 50 mg once, while dosing for pulmonary hypertension (PAH) usually starts at 20 mg three times a day Our study shows that chronic administration (4 weeks) of sildenafil citrate is not sufficient to cause hearing loss in the mouse model. The results presented here gives no indication that sildenafil citrate will affect hearing in humans. However, to ultimately determine whether PDE5i induces SSHL in a human population, a prospective double-blind clinical study would need to be performed that thoroughly investigates the dosing and temporal relationship of a PDE5i and controls for age-and noise induced hearing loss in the adult population.


Mixtures of polypropylene containing different concentrations of POM or d2w® were prepared by extrusion. The properties of the mixtures (blends) were evaluated based on the melt index (MFI), tensile properties, Fourier transform infrared spectroscopy (FTIR), Time inductive oxidation (OIT) and Thermogravimetric analysis (TGA). The two additives (POM and d2w®) enhanced the oxidative thermal degradation of polypropylene and the degradation of the polypropylene/POM mixture could be controlled by altering the POM concentration.Intentional degradation by pro-oxidant agents, many of which are metal-based, can result in uncertainty as to the time of biodegradation. Polyacetal (POM) is a thermoplastic polymer commercially classified as an engineering polymer and contains carbon, hydrogen and oxygen. The depolymerization of POM during processing can enhance thermal decomposition. The aim of this study was to investigate the controlled degradation of polypropylene induced by the degradation of POM or d In the US, the predominant method of waste disposal is currently landfill packaging, followed by recycling, composting and incineration  was used in granulated form, as supplied by Braskem . The polyacetal copolymer  was used in powder form as supplied by Ticona . The commercial pro-oxidant additive d2w® was supplied by RES Brazil .Isotactic polypropylene (iPP) H603 (density: 0.905 g/cm2w® was incorporated in granular form and POM in powder form. The incorporation of POM or d2w® into polypropylene (PP) initially involved homogenization in Drais with a load capacity of ~100 g of material. The mixing time was ~30 s. Blends of PP with POM or d2w® were prepared as shown in Table The additive dThe MFI was determined in a plastometer  according to ASTM D-1238 ASTM . The tesType IV specimens (ASTM D-638-10) ASTM  were inj-1 to 400 cm-1 at a resolution of 4 cm-1. The influence of POM and d2w® on polypropylene oxidation was determined from the spectra by calculating the carbonyl (CI) and hydroxyl (HI) indices based on the relationships CI = A1725/A2722 and HI = A3500/A2722, respectively.Films 30 ± 2 μm thick were prepared at 190°C with a compression pressure of 2000 psi and compression time of 80 s. FTIR measurements were obtained using a Varian 660-IR FT-IR spectrometer operated in transmittance mode. Thirty-two scans were obtained in triplicate from 4000 cm-1 and 306 J.g-1 for 100% crystalline polypropylene and polyacetal, respectively , crystallization temperature (Tc) and degree of crystallinity were calculated using the enthalpy of fusion values of 209 J.gThe OIT was determined by exposing ~10 mg of each mixture to a nitrogen flow of 50 ml/min and a heating rate of 20°C/min. An oxygen flow of 50 ml/min was used after melting at 200°C.TGA was done in equipment from TA Instruments. Approximately 10 mg of each mixture was placed in a nitrogen atmosphere and heated at a rate of 10°C/min over a temperature range of 25–550°C. The nitrogen flow over the measurement cell was 50 ml/min. The activation energy of degradation (Ea) was determined according to ASTM E1641 ASTM .2, PP4, PP6 and PP8) and probably reflected the immiscibility between the phases (PP/POM) since the variation in the measurements was proportional to the mass of POM in the PP/POM mixture (blend)  and polypropylene (matrix) should also be considered  were quite stable in this parameter. The HI index showed similar behavior to the CI index shown in Figure 8 had high hydroxyl formation, probably indicating greater susceptibility to the formation of nOH (acetic acid) .Absorbance in the region of 1725 cmDe Paoli , respect1, POM and d2w®) and mixtures (blends) (PP/d2w® and PP/POM). Figure 2w®. In mixtures containing d2w®  there was a reduction in Tc that was proportional to the d2w® concentration, such that the Tc was lower than for pure polypropylene. In PP/POM blends  the Tc of PP2, PP4 and PP6 stabilized at ~5°C above the Tc of pure polypropylene. This is a desirable result in processing by injection because it allows a reduction in the cooling time. The Tc of the PP8 blend was lower than for the other mixtures and also in relation to pure POM. This finding suggests that