Codon Preference Optimization Increases Heterologous PEDF Expression

Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5′ thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes , respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ∼3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ∼4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ∼3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at −3 to +50 of the Trx site of both fusion genes were −3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo

Neurotrophins enhance retinal pigment epithelial cell survival through neuroprotectin D1 signaling

Integrity of retinal pigment epithelial cells is necessary for photoreceptor survival and vision. The essential omega-3 fatty acid, docosahexaenoic acid, attains its highest concentration in the human body in photoreceptors and is assumed to be a target for lipid peroxidation during cell damage. We have previously shown, in contrast, that docosahexaenoic acid is also the precursor of neuroprotectin D1 (NPD1), which now we demonstrate, acts against apoptosis mediated by A2E, a byproduct of phototransduction that becomes toxic when it accumulates in aging retinal pigment epithelial (RPE) cells and in some inherited retinal degenerations. Furthermore, we show that neurotrophins, particularly pigment epithelium-derived factor, induce NPD1 synthesis and its polarized apical secretion. Moreover, docosahexaenoic acid (DHA) elicits a concentration-dependent and selective potentiation of pigment epithelial-derived factor-stimulated NPD1 synthesis and release through the apical RPE cell surface. The bioactivity of signaling activated by pigment epithelium-derived factor and DHA uncovered synergistic cytoprotection with concomitant NPD1 synthesis when cells were challenged with oxidative stress. Also, DHA and pigment epithelium-derived factor synergistically modify the expression of Bcl-2 family members, activating antiapoptotic proteins and decreasing proapoptotic proteins, and by attenuating caspase 3 activation during oxidative stress. Thus, our findings demonstrate that DHA-derived NPD1 protects against RPE cell damage mediated by aging/disease-induced A2E accumulation. Also, our results identify neurotrophins as regulators of NPD1 and of its polarized apical efflux from RPE cells. Taken together, these findings imply NPD1 may elicit autocrine actions on RPE cells and paracrine bioactivity in cells located in the proximity of the interphotoreceptor matrix

Autologous transplantation of genetically modified iris pigment epithelial cells: A promising concept for the treatment of age-related macular degeneration and other disorders of the eye

Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population. Laser photocoagulation, photodynamic therapy and excision of neovascular membranes have met with limited success. Submacular transplantation of autologous iris pigment epithelial (IPE) cells has been proposed to replace the damaged retinal pigment epithelium following surgical removal of the membranes. We tested our hypothesis that the subretinal transplantation of genetically modified autologous IPE cells expressing biological therapeutics might be a promising strategy for the treatment of ARMD and other retinal disorders. Pigment epithelium-derived factor (PEDF) has strong antiangiogenic and neuroprotective activities in the eye. Subretinal transplantation of PEDF expressing IPE cells inhibited pathological choroidal neovascularization in rat models of laser-induced rupture of Bruch's membrane and of oxygen induced ischemic retinopathy. PEDF expressing IPE transplants also increased the survival and preserved rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration. These findings suggest a promising concept for the treatment of ARMD and other retinal disorders

P300 correlates with learning & memory abilities and fluid intelligence

BACKGROUND: Educational psychology research has linked fluid intelligence with learning and memory abilities and neuroimaging studies have specifically associated fluid intelligence with event related potentials (ERPs). The objective of this study is to find the relationship of ERPs with learning and memory recall and predict the memory recall score using P300 (P3) component. METHOD: A sample of thirty-four healthy subjects between twenty and thirty years of age was selected to perform three tasks: (1) Raven’s Advanced Progressive Matrices (RAPM) test to assess fluid intelligence; (2) learning and memory task to assess learning ability and memory recall; and (3) the visual oddball task to assess brain-evoked potentials. These subjects were divided into High Ability (HA) and Low Ability (LA) groups based on their RAPM scores. A multiple regression analysis was used to predict the learning & memory recall and fluid intelligence using P3 amplitude and latency. RESULTS: Behavioral results demonstrated that the HA group learned and recalled 10.89 % more information than did the LA group. ERP results clearly showed that the P3 amplitude of the HA group was relatively larger than that observed in the LA group for both the central and parietal regions of the cerebrum; particularly during the 300–400 ms time window. In addition, a shorter latency for the P3 component was observed at Pz site for the HA group compared to the LA group. These findings agree with previous educational psychology and neuroimaging studies which reported an association between ERPs and fluid intelligence as well as learning performance. CONCLUSION: These results also suggest that the P3 component is associated with individual differences in learning and memory recall and further indicate that P3 amplitude might be used as a supporting factor in standard psychometric tests to assess an individual’s learning & memory recall ability; particularly in educational institutions to aid in the predictability of academic skills

Feature extraction and classification for EEG signals using wavelet transform and machine learning techniques

This paper describes a discrete wavelet transform-based feature extraction scheme for the classification of EEG signals. In this scheme, the discrete wavelet transform is applied on EEG signals and the relative wavelet energy is calculated in terms of detailed coefficients and the approximation coefficients of the last decomposition level. The extracted relative wavelet energy features are passed to classifiers for the classification purpose. The EEG dataset employed for the validation of the proposed method consisted of two classes: (1) the EEG signals recorded during the complex cognitive task—Raven’s advance progressive metric test and (2) the EEG signals recorded in rest condition—eyes open. The performance of four different classifiers was evaluated with four performance measures, i.e., accuracy, sensitivity, specificity and precision values. The accuracy was achieved above 98 % by the support vector machine, multi-layer perceptron and the K-nearest neighbor classifiers with approximation (A4) and detailed coefficients (D4), which represent the frequency range of 0.53–3.06 and 3.06–6.12 Hz, respectively. The findings of this study demonstrated that the proposed feature extraction approach has the potential to classify the EEG signals recorded during a complex cognitive task by achieving a high accuracy rate.</p

Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE

The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch’s membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells. Here, we evaluate how two different types of BrM substrates; (1) a commercially-available polyester transwell membrane, and (2) a novel electrospun scaffold developed in our laboratory, could support the generation of realistic RPE tissues in culture. Our findings reveal that both substrates were capable of supporting long-lasting RPE monolayers with structural and functional specialisations of in-situ RPE cells. These cultures were used to study autofluorescence and barrier formation, as well as activities such as outer-segment internalisation/trafficking and directional secretion of key proteins; the impairment of which underlies retinal disease. Hence, both substrates fulfilled important criteria for generating authentic in-vitro cultures and act as powerful tools to study RPE pathophysiology. However, RPE grown on electrospun scaffolds may be better suited to studying complex RPE-BrM interactions such as the formation of drusen-like deposits associated with early retinal disease