Hyperspectral cytometry at the single-cell level using a 32-channel photodetector

Despite recent progress in cell-analysis technology, rapid classification of cells remains a very difficult task. Among the techniques available, flow cytometry (FCM) is considered especially powerful, because it is able to perform multiparametric analyses of single biological particles at a high flow rate-up to several thousand particles per second. Moreover, FCM is nondestructive, and flow cytometric analysis can be performed on live cells. The current limit for simultaneously detectable fluorescence signals in FCM is around 8-15 depending upon the instrument. Obtaining multiparametric measurements is a very complex task, and the necessity for fluorescence spectral overlap compensation creates a number of additional difficulties to solve. Further, to obtain well-separated single spectral bands a very complex set of optical filters is required. This study describes the key components and principles involved in building a next-generation flow cytometer based on a 32-channel PMT array detector, a phase-volume holographic grating, and a fast electronic board. The system is capable of full-spectral data collection and spectral analysis at the single-cell level. As demonstrated using fluorescent microspheres and lymphocytes labeled with a cocktail of antibodies (CD45/FITC, CD4/PE, CD8/ECD, and CD3/Cy5), the presented technology is able to simultaneously collect 32 narrow bands of fluorescence from single particles flowing across the laser beam in <5 μs. These 32 discrete values provide a proxy of the full fluorescence emission spectrum for each single particle (cell). Advanced statistical analysis has then been performed to separate the various clusters of lymphocytes. The average spectrum computed for each cluster has been used to characterize the corresponding combination of antibodies, and thus identify the various lymphocytes subsets. The powerful data-collection capabilities of this flow cytometer open up significant opportunities for advanced analytical approaches, including spectral unmixing and unsupervised or supervised classification.     

Excretion of glutamic acid in Citrobacter intermedius C3 associated with plasmid deoxyribonucleic acid.

Several mutants of Citrobacter intermedius C3 lacking both the ability to synthesize proline and the ability to excrete glutamic acid were isolated by treatment with nitrosoguanidine. No revertants for either characteristic were obtained from these mutants. The ability to excrete glutamic acid was transferred to those mutants with very high frequencies in mating experience by using auxotropic excreting strains as donors. Moreover, the ability to synthesize proline was transferred together with the ability to excrete glutamic acid when an excreting strain was used as donor. The transconjugants showed a rapid spontaneous curing of both genetic markers. It was shown by two different methods that a band of covalently closed circular deoxyribonucleic acid is present in the cesium chloride gradients corresponding to the wild type and excretor mutants. Nonexcretor mutants described herein lacked such a band. Pro + transformants that were also excretors were obtained with plasmid deoxyribonucleic acid isolated either from wild type or from an excretor mutant. These data strongly indicate that glutamic acid excretion in C. intermedius C3 is related to the presence of extrachromosomal deoxyribonucleic acid.     

Global diversity of crabs (Aeglidae: Anomura: Decapoda) in freshwater

The freshwater anomuran crabs of the family Aeglidae are all restricted to southern South America occurring in Chile, Brazil, Bolivia, Uruguay, Paraguay, and Argentina. The family consists of a single genus, Aegla, containing 63 currently described species. There are another 5–10 known yet undescribed species to complement this diversity. The aeglids occur in freshwater lakes, streams, rivers, and in caves with freshwater. The origin of the family appears to be from marine ancestors from the Pacific invading streams in Chile about 75 mya radiating both in Chile and again on the eastern side of the Andes, particularly in Brazil. Of the 63 species, 23 or 36.5% are considered under threat and are in need of conservation action. Guest editors: E. V. Balian, C. Lévêque, H. Segers & K. Martens Freshwater Animal Diversity Assessment     

Use of RmInt1, a group IIB intron lacking the intron-encoded protein endonuclease domain, in gene targeting

The group IIA intron Ll.LtrB from Lactococcus lactis and the group IIB intron EcI5 from Escherichia coli have intron-encoded proteins (IEP) with a DNA-binding domain (D) and an endonuclease domain (En). Both have been successfully retargeted to invade target DNAs other than their wild-type target sites. RmInt1, a subclass IIB3/D intron with an IEP lacking D and En domains, is highly active in retrohoming in its host, Sinorhizobium meliloti. We found that RmInt1 was also mobile in E. coli and that retrohoming in this heterologous host depended on temperature, being more efficient at 28°C than at 37°C. Furthermore, we programmed RmInt1 to recognize target sites other than its wild-type site. These retargeted introns efficiently and specifically retrohome into a recipient plasmid target site or a target site present as a single copy in the chromosome, generating a mutation in the targeted gene. Our results extend the range of group II introns available for gene targeting.     

