Design of artificial red blood cells using polymeric hydrogel microcapsules: hydrogel stability improvement and polymer selection.

PURPOSE: To improve the stability of pectin-oligochitosan hydrogel microcapsules under physiological conditions. METHODS: Two different approaches were examined: change of the cross-linker length and treatment of the hydrogel microcapsules with 150 Mm CaCl2. Replacement of pectin with alginate was also studied. RESULTS AND CONCLUSIONS: It was observed that the molecular weight of the cross-linker oligochiotsan had no significant improvement on microcapsule stability. On the other hand, the treatment of pectin-oligochitosan microcapsules with Ca2+ increased the microcapsule stability significantly. Different types of alginate were used; however, no red-blood-cell-shaped microcapsules could be produced, which is likely due to the charge-density difference between deprotonated pectin and alginate polymers.

Development of a microscale red blood cell-shaped pectin-oligochitosan hydrogel system using an electrospray-vibration method: preparation and characterization.

PURPOSE: To develop and characterize a microscale pectin-oligochitosan hydrogel microcapsule system that could be applied in such biological fields as drug delivery, cell immobilization/encapsulation, and tissue engineering. METHODS: Microscale pectin-oligochitosan hydrogel microcapsules were prepared by using the vibration/electrostatic spray method. The morphology and chemistry of the hydrogel microcapsules were characterized by using scanning electron microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR), respectively. The designed hydrogel microcapsule system was then used to study the responsiveness of the microcapsules to different simulated human body fluids as well as cell encapsulation. RESULTS: The designed hydrogel microcapsule system exhibited a large surface area-to-volume ratio (red blood cell-shaped) and great pH/enzymatic responsiveness. In addition, this system showed the potential for controlled drug delivery and three-dimensional cell culture. CONCLUSION: This system showed a significant potential not only for bioactive-agent delivery, especially to the lower gastrointestinal (GI) tract, but also as a three-dimensional niche for cell culture. In particular, the hydrogel microcapsule system could be used to create artificial red-blood-cells as well as blood substitutes.

Spatiotemporal characteristics of swallowing in Parkinson's disease.

OBJECTIVES/HYPOTHESIS: The aim of this study was to quantitatively investigate spatiotemporal movement abnormalities during the pharyngeal phase of swallowing in individuals with Parkinson's disease (PD) and to investigate relationships between disease and dysphagia severities. STUDY DESIGN: Retrospective study. METHODS: We performed two-dimensional motion analysis of the hyoid bone, epiglottis, and vocal folds using videofluoroscopic images from 33 PD patients and 33 age-matched, healthy controls. The outcome measures were spatial and temporal movement variables during swallowing. Additionally, three subgroups of patients were compared based on the Hoehn and Yahr scale to identify the relationship between disease and dysphagia severities. RESULTS: Individuals with PD showed a reduced anterior hyoid bone displacement after normalization for each individual. The maximal angle of the epiglottic rotation was smaller in PD patients. The time to maximal displacement of the hyoid bone, epiglottis, and vocal folds were significantly delayed in PD patients (P = 0.001, 0.002, and < 0.001, respectively); the mean velocities of the hyoid bone and epiglottic movements were also reduced in PD patients (P < 0.001 and < 0.001, respectively). The velocity curves of the hyoid bone revealed more peaks in individuals with PD, representing incoordination during swallowing. Clinical disease severity was related to level of diet and supervision but not with dysphagia severity. CONCLUSION: This study shows that oropharyngeal bradykinesia, incoordination, reduced anterior hyoid bone movement, and decreased epiglottic rotation angle during swallowing are the most noticeable findings in individuals with PD. LEVEL OF EVIDENCE: N/A.

Helix capping in RNA structure.

Helices are an essential element in defining the three-dimensional architecture of structured RNAs. While internal basepairs in a canonical helix stack on both sides, the ends of the helix stack on only one side and are exposed to the loop side, thus susceptible to fraying unless they are protected. While coaxial stacking has long been known to stabilize helix ends by directly stacking two canonical helices coaxially, based on analysis of helix-loop junctions in RNA crystal structures, herein we describe helix capping, topological stacking of a helix end with a basepair or an unpaired nucleotide from the loop side, which in turn protects helix ends. Beyond the topological protection of helix ends against fraying, helix capping should confer greater stability onto the resulting composite helices. Our analysis also reveals that this general motif is associated with the formation of tertiary structure interactions. Greater knowledge about the dynamics at the helix-junctions in the secondary structure should enhance the prediction of RNA secondary structure with a richer set of energetic rules and help better understand the folding of a secondary structure into its three-dimensional structure. These together suggest that helix capping likely play a fundamental role in driving RNA folding.

