Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer

BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.     

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50–200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex “work-in-progress” MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.     

Analysis of grape berry cell wall proteome: a comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.     

Plasma N-glycome composition associates with chronic low back pain

Background

Low back pain (LBP) is the symptom of a group of syndromes with heterogeneous underlying mechanisms and molecular pathologies, making treatment selection and patient prognosis very challenging. Moreover, symptoms and prognosis of LBP are influenced by age, gender, occupation, habits, and psychological factors. LBP may be characterized by an underlying inflammatory process. Previous studies indicated a connection between inflammatory response and total plasma N-glycosylation. We wanted to identify potential changes in total plasma N-glycosylation pattern connected with chronic low back pain (CLBP), which could give an insight into the pathogenic mechanisms of the disease.

Dairy wastewater polluting load and treatment performances of an industrial three-cascade-reactor plant

An industrial three-cascade-reactor plant treating 45 m³ d^?1 of dairy wastewater (DW) was monitored for approx. one year to investigate the effect of variable daily influent loads. It removed more than 85% COD, NH₄-N and non-ionic and anionic surfactants from DW within the loads 7–24, 0.4–2.3, 0.4–0.7 and 0.1–0.5 kg d^?1, respectively; NH₄-N removal, in particular, was almost quantitative. Although the degradation of the above parameters below the lower load thresholds declined to 78.7, 87.5, 50.2 and 64.7%, respectively, their residual concentrations met effluent discharge standards. The biomass settling properties, assessed as sludge volume index (SVI), were satisfactory (generally lower than 150 ml g^?1) regardless of the organic load of the influent. The depletion of the pollutant load took mainly place in the first reactor albeit a significant contribution to the removal of the slowly degradable organic matter fraction was given by the two subsequent reactors.     

Indigenous Vibrio cholerae strains from a non-endemic region are pathogenic

Of the 200+ serogroups of Vibrio cholerae, only O1 or O139 strains are reported to cause cholera, and mostly in endemic regions. Cholera outbreaks elsewhere are considered to be via importation of pathogenic strains. Using established animal models, we show that diverse V. cholerae strains indigenous to a non-endemic environment (Sydney, Australia), including non-O1/O139 serogroup strains, are able to both colonize the intestine and result in fluid accumulation despite lacking virulence factors believed to be important. Most strains lacked the type three secretion system considered a mediator of diarrhoea in non-O1/O13 V. cholerae. Multi-locus sequence typing (MLST) showed that the Sydney isolates did not form a single clade and were distinct from O1/O139 toxigenic strains. There was no correlation between genetic relatedness and the profile of virulence-associated factors. Current analyses of diseases mediated by V. cholerae focus on endemic regions, with only those strains that possess particular virulence factors considered pathogenic. Our data suggest that factors other than those previously well described are of potential importance in influencing disease outbreaks.     

Uptake of 5 9Fe from soluble 5 9Fe-humate complexes by cucumber and barley plants

The capability of cucumber (Cucumis sativus L., cv. Serpente cinese), a Strategy I plant and barley (Hordeum vulgaris L., cv. Europa), a Strategy II plant to use Fe complexed by a water-soluble humic fraction (WEHS) extracted from a peat, was studied. Uptake of ⁵⁹Fe from ⁵⁹Fe-WEHS by cucumber plants was higher at pH 6.0 than at pH 7.5. Roots of intact cucumber plants were able to reduce the Fe^III-WEHS complex either at pH 6.0 or 7.5, rates being higher in the assay medium buffered at pH 6.0. After supply of ⁵⁹Fe-WEHS, a large pool of root extraplasmatic ⁵⁹Fe was formed, which could be used to a large extent by Fe-deficient plants, particularly under acidic conditions. Uptake of ⁵⁹Fe from ⁵⁹Fe-WEHS by Fe-sufficient and Fe-deficient barley plants was examined during periods of high (morning) and low (evening) PS release. Uptake paralleled the diurnal rhythm of PS release. Furthermore, ⁵⁹Fe uptake was strongly enhanced by addition of PS to the uptake solution in both Fe-sufficient and Fe-deficient plants. High amount of root extraplasmatic ⁵⁹Fe was formed upon supply of Fe-WEHS, particularly in the evening experiment. Fe-deficient barley plants were able to utilize Fe from the root extraplasmatic pool, conceivably as a result of high rates of PS release. The results of the present work together with previous observations indicate that cucumber plants (Strategy I) utilize Fe complexed to WEHS, presumably via reduction of Fe^III-WEHS by the plasma membrane-bound reductase, while barley plants (Strategy II) use an indirect mechanism involving ligand exchange between WEHS and PS.     

Characterization of the major allergens purified from the venom of the paper wasp Polistes gallicus

Allergic reactions to vespid stings are one of the major causes of IgE-mediated anaphylaxis. Vespa and Vespula venoms are closely related; Polistes venom is more distantly related and its allergens are less well studied. There is limited cross-reactivity between Polistes and the other vespid venoms because of differences in the epitopes on the allergen molecules.In this study, the major allergens of Polistes gallicus are isolated and characterized. P. gallicus venom contains four major allergens: phospholipase, antigen 5 (Ag5), hyaluronidase and protease that were characterized by mass spectrometry and specific binding to IgE. The complete amino acid sequence of Ag5 and the sequence of the N-terminal region of phospholipase were also determined. The alignment of Ag5 from P. gallicus (European species) and Polistes annularis (American species) shows an 85% identity that increases to 98% within the same subgenus. This could suggest the presence of specific epitopes on Ag5 molecule being the variations on the superficial loops. The features of the P. gallicus allergens could explain the partial cross-reactivity found between the American and European Polistes venoms, and suggest that the use of European Polistes venoms would improve the diagnostic specificity and the therapy of European patients and of North American patients sensitized by European Polistes.     

The three-dimensional structures of the Mycobacterium tuberculosis dihydrodipicolinate reductase-NADH-2,6-PDC and -NADPH-2,6-PDC complexes. Structural and mutagenic analysis of relaxed nucleotide specificity

Dihydrodipicolinate reductase (DHPR) catalyzes the reduced pyridine nucleotide-dependent reduction of the alpha,beta-unsaturated cyclic imine, dihydrodipicolinate, to generate tetrahydrodipicolinate. This enzyme catalyzes the second step in the bacterial biosynthetic pathway that generates meso-diaminopimelate, a component of bacterial cell walls, and the amino acid L-lysine. The Mycobacterium tuberculosis dapB-encoded DHPR has been cloned, expressed, purified, and crystallized in two ternary complexes with NADH or NADPH and the inhibitor 2,6-pyridinedicarboxylate (2,6-PDC). The structures have been solved using molecular replacement strategies, and the DHPR-NADH-2,6-PDC and DHPR-NADPH-2,6-PDC complexes have been refined against data to 2.3 and 2.5 A, respectively. The M. tuberculosis DHPR is a tetramer of identical subunits, with each subunit composed of two domains connected by two flexible hinge regions. The N-terminal domain binds pyridine nucleotide, while the C-terminal domain is involved in both tetramer formation and substrate/inhibitor binding. The M. tuberculosis DHPR uses NADH and NADPH with nearly equal efficiency based on V/K values. To probe the nature of this substrate specificity, we have generated two mutants, K9A and K11A, residues that are close to the 2'-phosphate of NADPH. These two mutants exhibit decreased specificity for NADPH by factors of 6- and 30-fold, respectively, but the K11A mutant exhibits 270% of WT activity using NADH. The highly conserved structure of the nucleotide fold may permit other enzyme's nucleotide specificity to be altered using similar mutagenic strategies.