morphological changes in POM are probably the result of its thermal degradation.Table 2, PP4, PP6 and PP8) and PP/d2w® . Mixtures containing POM showed additional peaks between the peaks of pure POM and polypropylene. Degradation reactions usually appear as endothermic processes and exothermic behavior is generally a response to depolymerization .Figure 2w® accelerated the oxidation of polypropylene in the presence of oxygen. However, there was little change in the OIT values of blends containing different amounts of d2w® , i.e., the OIT values essentially reflected the amount of pure additive (d2w®) present in the mixtures. This finding indicates that there is little margin for controlling the thermo-oxidation of polypropylene during processing since all samples were processed under the same condition. In addition, changes in morphology arising from processing interfere with the diffusion of volatile degradation products.The oxidation induction time (OIT) is an accelerated aging test that allows comparison of the relative resistance of materials to thermal oxidation. Table 6 onwards. In contrast, there was an increase in the OIT values of blends PP2 and PP4, i.e., a stabilizing (antioxidant) effect. A similar delay in the kinetics of degradation was observed in the absence of oxygen in the TGA of these two blends, i.e., the Ti of the blends was greater than that of pure polypropylene (PP1). The OIT and TGA results indicated that d2w® concentrations ≥2% increased the thermal stability of the blends. In the case of POM, there was a decrease in the stabilizing synergistic effect at concentrations up to 3%; at higher concentrations, POM had an oxidizing effect on polypropylene.In PP/POM blends, there was a marked decrease in the OIT values from blend PP2 and PP4. The oxidation of polypropylene (in powder form) has been referred to as non-homogeneous (heterogeneous) kinetics that is characterized by chemiluminescence  had a lower Ti than polypropylene, with POM showing the lowest value. The Ti of the blends were not lower than those of POM and d2w®, except for blend PP8. The values of Ti varied with the concentration of d2w® and POM in polypropylene. In the case of d2w®, increasing the concentration of the additive led to an increase in Ti and therefore had a stabilizing effect. This enhanced response can be attributed to the greater thermal stability of the vehicle for d2w® relative to polypropylene. The MFI corroborated this stabilizing effect. Increasing the concentration of POM led to an increase in Ti up to the value seen with blend PP4, after which the Ti stabilized up to PP6 and then decreased in blend PP8.Figure 2w® to polypropylene increased the thermal stability in a manner dependent on the concentration of additive. The low production of volatile components and the reduced mobility (diffusion capacity) of the polypropylene matrix were probably important factors in this thermal stability. The addition of POM to polypropylene resulted in synergistic and antagonistic effects (stabilization and degradation), with the extent of stabilization and degradation depending on the amount of volatile components produced.Together, these results indicate that the addition of d8 and for the pure materials (PP1 and POM); a theoretical curve calculated curve calculated based on the weighted average of the experimental curves of the pure materials (PP and POM) is also shown. The region of the curve for blend PP8 close to the experimental curve of pure polypropylene (PP1) may reflect a greater stabilizing effect of polypropylene. The stabilizing effect decreases as the loss of mass increases and the theoretical curve approximates the observed curve, probably because of competition with degradation reactions. The region beyond the point where the theoretical and observed curves cross corresponds to additional degradation by shifting the equilibrium of the degradation reactions of the matrix phase (PP) induced by the dispersed phase (POM).Figure 2w® and POM showed opposite changes in Ea as a function of the additive concentration in the polypropylene matrix. Ea may be regarded as the energy needed to cause the diffusion of one mole of atoms such that high Ea results in a relatively small diffusion coefficient  is also suggestive of synergism.Table 2w® promoted the oxidative thermal degradation of polypropylene (PP), with the extent of degradation being regulated by the POM concentration in PP/POM blends. At concentrations <3% (w/w), POM enhanced the thermal stabilization of polypropylene under the conditions investigated, whereas at concentrations >3% POM stimulated the oxidation of polypropylene. These results suggest that the POM with a concentration >3% (w/w), may act as a pro-oxidant agent of the PP, and the synergistic effect of degradation can be maximized by increasing the miscibility at the interface of the blend PP/POM.The addition of POM or d


Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific in vitro interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of Achaea janata. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of A. janata showed high structural similarity to the Cry1Aa toxin binding midgut APN of Bombyx mori, especially in the toxin binding region. Structural similarity was further substantiated by in vitro binding of Cry1Aa toxin. RNA interference (RNAi) resulted in significant down-regulation of AjAPN1 transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development of A. janata. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation.Insect midgut membrane-anchored aminopeptidases N  The activated toxins then bind to specific midgut receptors resulting in oligomerization and insertion of toxins into the membranes to generate pores leading to cell lysis and finally, the death of the insect in vitro cultured fat body cells indicated the possibility of Cry toxins binding to fat body membrane proteins and causing toxic effects to the cells Manduca sexta midgut APN in Drosophila melanogaster induced sensitivity to the lepidopteran-specific insecticidal Cry1Ac which otherwise is not toxic et al also demonstrated that Sf21 insect cells expressing Helicoverpa armigera midgut APN which allowed high sensitivity to Cry1Ac, upon down-regulation by RNA interference (RNAi) resulted in reduced sensitivity Insect midgut aminopeptidases N (APNs) are ZnM. sextaSpodoptera lituraS. lituraH. armigeraA. janata, AjAPN1 is the APN isoform which is predominantly expressed in fat body, Malpighian tubule and salivary gland In cases where the experimental determination of protein three-dimensional (3D) structure is not possible, homology modeling is the most widely used approach. To date, there are no reports on crystal structure of insect APNs. However, molecular models of midgut-specific APNs from A. janata larvae. We demonstrated specific in vitro interaction of Cry1Aa toxin with the 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland. High similarity of 3D molecular structure of AjAPN1 of A. janata with that of Bombyx mori midgut APN (Genbank AAC33301), especially in the Cry1Aa toxin binding region as well as in vitro binding of Cry1Aa toxin to it further supported its potential role in Cry toxin interaction and toxicity. RNAi-mediated silencing not only down-regulated AjAPN1 expression in fat body and Malpighian tubule but also induced adverse physiological effects, which suggest that it plays important physiological role during growth, development as well as metamorphosis in A. janata. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed drastically reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significantly reduced levels of AjAPN1 transcript and its encoded protein expression. Findings from the present study suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development and metamorphosis of A. janata. Though specific interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor in A. janata will require further investigation.In the present study, we employed homology modeling and RNAi strategies to decipher the functional role of AjAPN1 expression in non-gut hemocoelic tissues of The observations from ligand binding studies presented in A. janata fat body APN polyclonal antibody The specificity of interaction between the 113 kDa membrane protein  and Cry1Aa toxin was confirmed by co-immunoprecipitation. Analysis of the pull-down Cry1Aa toxin-binding protein complex by western blot using Thermoplasma acidophilum and human endoplasmic reticulum aminopeptidase-1 (Erap1) to have the best sequence identity with AjAPN1 (Genbank ABE02186). The sequence identity of AjAPN1 with tricorn interacting factor F3 and human Erap1 was 28% and 27% respectively  was in the disallowed region while 705 residues (86.5%) were in the most favored region, 97 residues (11.9%) in the additionally allowed region and 12 residues (1.5%) in the generously allowed region  against Protein Data Bank (PDB) revealed tricorn interacting factor F3 from ectively . The 3D d region . The ERRB. mori midgut APN to find out if the Cry1Aa toxin binding region is structurally conserved. The overall fold of the two APN structures was found to be conserved (++ binding motifs of AjAPN1 (located in domain II) showing high similarity with B. mori midgut APN and templates are shown in The 3D structure of AjAPN1 was compared with that of onserved a and 3BaB. mori midgut APN and Plutella xylostella midgut APN (Genbank AAF01259) were aligned and the sequence identity in the Cry1Aa toxin binding region was compared. Multiple sequence alignment of the 64 amino acid residues of Cry1Aa toxin binding region is represented