The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum.

BACKGROUND: The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. PRINCIPAL FINDINGS: The identification of 14 additional small mitochondrial transcripts from P. falciparum and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. SIGNIFICANCE: All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered.

A comparison of the crystal structures of eukaryotic and bacterial SSU ribosomal RNAs reveals common structural features in the hypervariable regions.

While the majority of the ribosomal RNA structure is conserved in the three major domains of life--archaea, bacteria, and eukaryotes, specific regions of the rRNA structure are unique to at least one of these three primary forms of life. In particular, the comparative secondary structure for the eukaryotic SSU rRNA contains several regions that are different from the analogous regions in the bacteria. Our detailed analysis of two recently determined eukaryotic 40S ribosomal crystal structures, Tetrahymena thermophila and Saccharomyces cerevisiae, and the comparison of these results with the bacterial Thermus thermophilus 30S ribosomal crystal structure: (1) revealed that the vast majority of the comparative structure model for the eukaryotic SSU rRNA is substantiated, including the secondary structure that is similar to both bacteria and archaea as well as specific for the eukaryotes, (2) resolved the secondary structure for regions of the eukaryotic SSU rRNA that were not determined with comparative methods, (3) identified eukaryotic helices that are equivalent to the bacterial helices in several of the hypervariable regions, (4) revealed that, while the coaxially stacked compound helix in the 540 region in the central domain maintains the constant length of 10 base pairs, its two constituent helices contain 5+5 bp rather than the 6+4 bp predicted with comparative analysis of archaeal and eukaryotic SSU rRNAs.

Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes.

BACKGROUND: Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent forms result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA) genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters. RESULTS: In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters C. virginica, C. gigas, and C. hongkongensis. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes. CONCLUSIONS: Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.

Placement of attine ant-associated Pseudonocardia in a global Pseudonocardia phylogeny (Pseudonocardiaceae, Actinomycetales): a test of two symbiont-association models.

We reconstruct the phylogenetic relationships within the bacterial genus Pseudonocardia to evaluate two models explaining how and why Pseudonocardia bacteria colonize the microbial communities on the integument of fungus-gardening ant species (Attini, Formicidae). The traditional Coevolution-Codivergence model views the integument-colonizing Pseudonocardia as mutualistic microbes that are largely vertically transmitted between ant generations and that supply antibiotics that specifically suppress the garden pathogen Escovopsis. The more recent Acquisition model views Pseudonocardia as part of a larger integumental microbe community that frequently colonizes the ant integument from environmental sources (e.g., soil, plant material). Under this latter model, ant-associated Pseudonocardia may have diverse ecological roles on the ant integument (possibly ranging from pathogenic, to commensal, to mutualistic) and are not necessarily related to Escovopsis suppression. We test distinct predictions of these two models regarding the phylogenetic proximity of ant-associated and environmental Pseudonocardia. We amassed 16S-rRNA gene sequence information for 87 attine-associated and 238 environmental Pseudonocardia, aligned the sequences with the help of RNA secondary structure modeling, and reconstructed phylogenetic relationships using a maximum-likelihood approach. We present 16S-rRNA secondary structure models of representative Pseudonocardia species to improve sequence alignments and identify sequencing errors. Our phylogenetic analyses reveal close affinities and even identical sequence matches between environmental Pseudonocardia and ant-associated Pseudonocardia, as well as nesting of environmental Pseudonocardia in subgroups that were previously thought to be specialized to associate only with attine ants. The great majority of ant-associated Pseudonocardia are closely related to autotrophic Pseudonocardia and are placed in a large subgroup of Pseudonocardia that is known essentially only from cultured isolates (rather than cloned 16S sequences). The preponderance of the known ant-associated Pseudonocardia in this latter clade of culturable lineages may not necessarily reflect abundance of these Pseudonocardia types on the ants, but isolation biases when screening for Pseudonocardia (e.g., preferential isolation of autotrophic Pseudonocardia with minimum-nutrient media). The accumulated phylogenetic patterns and the possibility of isolation biases in previous work further erode support for the traditional Coevolution-Codivergence model and calls for continued revision of our understanding how and why Pseudonocardia colonize the microbial communities on the integument of fungus-gardening ant species.

