Specificity and Strain-Typing Capabilities of Nanorod Array-Surface Enhanced Raman Spectroscopy for Mycoplasma pneumoniae Detection

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for > 20% of all community-acquired pneumonia (CAP). At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use. We have developed and previously described a silver nanorod array-surface enhanced Raman Spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae with statistically significant specificity and sensitivity in simulated and true clinical throat swab samples, and the ability to distinguish between reference strains of the two main genotypes of M. pneumoniae. Furthermore, we have established a qualitative lower endpoint of detection for NA-SERS of < 1 genome equivalent (cell/μl) and a quantitative multivariate detection limit of 5.3 ± 1 cells/μl. Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy. Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.     

IDENTIFICATION OF LECTINS IN THE KINETIDS OF TETRAHYMENA PYRIFORMIS

Previously we described lectin-like molecules in the ciliate Tetrahymena pyriformis; by application of synthetic neoglycoconjugates it is now shown that T. pyriformis contains considerable amounts of both a β-d-glucose- and a lactose-specific lectin. No evidence for the presence of α-d-mannose-, α-d-galactose- or of α-l-fucose-specific lectins could be obtained. The two lectins, identified in T. pyriformis, are associated with the kinetids. During cell division the lectins disappear or become masked in the fission furrow. Therefore, we assume that these lectins are involved in the organization of the distribution pattern of the kinetids during cell division perhaps due to lectin—glycoprotein interactions.     

Microsatellite variation and microevolution in the critically endangered San Clemente Island loggerhead shrike (Lanius ludovicianus mearnsi)

Polymorphic nuclear microsatellite loci were used to characterize genetic variation in contemporary and historic populations of the San Clemente Island loggerhead shrike (Lanius ludovicianus mearnsi), an endangered bird with a current population of 30 individuals that is endemic to to one of the California Channel Islands. We also compared the population of the shrike with two contemporary populations of the still abundant subspecies, L. l. gambeli, which live 120 km away on the adjacent mainland. The current population of L. l. mearnsi has 60 per cent of the genetic variation of the mainland shrike populations and is strongly differentiated from them. Comparison of living birds with 19 birds collected in 1915 shows that most of the variation within the island population was lost before the recent 90 per cent decline in population size, and the 20 per cent decrease in variation this century is probably attributable to genetic drift. Mitochondrial DNA control region sequence data from 80 year old specimens show that there may have been limited introgression to L. l. mearnsi, this century, from another island subspecies, L. l. anthonyi, found in the northern Channel Islands. Today, gene flow between L. l. mearnsi and mainland L. l. gambel is very low, even though a few mainland birds visit the island annually. The island subspecies population has evolved sufficient genetic independence to justify ongoing conservation efforts to counter demographic collapse and genetic erosion; the course of genetic erosion can now be monitored non-invasively, as demonstrated by this study, based on DNA amplified from feathers.     

Inhibitory effects of some esters of 2- and 3-substituted alkoxyphenylcarbamic acids on photosynthetic characteristics

The inhibitory effect of 23N-alkyl-4-piperidylesters (alkyl = ethyl-butyl) (APEA) and 8N-ethyl-2-pyrrolidinylmethylesters (EPMEA) of 2- and 3-substituted alkoxyphenylcarbamic acids (alkoxy = butoxy-heptyloxy-) on photosynthetic Hill reaction activity of spinach chloroplasts and on chlorophyll (Chl) synthesis in green algaeChlorella vulgaris was investigated. Inhibitory activities of these compounds were strongly connected with the lipophilicity of the whole molecule. A lower inhibitory activity of 2-alkoxy-substituted derivatives in relation to the corresponding 3-substituted ones was confirmed. Electron spin resonance (ESR) spectra of spinach chloroplasts demonstrated that the studied compounds affected the structure of photosystem (PS) 2 with the release of Mn²⁺ ions into interior of thylakoid membranes.     

Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.     

