Exosomes for angiogenesis induction in ischemic disorders

Ischaemic disorders are leading causes of morbidity and mortality worldwide. While the current therapeutic approaches have improved life expectancy and quality of life, they are unable to “cure” ischemic diseases and instate regeneration of damaged tissues. Exosomes are a class of extracellular vesicles with an average size of 100–150 nm, secreted by many cell types and considered a potent factor of cells for paracrine effects. Since exosomes contain multiple bioactive components such as growth factors, molecular intermediates of different intracellular pathways, microRNAs and nucleic acids, they are considered as cell-free therapeutics. Besides, exosomes do not rise cell therapy concerns such as teratoma formation, alloreactivity and thrombotic events. In addition, exosomes are stored and utilized more convenient. Interestingly, exosomes could be an ideal complementary therapeutic tool for ischemic disorders. In this review, we discussed therapeutic functions of exosomes in ischemic disorders including angiogenesis induction through various mechanisms with specific attention to vascular endothelial growth factor pathway. Furthermore, different delivery routes of exosomes and different modification strategies including cell preconditioning, gene modification and bioconjugation, were highlighted. Finally, pre-clinical and clinical investigations in which exosomes were used were discussed.     

In silico design and in vitro expression of novel multiepitope DNA constructs based on HIV-1 proteins and Hsp70 T-cell epitopes

Objectives

Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection.

Methods

Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting.

Results

The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56–60% as the bands of?~?63 and?~?72 kDa confirmed in western blotting, respectively.

Conclusion

The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.

     

Local FK506 dose-dependent study using a novel three-dimensional organotypic assay

Local administration of FK506, an FDA approved immunosuppressant with neuroregenerative properties, is a promising technique to achieve improved peripheral nerve regeneration while preventing the side effects associated with the systemic administration of this drug. Although considerable research has been devoted to the development of clinically suitable systems for local delivery of FK506 to the site of nerve injury and repair, the optimal dose of FK506 for enhancement of axon regeneration in the peripheral nerve has not yet been established. To this end, we devised a three-dimensional (3D) organotypic assay capable of mimicking the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into the native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). A novel 3D compartmented cell culture system was adapted from the 3D organotypic assay to achieve local delivery of FK506 just to the growing neurites in vitro and establish the required local dose of FK506 for peripheral nerve regeneration. A bimodal dose response was observed by culturing the entire DRG–ANA construct with media containing different concentrations of FK506. Low drug concentration of 1 pg/ml and high drug concentration of 100 ng/ml lead to the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, concentrating the drug to the growing neurites resulted in significant increase in both neurite extension and neurite density, an effect that was not observed with the FK506 delivery to both neurites and neural cell bodies within DRG. The findings in this study provide valuable insight into the optimal local dose of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study suggests the potential interaction of FK506 with axons at the level of the growth cone.     

A study of the protein secretory pathway of Aspergillus niger using a glucoamylase-GFP fusion protein

The effect of various treatments that block protein secretion was visualized in Aspergillus niger using a strain expressing a glucoamylase-GFP fusion protein. Cold shock caused the retention of the fusion protein in a reticulate network (ER) with brighter nodes that may represent Golgi bodies. Treatment of germlings with brefeldin A (BFA) also initially caused accumulation within the ER but prolonged exposure led to the formation and targeting of the fusion protein to vacuoles from the ER. Disruption of actin with cytochalasin A initially led to a faint diffuse accumulation and ultimately to the formation of aggregated bodies which were not vacuoles, suggesting that the actin cytoskeleton is important in secretory vesicle transport. Disruption of microtubules with nocodazole led to hyperbranching but did not cause intracellular accumulation, suggesting that microtubules play a role in directing vesicle transport rather than vesicle movement per se. Treatment of regenerating protoplasts confirmed that BFA and cytochalasin but not nocodazole inhibited protein secretion. When germlings were subjected to carbon starvation, vacuolation was rapidly initiated throughout the hyphae and GFP fluorescence was visible in some of the vacuoles, indicating retargeting of the fusion protein from the secretory pathway to the vacuoles.     

Effect of electroacupuncture on pressor reflex during gastric distension

The effect of electroacupuncture (EA) on the reflex cardiovascular response induced by mechanical distension of the stomach was studied in ventilated male Sprague-Dawley rats anesthetized by ketamine and alpha-chloralose. Repeated balloon inflation of the stomach to produce 20 mmHg tension on the gastric wall induced a consistent rise in mean arterial pressure, while heart rate (372 +/- 22 beats/min) was unchanged. This response was reversed by transection of the splanchnic nerves. Bilateral application of EA (1-2 mA, 2 Hz) at Neiguan-Jianshi acupoints (pericardial meridian, Pe 5-6) over the median nerve for 30 min significantly decreased the pressor response from 33 +/- 6 to 18 +/- 4 mmHg (n = 7, P < 0.05). This effect began after 10 min of EA and continued for 40 min after termination of EA. EA at Zusanli-Shangquxu acupoints (stomach meridian, St 36-37) over the deep peroneal nerve similarly inhibited the pressor response. The effect lasted for 10 min after EA was stopped (n = 6, P < 0.05), while EA at Guangming-Xuanzhong acupoints (gallbladder meridian, GB 37-39) over the superficial peroneal nerve did not inhibit the pressor response. Naloxone injected intravenously (n = 6) immediately after termination of EA or administered by microinjection into the rostral ventrolateral medulla (rVLM) 25 min after initiation of EA (n = 6) reversed the inhibition by EA, suggesting an opiate mechanism, including the rVLM, was involved.     

The relation of omentin gene expression and glucose homeostasis of visceral and subcutaneous adipose tissues in non-diabetic adults

Molecular Biology Reports - Adipose tissue (AT) is a passive reservoir for&nbsp;energy storage&nbsp;and an active endocrine organ responsible for synthesizing bioactive molecules called...     

On the optimality of the genetic code, with the consideration of coevolution theory by comparison of prominent cost measure matrices

Statistical and biochemical studies have revealed non-random patterns in codon assignments. The canonical genetic code is known to be highly efficient in minimizing the effects of mistranslation errors and point mutations, since it is known that when an amino acid is converted to another due to error, the biochemical properties of the resulted amino acid are usually very similar to those of the original one. In this study, using altered forms of the fitness functions used in the prior studies, we have optimized the parameters involved in the calculation of the error minimizing property of the genetic code so that the genetic code outscores the random codes as much as possible. This work also compares two prominent matrices, the Mutation Matrix and Point Accepted Mutations 74-100 (PAM(74-100)). It has been resulted that the hypothetical properties of the coevolution theory of the genetic code are already considered in PAM(74-100), giving more evidence on the existence of bias towards the genetic code in this matrix. Furthermore, our results indicate that PAM(74-100) is biased towards the single base mistranslation occurrences in second codon position as well as the frequency of amino acids. Thus PAM(74-100) is not a suitable substitution matrix for the studies conducted on the evolution of the genetic code.     

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin

Hymeglusin (1233A, F244, L-659-699) is established as a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 ?) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.     

Diversity in the C3b [corrected] contact residues and tertiary structures of the staphylococcal complement inhibitor (SCIN) protein family

To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B^18–85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B^18–85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg⁴⁴ and Tyr⁵¹ as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.