in B. mori midgut APN and P. xylostella midgut APN was 81% (52 amino acid residues) and 48% (31 amino acid residues) respectively, which included the 27 amino acid residues common to both B. mori midgut APN and P. xylostella midgut APN  1.9 and 2.8 in fat body and Malpighian tubule respectively. Correspondingly, western blot analysis showed a substantially reduced expression level of the target protein in fat body and Malpighian tubule of target siRNA injected insects as compared to the control insects (GFP siRNA) were a significant (P<0.05) 50% and 56% for fat body and Malpighian tubule respectively  42% larval deaths in the target gene siRNA injected insects, while in the control group, it was only 5% . We obseIn vitro Cry1Aa toxin binding analysis revealed a drastically reduced interaction of Cry1Aa toxin with the 113 kDa membrane protein of fat body and Malpighian tubule of the AjAPN1 knockdown insects  and Neobellieria bullata (Diptera) A. janatain vitro binding of Cry1Aa toxin predominantly to a 113 kDa membrane protein not only in fat body but also in Malpighian tubule and salivary gland of A. janata. Co-immunoprecipitation analysis demonstrated that the interaction was specific and further; using A. janata fat body APN polyclonal antibody, we indicated that the 113 kDa interacting membrane protein of these tissues could possibly be an APN. Therefore, this in vitro Cry1Aa toxin-113 kDa membrane protein interaction in these tissues could very well represent Cry toxin-APN interaction under in vivo condition. Further in the larval hemocoel, AjAPN1 expressing fat body, Malpighian tubules and salivary glands are bathed in hemolymph, which would likely facilitate the interaction of Cry1Aa toxin with the AjAPN1 protein associated with these tissues.With the ever increasing world population and pronounced toxicological effects of chemical pesticides, the need for more effective and eco-friendly insect pest control approach in agriculture has become more urgent. Developing alternate approaches that could target novel molecules of non-gut hemocoelic tissues along with known molecular targets of the gut would not only facilitate effective management of insect pests but would also help in tackling the problem of increasing pests resistance to B. mori, high susceptibility to Cry1Aa toxin and low susceptibility to Cry1Ac toxin was shown to be associated with high and low binding specificities to APN respectively M. sexta APN binds to Cry1Aa and Cry1Ac with equally high affinities and subsequently, the larvae were found to be highly sensitive to both Cry1Aa toxin and Cry1Ac toxin B. mori midgut APN were shown to be different et al identified the region between 135-Ile and 198-Pro in B. mori midgut APN as the Cry1Aa toxin binding region B. mori midgut APN P. xylostella midgut APN B. mori and P. xylostella midgut APNs were suggested to be important for Cry1Aa toxin binding B. mori midgut APN, 52 amino acid residues including the vital 27 amino acid residues were identical. AjAPN1 and P. xylostella midgut APN also shared 31 amino acid residues which also included the important 27 amino acid residues. Hence we assume the interaction between Cry1Aa toxin and 113 kDa membrane protein of fat body, Malpighian tubule and salivary gland of A. janata to be mediated by the presence of Cry1Aa toxin binding region in AjAPN1. In our earlier study, we demonstrated high Cry1A toxicity upon oral ingestion in A. janata and subsequently, the identification of Cry1A toxin binding region in the midgut APN of this species further strengthened this notion L. dispar BBMV In B. mori midgut APN 3D model was also constructed for comparison. The overall folding pattern of AjAPN1 and B. mori midgut APN 3D structures was highly conserved with the available 3D structures of the experimentally determined gluzincin aminopeptidases. This suggests that the structure of different APNs share a conserved conformation but the Cry toxin recognition or binding site could vary. Interestingly, AjAPN1 and B. mori midgut APN shared high sequence and structural similarity in the Cry1Aa toxin binding region, and therefore it is highly probable that both the APNs are Cry1Aa toxin receptors. Besides, the ability of Cry1 toxins to easily induce toxicity in a broad spectrum of insect pests by recognizing the conserved structures of APNs further strengthened the present case in vitro interaction of Cry1Aa toxin to the 113 kDa membrane protein of Malpighian tubule where AjAPN1 of theoretical molecular weight of 111 kDa was expressed.It is reported that different Cry1A toxins have similar primary sequences as well as 3D structures and therefore recognize APNs with similar structure; the specificity of interaction being mediated by the structure of Cry1A toxin binding region of APN A. janata, we employed RNAi