RDC derived protein backbone resonance assignment using fragment assembly

Experimental residual dipolar couplings (RDCs) in combination with structural models have the potential for accelerating the protein backbone resonance assignment process because RDCs can be measured accurately and interpreted quantitatively. However, this application has been limited due to the need for very high-resolution structural templates. Here, we introduce a new approach to resonance assignment based on optimal agreement between the experimental and calculated RDCs from a structural template that contains all assignable residues. To overcome the inherent computational complexity of such a global search, we have adopted an efficient two-stage search algorithm and included connectivity data from conventional assignment experiments. In the first stage, a list of strings of resonances (CA-links) is generated via exhaustive searches for short segments of sequentially connected residues in a protein (local templates), and then ranked by the agreement of the experimental ¹³C_α chemical shifts and ¹⁵N-¹H RDCs to the predicted values for each local template. In the second stage, the top CA-links for different local templates in stage I are combinatorially connected to produce CA-links for all assignable residues. The resulting CA-links are ranked for resonance assignment according to their measured RDCs and predicted values from a tertiary structure. Since the final RDC ranking of CA-links includes all assignable residues and the assignment is derived from a “global minimum”, our approach is far less reliant on the quality of experimental data and structural templates. The present approach is validated with the assignments of several proteins, including a 42 kDa maltose binding protein (MBP) using RDCs and structural templates of varying quality. Since backbone resonance assignment is an essential first step for most of biomolecular NMR applications and is often a bottleneck for large systems, we expect that this new approach will improve the efficiency of the assignment process for small and medium size proteins and will extend the size limits assignable by current methods for proteins with structural models.     

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy

Objective

There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1).

Materials and Methods

pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis.

Results

After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation.

Conclusion

This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.     

Long term remission of metastatic placental site trophoblastic tumor (PSTT): Case report and review of literature

BACKGROUND: Placental site trophoblastic tumor (PSTT) is a rare and unique form of gestational trophoblastic disease (GTD). This tumor represents a neoplastic transformation of intermediate trophoblastic cells. We document a case of long term remission in a patient with metastatic PSTT. CASE PRESENTAION: A 27-year-old patient with metastatic PSTT was treated with combination therapy (chemotherapy and surgery). Patient is alive after 10 years without any evidence of recurrence. Literature on PSTT was searched using Medline and cross references, and pertinent articles were reviewed. CONCLUSION: With surgery and chemotherapy it is possible to achieve long-term remission in metastatic PSTT. Only a handful of previously reported cases with prolonged remission had been treated with the described combined chemotherapy and surgical approach. We suggest that this approach may be recommended for metastatic PSTT.     

Leishmania major: In vitro and in vivo anti-leishmanial effect of cantharidin

Cantharidin is a natural poisonous compound secreted by male blister beetles. The effect of different doses of cantharidin on Leishmania major (MRHO/IR/75/ER) were investigated both in vitro (promastigote and amastigote viability) and in experimentally-infected BALB/c mice (skin lesions) using ointment or soluble cantharidin. In this study, cantharidin with concentrations of 0.5, 1, 2, 5, 10, 20 and 50 μg/ml inhibited the growth of L. major promastigotes after 24 h and the resultant inhibition levels were 39.22%, 41.95%, 49.88%, 54.78%, 58.01%, 68.30% and 80.04%, respectively. After 72 h, the mean number of amastigotes per macrophage in the culture using 2 μg/ml of cantharidin, (the 50% inhibitory concentration dose (IC₅₀)), was 1.2 while in the control group it was 2.7. In order to perform the inflammatory blister technique, 500 μg of cantharidin were solved in 25 μl of DMSO to show the formation of the blister which leads to treatment of cutaneous leishmaniasis. Using the blister technique, the small lesions (<5 mm) healed after one session. Two weeks of topical treatment with 0.1% cantharidin ointment was an effective method for treating cutaneous leishmaniasis in infected BALB/c mice.     

