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

A chronic fatigue syndrome – related proteome in human cerebrospinal fluid

BACKGROUND: Chronic Fatigue Syndrome (CFS), Persian Gulf War Illness (PGI), and fibromyalgia are overlapping symptom complexes without objective markers or known pathophysiology. Neurological dysfunction is common. We assessed cerebrospinal fluid to find proteins that were differentially expressed in this CFS-spectrum of illnesses compared to control subjects. METHODS: Cerebrospinal fluid specimens from 10 CFS, 10 PGI, and 10 control subjects (50 μl/subject) were pooled into one sample per group (cohort 1). Cohort 2 of 12 control and 9 CFS subjects had their fluids (200 μl/subject) assessed individually. After trypsin digestion, peptides were analyzed by capillary chromatography, quadrupole-time-of-flight mass spectrometry, peptide sequencing, bioinformatic protein identification, and statistical analysis. RESULTS: Pooled CFS and PGI samples shared 20 proteins that were not detectable in the pooled control sample (cohort 1 CFS-related proteome). Multilogistic regression analysis (GLM) of cohort 2 detected 10 proteins that were shared by CFS individuals and the cohort 1 CFS-related proteome, but were not detected in control samples. Detection of ≥1 of a select set of 5 CFS-related proteins predicted CFS status with 80% concordance (logistic model). The proteins were α-1-macroglobulin, amyloid precursor-like protein 1, keratin 16, orosomucoid 2 and pigment epithelium-derived factor. Overall, 62 of 115 proteins were newly described. CONCLUSION: This pilot study detected an identical set of central nervous system, innate immune and amyloidogenic proteins in cerebrospinal fluids from two independent cohorts of subjects with overlapping CFS, PGI and fibromyalgia. Although syndrome names and definitions were different, the proteome and presumed pathological mechanism(s) may be shared

Succesvolle implementatie van een telefonisch verloskundig triagesysteem: De Nederlandse Triagewijzer Verloskunde

Er is op dit moment een toename van het aantal ongeplande verloskundige (spoed)contacten. Daarom is er vraag naar een goedwerkend telefonisch verloskundig triagesysteem. De Nederlandse Triagewijzer Verloskunde is de afgelopen jaren succesvol ontwikkeld en geïmplementeerd in verschillende Nederlandse ziekenhuizen. Het systeem levert patiëntveiligheid en doelmatigheid. In dit artikel wordt de totstandkoming van het huidige verloskundige triagesysteem beschreven. Daarnaast wordt beschreven welke procesmatige acties hebben bijgedragen aan de succesvolle implementatie erva

Pigment epithelium-derived factor overexpression inhibits orthotopic osteosarcoma growth, angiogenesis and metastasis

Despite significant improvements, the current management of primary osteosarcoma is still limited by the development of metastatic disease, which occurs in approximately 30% of patients despite aggressive multiagent chemotherapy and tumor-ablative surgery. Therefore, there is a need for the development of novel agents to improve the outcome of these patients. Pigment epithelium-derived factor (PEDF) has been shown to be one of the most potent inhibitors of angiogenesis, and more recently has demonstrated a functional role in tumor growth, invasion and metastasis. In this study we report, for the first time, the multitargeted role of PEDF in the inhibition of growth, angiogenesis and metastasis of two orthotopic models of osteosarcoma (rat UMR 106-01 and human SaOS-2). Through stable plasmid-mediated gene transfer of full-length human PEDF, we show that PEDF overexpression significantly reduced tumor cell proliferation (P<0.05) and Matrigel invasion (UMRPEDF, P<0.001; SaOSPEDF, P<0.05) and increased adhesion to collagen type-1 (P<0.01), in vitro. In vivo, PEDF overexpression dramatically suppressed orthotopic osteosarcoma growth (P<0.05) and the development of spontaneous pulmonary metastases (UMRPEDF, P<0.05; SaOSPEDF, P<0.001). Furthermore, PEDF-overexpressing tumors exhibited reduced intratumoral angiogenesis, evidenced by a significant decrease in microvessel density (P<0.05). Therefore, together these results suggest that PEDF may be a new and promising approach for the treatment of osteosarcoma