approach for silencing the expression of the protein. Recently, Terenius et al suggested that either for feeding or hemocoelic injection, fairly high concentrations of dsRNA are required to achieve high degree of silencing in lepidopteran insects H. armigera, feeding of short dsRNAs rather than long dsRNA produced higher level of silencing AjAPN1 siRNA duplexes at concentrations of 1, 2.5 and 5 µg/100 mg body weight either to fourth or fifth instar larvae did not yield any desired result. When fourth and fifth instar larvae were used, we found that there was a minor decline in AjAPN1 transcript levels in all the three tissues at 3–4 day post-injection, which possibly could be due to normal developmental regulation of the gene rather than the consequence of RNAi. Earlier reports showed that hemocoelic injection of dsRNA to fifth instar larvae of S. lituraH. armigeraOstrinia nubilalis, Spodoptera exigua and Epiphyas postvittanaA. janata where AjAPN1 gene silencing could not be achieved when target double-stranded siRNA was injected into fifth instar larvae. When third instar larvae were injected with 5 µg/100 mg body weight of target siRNA duplexes and analyzed at different time points, we observed a significant 1.9 and 2.8-fold decrease in AjAPN1 transcript level of fat body and Malpighian tubule respectively at 66 h post-injection. Consequently, there was a corresponding substantial decrease in the AjAPN1 protein expression level and significant reductions of 50% and 56% in the APN activity levels of fat body and Malpighian tubule respectively. The control (GFP siRNA) used in the experiment further confirmed that the responses were specific to the gene for which it was employed. Our results demonstrated that the susceptibility of an insect species to siRNA could also depend on the nature of the tissue and larval stage of development. This observation is not surprising as earlier studies have shown that regardless of the delivery methods, RNAi effects may vary in different species, instars or even among individuals within the same species of lepidopteran insects To further substantiate AjAPN1 as a potential Cry1Aa toxin receptor in non-gut hemocoelic tissues of AjAPN1 gene silencing may be lethal to A. janata development. The larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults as a result of AjAPN1 gene silencing clearly indicated that this gene product plays important physiological role(s) during post-embryonic development and metamorphosis of A. janata. In Diatraea saccharalis, only a partial suppression of APN expression was enough to bring about significant decrease in Cry1Ab susceptibility in vitro Cry1Aa toxin binding assay which revealed a drastically reduced interaction of Cry1Aa toxin with the 113 kDa membrane protein of fat body and Malpighian tubule of the AjAPN1 knockdown larvae correlating well with significantly inhibited expression of the target gene. These results further demonstrated that the interaction of Cry1Aa toxin with the 113 kDa membrane protein of non-gut hemocoelic tissues of A. janata is specific.High percentage of larval mortality as a consequence of target siRNA injection indicated that A. janata. Secondly, we demonstrated specific interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein of non-gut hemocoelic tissues and indicated its potential role as a Cry1Aa toxin receptor in these tissues of A. janata. However, its functional role as a Cry1Aa toxin receptor in A. janata still remains to be proved.In conclusion, findings from the present study suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development of A. janata neonate larvae were obtained from Directorate of Oilseeds Research, Hyderabad, India and reared on fresh castor leaves (Ricinus communis) as diet under photoperiod of 14∶10 h (light:dark) and 60–70% relative humidity at 27±2°C in the insect culture facility of our laboratory. The larval development proceeds through five instars and the whole life cycle is completed in about 45–50 days. Each instar from first to fourth lasts for 2 days while the last or fifth instar lasts 4–5 days. The fifth instar is further classified into early (5E) and late (5L) fifth instar which lasts 2 and 2–3 days respectively, then followed by non-feeding pre-pupal (PP) stage. For RNAi studies, third, fourth and 5E instars while for all other studies, only 5E instar larvae were used. Tissues were dissected out in ice-cold insect Ringer solution  and used immediately.Escherichia coli JM103 strain ECE52 harboring cry1Aa gene Bacillus Genetic Stock Centre . The protoxin was activated Cry1Aa protoxin was prepared from recombinant et alet alBrush border membrane vesicles (BBMVs) of midgut were prepared according to the method described by Wolfersberger 2) for 12 h at 4°C. This was followed by addition of 5 µl of purified