Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.Download video file.^{(61M, mov)}     

Geographically structured genetic variation in the Medicago lupulina–Ensifer mutualism

Gene flow between genetically differentiated populations can maintain variation in species interactions, especially when population structure is congruent between interacting species. However, large‐scale empirical comparisons of the population structure of interacting species are rare, particularly in positive interspecific interactions (mutualisms). One agriculturally and ecologically important mutualism is the partnership between legume plants and rhizobia. Through characterizing and comparing the population genomic structure of the legume Medicago lupulina and two rhizobial species (Ensifer medicae and E. meliloti), we explored the spatial scale of population differentiation between interacting partners in their introduced range in North America. We found high proportions of E. meliloti in southeastern populations and high proportions of E. medicae in northwestern populations. Medicago lupulina and the Ensifer genus showed similar patterns of spatial genetic structure (isolation by distance). However, we detected no evidence of isolation by distance or population structure within either species of bacteria. Genome‐wide nucleotide diversity within each of the two Ensifer species was low, suggesting limited introduction of strains, founder events, or severe bottlenecks. Our results suggest that there is potential for geographically structured coevolution between M. lupulina and the Ensifer genus, but not between M. lupulina and either Ensifer species.     

Dynamic diversification history with rate upshifts in Holarctic bell‐flowers (Campanula and allies)

Campanula s.l. is one of the most speciose flowering plant lineages of the Holarctic (ca. 600 species). In the present study we sequenced three regions of the plastid genome (petD, rpl16 and trnK/matK) across a broad sample of Campanula s.l., which markedly improved phylogenetic resolution and statistical support compared to previous studies. Based on this robust phylogenetic hypothesis we estimated divergence times using BEAST, diversification rate shifts using Bayesian Analysis of Macroevolutionary Mixture (BAMM) and TreePar, and ancestral ranges using Biogeography with Bayesian (and likelihood) Evolutionary Analyses in R. Campanula s.l. is estimated to have originated during the Early Eocene but the major diversification events occurred between the Late Oligocene and Middle Miocene. Two upward diversification rate shifts were revealed by BAMM, specific to the crown nodes of two Campanula clades: CAM17, a mostly South European‐SW Asian lineage originating during the Middle Miocene and containing nearly half of all known Campanula species; and CAM15B, a SW Asian–Sino‐Himalayan lineage of nine species originating in the early Pleistocene. The dynamic diversification history of Campanula and the inferred rate shifts are discussed in a geo‐historical context.     

CtrA controls cell division and outer membrane composition of the pathogen Brucella abortus

Brucella abortus is a pathogen infecting cattle, able to survive, traffic, and proliferate inside host cells. It belongs to the Alphaproteobacteria, a phylogenetic group comprising bacteria with free living, symbiotic, and pathogenic lifestyles. An essential regulator of cell cycle progression named CtrA was described in the model bacterium Caulobacter crescentus. This regulator is conserved in many alphaproteobacteria, but the evolution of its regulon remains elusive. Here we identified promoters that are CtrA targets using ChIP‐seq and we found that CtrA binds to promoters of genes involved in cell cycle progression, in addition to numerous genes encoding outer membrane components involved in export of membrane proteins and synthesis of lipopolysaccharide. Analysis of a conditional B. abortus ctrA loss of function mutant confirmed that CtrA controls cell division. Impairment of cell division generates elongated and branched morphologies, that are also detectable inside HeLa cells. Surprisingly, abnormal bacteria are able to traffic to the endoplasmic reticulum, the usual replication niche of B. abortus in host cells. We also found that CtrA depletion affected outer membrane composition, in particular the abundance and spatial distribution of Omp25. Control of the B. abortus envelope composition by CtrA indicates the plasticity of the CtrA regulon along evolution.     

Bilobalide, a component of the Ginkgo biloba extract (EGb 761), protects against neuronal death in global brain ischemia and in glutamate-induced excitotoxicity.

In this study, the effect of bilobalide, a purified terpene lactone component of the Ginkgo biloba extract (EGb 761), and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death was compared. In the case of ischemic injury, neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in the hippocampal regions of gerbils was measured. A significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons at 7-days of reperfusion after 5 min of transient global forebrain ischemia. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected hippocampal CA1 neurons against ischemia-induced neuronal death and reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death [effective concentration (EC50) = 5 microg/ml (12 microM) forbilobalide and 100 microg/ml for EGb 761]. These results suggest thatboth EGb 761 and bilobalide protect against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.     