Hemoperfusion using dual pulsatile pump in paraquat poisoning.

INTRODUCTION: Hemoperfusion is an effective method for removing paraquat from the body. However, the need for special equipment and personnel to perform hemoperfusion limits its applicability in the emergency department. A portable, user-friendly extracorporeal life support system (Twin Pulse Life Support [T-PLS]) capable of producing dual pulsatile flow by an electromechanical blood pump has recently been developed. We compared the effects of hemoperfusion using traditional and dual-pulsatile pumps in a paraquat-intoxicated canine model. We hypothesized that T-PLS would be as effective as nonpulsatile hemoperfusion in reducing plasma and tissue paraquat levels. METHODS: Twelve adult male mongrel dogs were randomly assigned to hemoperfusion using a standard nonpulsatile pump (n = 6) or the T-PLS (n = 6). Paraquat intoxication was induced by intramuscular injection of paraquat (30 mg/kg). One hour after paraquat administration, hemoperfusion was performed for 4 hours at a flow rate of 125 mL/min. Periodic hemodynamic, chemical, and hematologic parameters as well as paraquat blood levels were obtained before and during the experiment. All animals were euthanized after completing 4 hours of hemoperfusion, and tissue levels of paraquat were determined. RESULTS: During hemoperfusion, hemodynamic parameters including aortic blood pressure, heart rate, rectal temperature, and SaO(2) showed no significant difference between the T-PLS group and the nonpulsatile pump groups. Chemical and hematologic parameters such as serum electrolytes, platelet level, creatinine concentration, the ratio of blood urea nitrogen (BUN) to creatinine, alanine aminotransferase/aspartate aminotransferase (ALS/AST) levels, and plasma hemoglobin concentration as an indicator of hemolysis also showed no between-group differences. Plasma paraquat concentrations and lung and kidney tissue paraquat concentrations were also similar in both groups. CONCLUSIONS: In this experimental canine study, T-PLS was as effective as traditional nonpulsatile hemoperfusion in reducing plasma and tissues paraquat levels. There were also no differences in hemodynamic, chemical, and hematologic parameters between the groups. Hemoperfusion using T-PLS may play a role as an alternative method for treating patients with paraquat poisoning.

The origin and evolution of the ribosome.

BACKGROUND: The origin and early evolution of the active site of the ribosome can be elucidated through an analysis of the ribosomal proteins' taxonomic block structures and their RNA interactions. Comparison between the two subunits, exploiting the detailed three-dimensional structures of the bacterial and archaeal ribosomes, is especially informative. RESULTS: The analysis of the differences between these two sites can be summarized as follows: 1) There is no self-folding RNA segment that defines the decoding site of the small subunit; 2) there is one self-folding RNA segment encompassing the entire peptidyl transfer center of the large subunit; 3) the protein contacts with the decoding site are made by a set of universal alignable sequence blocks of the ribosomal proteins; 4) the majority of those peptides contacting the peptidyl transfer center are made by bacterial or archaeal-specific sequence blocks. CONCLUSION: These clear distinctions between the two subunit active sites support an earlier origin for the large subunit's peptidyl transferase center (PTC) with the decoding site of the small subunit being a later addition to the ribosome. The main implications are that a single self-folding RNA, in conjunction with a few short stabilizing peptides, formed the precursor of the modern ribosomal large subunit in association with a membrane.

The UAA/GAN internal loop motif: a new RNA structural element that forms a cross-strand AAA stack and long-range tertiary interactions.

Analysis of aligned RNA sequences and high-resolution crystal structures has revealed a new RNA structural element, termed the UAA/GAN motif. Found in internal loops of the 23 S rRNA, as well as in RNase P RNA and group I and II introns, this six-nucleotide motif adopts a distinctive local structure that includes two base-pairs with non-canonical conformations and three conserved adenine bases, which form a cross-strand AAA stack in the minor groove. Most importantly, the motif invariably forms long-range tertiary contacts, as the AAA stack typically forms A-minor interactions and the flipped-out N nucleotide forms additional contacts that are specific to the structural context of each loop. The widespread presence of this motif and its propensity to form long-range contacts suggest that it plays a critical role in defining the architectures of structured RNAs.