Cry1Aa polyclonal antibody (2.5 µg) and incubated for another 3 h. Subsequently, 50 µl of Protein A agarose beads was added to the mixture and incubated for another 2 h at 4°C on a rotary shaker. The agarose beads were pelleted, washed six times with binding buffer, re-suspended in equal volume of 2X SDS sample buffer containing 2-mercaptoethanol  and heated at 100°C for 5 min. The pull-down Cry1Aa toxin-interacting proteins were separated by 7.5% SDS-PAGE, electro-blotted onto nitrocellulose membrane , incubated with A. janata fat body APN polyclonal antibody (1∶10000 dilutions) Triton X-100 solubilized Malpighian tubule and salivary gland membrane protein preparations (200 µg each) were incubated with purified activated Cry1Aa toxin (5 µg) in binding buffer  A. janata and midgut APN of B. mori were obtained from NCBI (http://www.ncbi.nih.gov). For model construction, we used MODELLER program B. mori midgut APN 3D model was constructed using the crystal structure of the soluble domain of human Erap1 (PDB code: 2YD0) as the template.The crystal structures of tricorn interacting factor F3 from AjAPN1 gene siRNA duplexes of 19-nt each of sense and antisense strands was custom-designed, synthesized and commercially supplied by Sigma-Aldrich  and then reverse transcribed to corresponding cDNAs using SuperScript III first strand synthesis kit (Invitrogen). For semi-quantitative PCR analysis, a primer pair of 5′TGGCTGGATATGGTATTACT3′ and 5′GATATTGAATGCTCTGGTGTA3′ was used as forward and reverse primer respectively to amplify a 476 bp fragment of AjAPN1 transcript levels. Insect 18S rRNA (Applied Biosystems) was used as an internal reference to normalize the AjAPN1 transcript levels. AjAPN1 transcript levels of the target siRNA injected larvae are presented as change in the transcript levels relative to the control siRNA injected larvae using the 2−ΔΔCt method described by Livak and Schmittgen Real-time PCR analysis was performed in a 20 µl reaction volume using a custom-made Taqman gene expression assay (Applied Biosystems). A forward primer 5′AGGAATACACAGGCTATCCGTACT3′, a reverse primer 5′GGCTGCTTGCTGCATGAT3′ and a Taqman probe “CAATGACCGAGAACATC” were used for the analysis of A. janata fat body APN polyclonal antibody (1∶10000 dilutions) p-nitroanilide was taken as 9.9×10−3 mol/L p-nitroanilide released/min/mg protein.The membrane protein fractions of fat body, Malpighian tubule and salivary gland from target and control gene siRNA injected larvae were electrophoretically separated by 7.5% SDS-PAGE and electro-blotted onto nitrocellulose membrane  The phenotypic differences between target and control gene siRNA injected insect groups were investigated by examining larval mortality, larval growth rate, pupal weights and analysis of larval-pupal as well as pupal-adult transformations. The larval weight was recorded every 24 h for 5 days. Larval mortality was calculated 2 day post-injection. Phenotypic features were analyzed every 2 day until all adults eclosed. Photographs were taken with FinePix S9600 digital camera (Nikon).Data are expressed as mean±standard deviation of three independent experiments (n = 3). Differences between groups were analyzed for statistical significance by One-Way ANOVA followed by Students-Newman-Keuls’ (SNK) test using SigmaPlot 11.0 software . A probability of P<0.05 was considered statistically significant.Figure S1Sequence alignment. AjAPN1 amino acid sequence was alligned with tricorn interacting factor F3 from Thermoplasma acidophilum (PDB code: 1Z1W) and human endoplasmic reticulum aminopeptidase-1 (Erap1) (PDB code: 3QNF) templates using S-alignment software (MODELLER).(DOC)Click here for additional data file.Figure S2Ramachandran plot of AjAPN1 molecular model. Amino acid residues in red, yellow, cream and white shaded regions are in most favorable, additionally allowed, generously allowed and disallowed regions, respectively.(DOC)Click here for additional data file.Figure S3Ramachandran plot of B. mori midgut APN molecular model. Amino acid residues in red, yellow, cream and white shaded regions are in most favorable, additionally allowed, generously allowed and disallowed regions, respectively.(DOC)Click here for additional data file.Figure S4Integrity of double-stranded AjAPN1 siRNA oligonucleotides. Analysis of integrity of the double-stranded siRNA duplexes was performed by non-denaturing polyacrylamide gel electrophoresis and visualized by UV shadowing. Lanes M: oligonucleotide marker, Lanes 1 and 2: double-stranded AjAPN1 siRNA duplexes.(DOC)Click here for additional data file.Figure S5Home-assembled microsyringe set-up. Hamilton microsyringe holder was fitted to a glass needle through a sterile plastic tube. The glass needles were prepared using a micropipette puller .(DOC)Click here for additional data file.