Leishmania major: Disruption of signal peptidase type I and its consequences on survival, growth and infectivity

Leishmania major (L. major) signal peptidase type I (SPase I) is an endopeptidase encoded by a single-copy gene. In all organisms, SPase I is responsible for removing the signal peptide from secretory pre-proteins and releasing mature proteins to cellular or extra-cellular space. In this study, the role of SPase I in L. major is investigated by gene deletion using homologous recombination (HR). The null mutant of SPase I was not possible to create, suggesting that SPase I is an essential gene for parasite survival.The obtained heterozygote mutant by disrupting one allele of SPase I in L. major showed significantly reduced level of infectivity in bone marrow-derived macrophages. In addition, the heterozygote mutants are unable to cause cutaneous lesion in susceptible BALB/c mice. This is the first report showing that SPase I may have an important role in Leishmania infectivity, e.g. in differentiation and survival of amastigotes. Apparently, the SPase I expression is not essential for in vitro growth of the parasite.     

Composition and antimicrobial activity of the essential oil of Dicyclophora persica Boiss. from Iran

The chemical composition of the essential oil of Dicyclophora persica Boiss. was identified by GC and GC-MS analysis. The analysis of the oil resulted in the identification of forty-five components constituting 98.6% of the total oil. The main constituents were a-pinene (31.5%), (Z)-beta-ocimene (23.3%), p-cymene (6.7%) and (E)-beta-ocimene (5.4%). The antimicrobial activity of the oil was tested by the disk diffusion method against four Gram-positive (Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis) and three Gram-negative (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) bacteria together with a fungus (Aspergillus niger). The oil showed strong inhibition activity toward all the tested microorganisms except for Pseudomonas aeruginosa.     

Bioengineered chimeric tRNA/pre-miRNAs as prodrugs in cancer therapy

Today, biologic prodrugs have led to targeting specific tumor markers and have increased specificity and selectivity in cancer therapy. Various studies have shown the role of ncRNAs in cancer pathology and tumorigenesis and have suggested that ncRNAs, especially miRNAs, are valuable molecules in understanding cancer biology and therapeutic processes. Most miRNAs-based research and treatment are limited to chemically synthesized miRNAs. Synthetic alterations in these miRNA mimics may affect their folding, safety profile, and even biological activity. However, despite synthetic miRNA mimics produced by automated systems, various carriers could be used to achieve efficient production of bioengineered miRNAs through economical microbial fermentation. These bioengineered miRNAs as biological prodrugs could provide a new approach for safe therapeutic methods and drug production. In this regard, bioengineered chimeric miRNAs could be selectively processed to mature miRNAs in different types of cancer cells by targeting the desired gene and regulating cancer progression. In this article, we aim to review bioengineered miRNAs and their use in cancer therapy, as well as offering advances in this area, including the use of chimeric tRNA/pre-miRNAs.     

The association between adiponectin (+45T/G) and adiponectin receptor-2 (+795G/A) single nucleotide polymorphisms with cirrhosis in Iranian population

Adiponectin which possesses anti-inflammatory and insulin-sensitizing properties is elevated in blood circulation of liver cirrhosis patients. The genetic variations in the adiponectin gene can affect the circulating adiponectin level and stimulation of adiponectin receptor that may affect the activity of adiponectin. We investigated the effect of adiponectin single nucleotide polymorphisms (SNP) 45 T/G and adiponectin receptor-2 gene SNP 795G/A in cirrhotic Iranian population. A total of 97 cirrhotic patients and 128 healthy controls from Iranian population were genotyped for the adiponectin and adiponectin receptor 2 gene (+45T>G and 795G/A) by polymerase chain reaction-restriction fragment length polymorphism. G frequency was 21.1% versus 12.89% (P = 0.001) for SNP45, and G frequency was 75.8% versus 76.2% (P = 0.526) for SNP795G/A in the patients and control group, respectively. Based on our findings, the expression of the G allele at SNP45 is higher in the patient group compared with healthy subjects, suggesting that it may affect liver injury through changes in the plasma adiponectin level.