Diversity of base-pair conformations and their occurrence in rRNA structure and RNA structural motifs.

In addition to the canonical base-pairs comprising the standard Watson-Crick (C:G and U:A) and wobble U:G conformations, an analysis of the base-pair types and conformations in the rRNAs in the high-resolution crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits has identified a wide variety of non-canonical base-pair types and conformations. However, the existing nomenclatures do not describe all of the observed non-canonical conformations or describe them with some ambiguity. Thus, a standardized system is required to classify all of these non-canonical conformations appropriately. Here, we propose a new, simple and systematic nomenclature that unambiguously classifies base-pair conformations occurring in base-pairs, base-triples and base-quadruples that are associated with secondary and tertiary interactions. This system is based on the topological arrangement of the two bases and glycosidic bonds in a given base-pair. Base-pairs in the internal positions of regular secondary structure helices usually form with canonical base-pair groups (C:G, U:A, and U:G) and canonical conformations (C:G WC, U:A WC, and U:G Wb). In contrast, non-helical base-pairs outside of regular structure helices usually have non-canonical base-pair groups and conformations. In addition, many non-helical base-pairs are involved in RNA motifs that form a defined set of non-canonical conformations. Thus, each rare non-canonical conformation may be functionally and structurally important. Finally, the topology-based isostericity of base-pair conformations can rationalize base-pair exchanges in the evolution of RNA molecules.

The lonepair triloop: a new motif in RNA structure.

The lonepair triloop (LPTL) is an RNA structural motif that contains a single ("lone") base-pair capped by a hairpin loop containing three nucleotides. The two nucleotides immediately outside of this motif (5' and 3' to the lonepair) are not base-paired to one another, restricting the length of this helix to a single base-pair. Four examples of this motif, along with three tentative examples, were initially identified in the 16S and 23S rRNAs with covariation analysis. An evaluation of the recently determined crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits revealed the authenticity for all of these proposed interactions and identified 16 more LPTLs in the 5S, 16S and 23S rRNAs. This motif is found in the T loop in the tRNA crystal structures. The lonepairs are positioned, in nearly all examples, immediately 3' to a regular secondary structure helix and are stabilized by coaxial stacking onto this flanking helix. In all but two cases, the nucleotides in the triloop are involved in a tertiary interaction with another section of the rRNA, establishing an overall three-dimensional function for this motif. Of these 24 examples, 14 occur in multi-stem loops, seven in hairpin loops and three in internal loops. While the most common lonepair, U:A, occurs in ten of the 24 LPTLs, the remaining 14 LPTLs contain seven different base-pair types. Only a few of these lonepairs adopt the standard Watson-Crick base-pair conformations, while the majority of the base-pairs have non-standard conformations. While the general three-dimensional conformation is similar for all examples of this motif, characteristic differences lead to several subtypes present in different structural environments. At least one triloop nucleotide in 22 of the 24 LPTLs in the rRNAs and tRNAs forms a tertiary interaction with another part of the RNA. When a LPTL containing the GNR or UYR triloop sequence forms a tertiary interaction with the first (and second) triloop nucleotide, it recruits a fourth nucleotide to mediate stacking and mimic the tetraloop conformation. Approximately half of the LPTL motifs are in close association with proteins. The majority of these LPTLs are positioned at sites in rRNAs that are conserved in the three phylogenetic domains; a few of these occur in regions of the rRNA associated with ribosomal function, including the presumed site of peptidyl transferase activity in the 23S rRNA.

The accuracy of ribosomal RNA comparative structure models.

The determination of the 16S and 23S rRNA secondary structure models was initiated shortly after the first complete 16S and 23S rRNA sequences were determined in the late 1970s. The structures that are common to all 16S rRNAs and all 23S rRNAs were determined using comparative methods from the analysis of thousands of rRNA sequences. Twenty-plus years later, the 16S and 23S rRNA comparative structure models have been evaluated against the recently determined high-resolution crystal structures of the 30S and 50S ribosomal subunits. Nearly all of the predicted covariation-based base pairs, including the regular base pairs and helices, and the irregular base pairs and tertiary interactions, were present in the 30S and 50S crystal structures.