Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spinal cord injury

Background aimsSpinal cord injury (SCI) is a medically untreatable condition for which stem cells have created hope. Pre-clinical and clinical studies have established that these cells are safe for transplantation. The dose dependency, survivability, route of administration, cell migration to injury site and effect on sensory and motor behavior in an SCI-induced paraplegic model were studied.MethodsA spinal cord contusion injury model was established in rats. Bone marrow (BM) mesenchymal stromal cells (MSC) were tagged to facilitate tracing in vivo. Two different doses (2 and 5 million cells/kg body weight) and two different routes of infusion (site of injury and lumbar puncture) were tested during and after the spinal shock period. The animals were tested post-transplantation for locomotor capacity, motor control, sensory reflex, posture and body position. Stem cell migration was observed 1 month post-transplantation in spinal cord sections.ResultsThe overall results demonstrated that transplantation of BM MSC significantly improved the locomotor and sensory behavior score in the experimental group compared with the sham control group, and these results were dose dependent. All the infused stem cells could be visualized at the site of injury and none was visualized at the injected site. This indicated that the cells had survived in vivo, were probably chemoattracted and had migrated to the lesion site.ConclusionsMSC transplanted with a lumbar puncture method migrate to the site of injury and are the most suitable for SCI healing. These cells demonstrate a dose-dependent effect and promote functional recovery when injected during or after the spinal shock period.     

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.     

Appreciating the role of carbon nanotube composites in preventing biofouling and promoting biofilms on material surfaces in environmental engineering: A review

The ability of carbon nanotubes (CNTs) to undergo surface modification allows them to form nanocomposites (NCs) with materials such as polymers, metal nanoparticles, biomolecules, and metal oxides. The biocidal nature, protein fouling resistance, and fouling release properties of CNT-NCs render them the perfect material for biofouling prevention. At the same time, the cytotoxicity of CNT-NCs can be reduced before applying them as substrates to promote biofilm formation in environmental biotechnology applications. This paper reviews the potential prospects of CNT-NCs to accomplish two widely varying objectives in environmental engineering applications: (i) preventing biofouling, and (ii) promoting the formation of desirable biofilms on materials surface. This paper addresses practical issues such as costs, risks to human health, and ecological impacts that are associated with the application, development and commercialization of CNT-NC technology.     

Characterization of Indian bred rose cultivars using morphological and molecular markers for conservation and sustainable management

Rose (Rosa × hybrid L.) is one of the most important commercial ornamental crops cultivated worldwide for its beauty, fragrance and nutraceutical values. Characterization of rose germplasm provides precise information about the extent of diversity present among the cultivars. It also helps in cultivar identification, intellectual property right protection, variety improvement and genetic diversity conservation. In the present study, 109 Indian bred rose cultivars were characterized using 59 morphological and 48 SSR markers. Out of 48 SSRs used, 31 markers exhibited polymorphism and 96 alleles were identified with an average of 3.9 alleles per locus. Nei’s expected heterozygosity value of each locus ranged from 0.08 (with SSR ABRII/RPU32) to 0.78 (SSR Rh58). The similarity coefficient values ranged from 0.42 to 0.90 which indicated presence of moderated diversity among Indian cultivars. The neighbor-joining tree based on morphological data grouped the cultivars into two major clusters and several minor clusters based on their morphological resemblance. However, UPGMA dendrogram constructed using matching coefficient values grouped the cultivars into eight different clusters. Interpopulation analysis revealed higher genetic similarities between Hybrid Tea and Floribunda cultivars. An analysis for presence of population sub-structure grouped the Indian cultivars into eight different genetic groups. Analysis of molecular variance revealed apportioning of 97.59% of the variation to within subgroup diversity and 3.07% to between the cultivar groups. We have demonstrated here successful utilization of robust SSR to distinguish cultivars and assess genetic diversity among Indian bred rose cultivars. The information provided here is useful for cultivar identification and protection, cultivar improvement and genetic diversity conservation.     

Resolution of experimental lung injury by monocyte-derived inducible nitric oxide synthase

Although early events in the pathogenesis of acute lung injury (ALI) have been defined, little is known about the mechanisms mediating resolution. To search for determinants of resolution, we exposed wild type (WT) mice to intratracheal LPS and assessed the response at intervals to day 10, when injury had resolved. Inducible NO synthase (iNOS) was significantly upregulated in the lung at day 4 after LPS. When iNOS(-/-) mice were exposed to intratracheal LPS, early lung injury was attenuated; however, recovery was markedly impaired compared with WT mice. iNOS(-/-) mice had increased mortality and sustained increases in markers of lung injury. Adoptive transfer of WT (iNOS(+/+)) bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS(-/-) mice restored resolution of ALI. Irradiated bone marrow chimeras confirmed the protective effects of myeloid-derived iNOS but not of epithelial iNOS. Alveolar macrophages exhibited sustained expression of cosignaling molecule CD86 in iNOS(-/-) mice compared with WT mice. Ab-mediated blockade of CD86 in iNOS(-/-) mice improved survival and enhanced resolution of lung inflammation. Our findings show that monocyte-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung repair.     

A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G

Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures.     

Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome

During translation, the ribosome and several of its constituent proteins undergo structural transitions between different functional states. Protein L12, present in four copies in prokaryotic ribosomes, forms a flexible "stalk" with key functions in factor-dependent GTP hydrolysis during translocation. Here we have used heteronuclear NMR spectroscopy to characterize L12 conformation and dynamics in solution and on the ribosome. Isolated L12 forms a symmetric dimer mediated by the N-terminal domains (NTDs), to which each C-terminal domain (CTD) is connected via an unstructured hinge segment. The overall structure can be described as three ellipsoids joined by flexible linkers. No persistent contacts are seen between the two CTDs, or between the NTD and CTD in the L12 dimer in solution. In the (1)H-(15)N HSQC spectrum of the Escherichia coli 70S ribosome, a single set of cross-peaks are observed for residues 40-120 of L12, the intensities of which correspond to only two of four protein copies. The structure of the CTDs observed on the ribosome is indistinguishable from that of isolated L12. These results indicate that two CTDs with identical average structures are mobile and extend away from the ribosome, while the other two copies most likely interact tightly with the ribosome even in the absence of translational factors.