Impact of the introduced yellow crazy ant Anoplolepis gracilipes on Bird Island, Seychelles

The introduced yellow crazy ant or long-legged ant Anoplolepis gracilipes was first reported in Seychelles in 1969 and now occurs on at least nine islands in the Central Seychelles. We describe the yellow crazy ant's effects on vegetation and invertebrate communities on one of these, Bird Island; in 2000, Anoplolepis (first reported in 1991) occurred there at densities at least 80 times higher than on other islands in the Central Seychelles. They were associated with high densities of coccid scale insects on foliage, especially of the native tree Pisonia grandis, in some instances causing tree death. Yellow crazy ants on Bird Island also significantly affected invertebrate communities on foliage and on the ground, both in terms of taxonomic composition and the density of specific taxa, apparently causing the local exclusion of some invertebrates.     

Greenhouse and field assessment of phytoremediation for petroleum contaminants in a riparian zone

Greenhouse and field studies were conducted to evaluate the feasibility of phytoremediation for clean-up of highly contaminated sediments from Indiana Harbor. In the greenhouse study, plant species evaluated were willow (Salix exigua), poplar (Populus spp.), eastern gamagrass (Tripsacum dactyloides), arrowhead (Sagitaria latifolia), switchgrass (Panicum virgatum), and sedge (Carex stricta). Sediments with sedge, switchgrass, and gamagrass had significantly less residual total petroleum hydrocarbons (TPH) after one year of growth (approximately 70% reduction) than sediments containing willow, poplar, or no plants (approximately 20% reduction). Although not all polycyclic aromatic hydrocarbons (PAH) had concentration differences due to the presence of plants, residual pyrene concentrations in the unvegetated pots were significantly higher than in pots containing sedge, switchgrass, arrowhead, and gamagrass. As evaluated by TPH dissipation in the upper section of the pots, the sedge, switchgrass, and gamagrass treatments had higher TPH degradation than the unvegetated, willow and poplar treatments. These trends were similar for soil at the bottom of the pots, with the exception that in the switchgrass treatment, degradation was not significantly different than in the unvegetated soil. Two target contaminants, pyrene and benzo[b]fluoranthene, showed differences in degradation between planted and unvegetated treatments. In the field study, phytoremediation plant species were eastern gamagrass (T. dactyloides), switchgrass (P. virgatum), and sedge (C. stricta). In addition, rhizosphere characteristics of arrowhead (S. latifolia) and sedge were assessed. Arrowhead- and sedge-impacted soils were found to contain significantly more PAH-degrading bacteria than unvegetated soils. However, over the 12-month field study, no significant differences in contamination were found between the planted and unplanted soils for TPH and PAH concentrations. TPH concentrations near the canal were greater than concentrations further from the canal, indicating that the canal may have served as a continuous source of contamination during the study.     

Seasonal and inter-annual drivers of yellow fever transmission in South America

In the last 20 years yellow fever (YF) has seen dramatic changes to its incidence and geographic extent, with the largest outbreaks in South America since 1940 occurring in the previously unaffected South-East Atlantic coast of Brazil in 2016–2019. While habitat fragmentation and land-cover have previously been implicated in zoonotic disease, their role in YF has not yet been examined. We examined the extent to which vegetation, land-cover, climate and host population predicted the numbers of months a location reported YF per year and by each month over the time-period. Two sets of models were assessed, one looking at interannual differences over the study period (2003–2016), and a seasonal model looking at intra-annual differences by month, averaging over the years of the study period. Each was fit using hierarchical negative-binomial regression in an exhaustive model fitting process. Within each set, the best performing models, as measured by the Akaike Information Criterion (AIC), were combined to create ensemble models to describe interannual and seasonal variation in YF. The models reproduced the spatiotemporal heterogeneities in YF transmission with coefficient of determination (R²) values of 0.43 (95% CI 0.41–0.45) for the interannual model and 0.66 (95% CI 0.64–0.67) for the seasonal model. For the interannual model, EVI, land-cover and vegetation heterogeneity were the primary contributors to the variance explained by the model, and for the seasonal model, EVI, day temperature and rainfall amplitude. Our models explain much of the spatiotemporal variation in YF in South America, both seasonally and across the period 2003–2016. Vegetation type (EVI), heterogeneity in vegetation (perhaps a proxy for habitat fragmentation) and land cover explain much of the trends in YF transmission seen. These findings may help understand the recent expansions of the YF endemic zone, as well as to the highly seasonal nature of YF.     

Beneficial mutations for carotenoid production identified from laboratory-evolved Saccharomyces cerevisiae

Journal of Industrial Microbiology & Biotechnology - Adaptive laboratory evolution (ALE) is a powerful tool used to increase strain fitness in the presence of environmental stressors. If...