Nitrogen use efficiency by a slow-growing species as affected by CO2 levels, root temperature, N source and availability

This study examines the importance of N source and concentration on plant response to distinct CO₂ concentrations and root temperatures. The experimental design of this work was a factorial combination of: CO₂ concentration, nitrogen concentration, nitrogen source and root temperature. Carob (Ceratonia siliqua L.) was assessed as a potential model of a slow growing Mediterranean species.

The results showed that: 1) biomass increment under high CO₂ varied between 13 and 100 percnt; in relation to plants grown under the same conditions but at ambient CO₂ concentrations, depending on the root temperature and nitrogen source; 2) nitrate-fed plants attained a larger increase in biomass production compared to ammonium-fed ones. This performance seems to be linked to the co-ordinated regulation of the activities of glutamine synthetase and sucrose phosphate synthase. The variations in the magnitude and nature of growth responses to elevated CO₂ observed resulted in substantial changes in the chemical composition of the plant material and consequently in plant nitrogen use efficiency.

Although performed with seedlings and under controlled conditions, this work emphasizes the importance of the nitrogen source used by the plants, a factor rarely taken into consideration when forecasting plant responses to global changes. Particularly, the results presented here, highlight the potential for uncoupling biomass accumulation from increment of air CO₂ concentration and show that more than nitrogen availability N source may offset positive plant growth responses under elevated CO₂ and root temperature.     

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.     

Influence of physico-chemical parameters of the aquatic medium on germination of Eichhornia crassipes seeds

The germination of seeds of Eichhornia crassipes in locations distant from the foci of infestation may be a means of dispersal of this invasive plant. Nonetheless, no modern studies have examined the influence of single components on germination, although the influence of temperature, oxygen and redox potential was examined in earlier studies. The effects of pH, conductivity, nitrate, phosphate, potassium, calcium, iron and boron on germination of E. crassipes were determined in seed germination tests. The results showed that external input of nutrients influences germination, since neither pH nor conductivity alone had any effect, but there was a significant increase in germination in growth media containing phosphorus at 3.95 mg·l(-1) (P/L; 95.5%) and boron at 10 mg·l(-1) (B/L; 97.5%); at higher concentrations, the latter was toxic. These findings contribute to knowledge of factors controlling the germination of E. crassipes seeds. Consequently, E. crassipes seeds would find very good conditions for germination in water classified as hypereutrophic, which may play a decisive role in expansion of this plant.     

Mating Frequency, Fecundity and Fertilization Success in the Hide Beetle, Dermestes Maculatus

While the immediate benefits accrued to females through multiple mating are well documented, the effect of sperm depletion for multiply mating males is rarely considered. We show that, in small mixed-sex laboratory aggregations, both male and female hide beetles, Dermestes maculatus (De Geer) mated multiply. There was considerable variation in the mating frequency of both sexes; however the skew in mating success was comparable for males and females. Several individuals that mated multiply also re-mated with a previous partner, but in a competitive environment no male copulated more than seven times. Mating success was unrelated to an individual's size, but males that had the most inter-sexual matings also engaged in the most intra-sexual mating attempts. In a second experiment, we show that, even in the absence of rivals, only a small number of males mated with all available virgin females. Moreover, even though males were mated twice to each female, males that copulated more than eight times failed to fertilize any eggs. We suggest that under natural conditions male hide beetles may refrain from mating either prior to, or at the point of, sperm depletion thereby reducing the selection pressure for females to discriminate against sperm depleted males. However, fecundity and fertilization success varied considerably across females and even those mating with sperm-replete males were unable to fertilize 100% of their egg batch. Thus, direct fertilization benefits accrued by females through mating more than once with the same male may play a key role in the maintenance of polyandry in this species.