Temporal variability in the spatial and environmental determinants of functional metacommunity organization – stream fish in a human‐modified landscape

1. Quantifying the relative importance of environmental filtering versus regional spatial structuring has become an intensively studied area in the context of metacommunity ecology. However, most studies have evaluated the role of environmental and spatial processes using taxonomic data sets of single snapshot surveys. 2. Here, we examined temporal changes in patterns and possible processes behind the functional metacommunity organization of stream fishes in a human‐modified landscape. Specifically, we (i) studied general changes in the functional composition of fish assemblages among 40 wadeable stream sites during a 3‐year study period in the catchment area of Lake Balaton, Hungary, (ii) quantified the relative importance of spatial and environmental factors as determinants of metacommunity structure and (iii) examined temporal variability in the relative role of spatial and environmental processes for this metacommunity. 3. Partial triadic analysis showed that assemblages could be effectively ordered along a functional gradient from invertebrate consuming species dominated by the opportunistic life‐history strategy, to assemblages with a diverse array of functional attributes. The analysis also revealed that functional fish assemblage structure was moderately stable among the sites between the sampling periods. 4. Despite moderate stability, variance partitioning using redundancy analyses (RDA) showed considerable temporal variability in the contribution of environmental and spatial factors to this pattern. The analyses also showed that environmental variables were, in general, more important than spatial ones in determining metacommunity structure. Of these, natural environmental variables (e.g. altitude, velocity) proved to be more influential than human‐related effects (e.g. pond area, % inhabited area above the site, nutrient enrichment), even in this landscape with relatively low variation in altitude and stream size. 5. Pond area was, however, the most important human stressor variable that was positively associated with the abundance of non‐native species with diverse functional attributes. The temporal variability in the relative importance of environmental and spatial factors was probably shaped by the release of non‐native fish from fish ponds to the stream system during flood events. 6. To conclude, both spatial processes and environmental control shape the functional metacommunity organization of stream fish assemblages in human‐modified landscapes, but their importance can vary in time. We argue, therefore, that metacommunity studies should better consider temporal variability in the ecological mechanisms (e.g. dispersal limitation, species sorting) that determine the dynamics of landscape‐level community organization.     

Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales

Powerful analyses of population structure require information from multiple genetic loci. To help develop a molecular toolbox for obtaining this information, we have designed universal oligonucleotide primers that span conserved intron-exon junctions in a wide variety of animal phyla. We test the utility of exon-primed, intron-crossing amplifications by analyzing the variability of actin intron sequences from humpback, blue, and bowhead whales and comparing the results with mitochondrial DNA (mtDNA) haplotype data. Humpback actin introns fall into two major clades that exist in different frequencies in different oceanic populations. It is surprising that Hawaii and California populations, which are very distinct in mtDNAs, are similar in actin intron alleles. This discrepancy between mtDNA and nuclear DNA results may be due either to differences in genetic drift in mitochondrial and nuclear genes or to preferential movement of males, which do not transmit mtDNA to offspring, between separate breeding grounds. Opposing mtDNA and nuclear DNA results can help clarify otherwise hidden patterns of structure in natural populations.      

Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8- fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400- fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.      

Defensive behaviour and its inheritance in the anabantoid fish, Macropodus opercularis and Macropodus opercularis concolor

In this preliminary study defense behaviour patterns (fear responses) are described in two closely related, behaviourally different inbred labyrinth fish subspecies and in their F₁ generation. The subspecies M. opercularis (characterized briefly by “active escape”) and M. opercularis concolor (characterized by “passive escape”) showed specific differences in the manifestation of certain defense behaviour patterns. In the F₁ hybrid generation dominance and overdominance of M. opercularis was found in most defense behaviour patterns. Analysing the frequencies and sequences of movement patterns it could be shown that defensive behaviour is not a random or entirely “plastic” process but that there is sequential linkage between the patterns and they form characteristic clusters. Our results suggest that manifestations of different patterns are under genetic control and presumably, genetic determination of certain patterns is not very complex. Attempts were made to determine whole brain noradrenaline, serotonine and dopamine levels of the two subspecies and a significant difference was found in the noradrenaline content.