Testing for Starch,Respiring Tissues,and Vascular Bundles: Inquiry-Based Seed and Stem Anatomy Labs

In this article, the authors present lab exercises in which students use the microscope to study cells. After learning how to recognize the parts of a cell, differentiate between types of cells, and identify the nucleus, the students study blood samples from both familiar and “unknown” animals. The activities allow students to work independently and to become more proficient at microscope use. Students also gain a deeper understanding of the more “invisible” world of science.     

Stratifying type 2 diabetes cases by BMI identifies genetic risk variants in LAMA1 and enrichment for risk variants in lean compared to obese cases

Common diseases such as type 2 diabetes are phenotypically heterogeneous. Obesity is a major risk factor for type 2 diabetes, but patients vary appreciably in body mass index. We hypothesized that the genetic predisposition to the disease may be different in lean (BMI<25 Kg/m²) compared to obese cases (BMI≥30 Kg/m²). We performed two case-control genome-wide studies using two accepted cut-offs for defining individuals as overweight or obese. We used 2,112 lean type 2 diabetes cases (BMI<25 kg/m²) or 4,123 obese cases (BMI≥30 kg/m²), and 54,412 un-stratified controls. Replication was performed in 2,881 lean cases or 8,702 obese cases, and 18,957 un-stratified controls. To assess the effects of known signals, we tested the individual and combined effects of SNPs representing 36 type 2 diabetes loci. After combining data from discovery and replication datasets, we identified two signals not previously reported in Europeans. A variant (rs8090011) in the LAMA1 gene was associated with type 2 diabetes in lean cases (P = 8.4×10⁻⁹, OR = 1.13 [95% CI 1.09–1.18]), and this association was stronger than that in obese cases (P = 0.04, OR = 1.03 [95% CI 1.00–1.06]). A variant in HMG20A—previously identified in South Asians but not Europeans—was associated with type 2 diabetes in obese cases (P = 1.3×10⁻⁸, OR = 1.11 [95% CI 1.07–1.15]), although this association was not significantly stronger than that in lean cases (P = 0.02, OR = 1.09 [95% CI 1.02–1.17]). For 36 known type 2 diabetes loci, 29 had a larger odds ratio in the lean compared to obese (binomial P = 0.0002). In the lean analysis, we observed a weighted per-risk allele OR = 1.13 [95% CI 1.10–1.17], P = 3.2×10⁻¹⁴. This was larger than the same model fitted in the obese analysis where the OR = 1.06 [95% CI 1.05–1.08], P = 2.2×10⁻¹⁶. This study provides evidence that stratification of type 2 diabetes cases by BMI may help identify additional risk variants and that lean cases may have a stronger genetic predisposition to type 2 diabetes.     

Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. The lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and CHIKV-host interactions. Infectious cDNA clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. Existing CHIKV cDNA clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. To obtain a virus expected to have the general characteristics of the recent E1-226V CHIKV isolates, we have constructed a new CHIKV full-length cDNA clone, CHIKV LS3, based on the consensus sequence of their aligned genomes. Here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (CHIKV LS3-GFP). Their characteristics were compared to those of natural strain ITA07-RA1, which was isolated during the 2007 outbreak in Italy. In cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. Compared to ITA07-RA1 and clinical isolate NL10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of CHIKV replication. Cyclosporin A had no effect on CHIKV replication, suggesting that cyclophilins -opposite to what was found for other +RNA viruses- do not play an essential role in CHIKV replication. The characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that CHIKV LS3 and LS3-GFP are suitable and representative tools to study CHIKV-host interactions, screen for antiviral compounds and unravel their mode of action.     

A Translocated Effector Required for Bartonella Dissemination from Derma to Blood Safeguards Migratory Host Cells from Damage by Co-translocated Effectors

Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepE_Bhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepE_Bhe (BID2.E_Bhe). Ectopic expression of BID2.E_Bhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepE_Btr, BepE_Bhe or BIDs.E_Bhe restored bacteremia. Given that we observed a similar protective effect of BepE_Bhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that infected dermal dendritic cells may be involved in disseminating Bartonella towards the blood stream in a BepE-dependent manner.     

Diversity of Bacterial Communities in a Profile of a Winter Wheat Field: Known and Unknown Members

In soils, bacteria are very abundant and diverse. They are involved in various agro-ecosystem processes such as the nitrogen cycle, organic matter degradation, and soil formation. Yet, little is known about the distribution and composition of bacterial communities through the soil profile, particularly in agricultural soils, as most studies have focused only on topsoils or forest and grassland soils. In the present work, we have used bar-coded pyrosequencing analysis of the V3 region of the 16S rRNA gene to analyze bacterial diversity in a profile (depths 10, 25, and 45 cm) of a well-characterized field of winter wheat. Taxonomic assignment was carried out with the Ribosomal Database Project (RDP) Classifier program with three bootstrap scores: a main run at 0.80, a confirmation run at 0.99, and a run at 0 to gain information on the unknown bacteria. Our results show that biomass and bacterial quantity and diversity decreased greatly with depth. Depth also had an impact, in terms of relative sequence abundance, on 81 % of the most represented taxonomic ranks, notably the ranks Proteobacteria, Bacteroidetes, Actinobacteridae, and Acidobacteria. Bacterial community composition differed more strongly between the topsoil (10 and 25 cm) and subsoil (45 cm) than between levels in the topsoil, mainly because of shifts in the carbon, nitrogen, and potassium contents. The subsoil also contained more unknown bacteria, 53.96 % on the average, than did the topsoil, with 42.06 % at 10 cm and 45.59 % at 25 cm. Most of these unknown bacteria seem to belong to Deltaproteobacteria, Actinobacteria, Rhizobiales, and Acidobacteria.     

Carbohydrate control over carotenoid build‐up is conditional on fruit ontogeny in clementine fruits

The final contents of primary and secondary metabolites of the ripe fruit depend on metabolic processes that are tightly regulated during fruit ontogeny. Carbohydrate supply during fruit development is known to influence these processes but, with respect to secondary metabolites, we do not really know whether this influence is direct or indirect. Here, we hypothesized that the sensitivity of clementine fruit metabolism to carbohydrate supply was conditional on fruit developmental stage. We applied treatments increasing fruit load reversibly or irreversibly at three key stages of clementine (Citrus clementina Hort. ex Tan.) fruit development: early after cell division, at the onset of fruit coloration (color break) and near maturity. The highest fruit load obtained by early defoliation (irreversible) had the highest impact on fruit growth, maturity and metabolism, followed by the highest fruit load obtained by early shading (reversible). Final fruit size decreased by 21 and 18% in these early irreversible and reversible treatments, respectively. Soluble sugars decreased by 18% in the early irreversible treatment, whereas organic acids increased by 46 and 29% in these early irreversible and reversible treatments, respectively. Interestingly, total carotenoids increased by 50 and 18%, respectively. Changes in leaf starch content and photosynthesis supported that these early treatments triggered a carbon starvation in the young fruits, with irreversible effects. Furthermore, our observations on the early treatments challenge the common view that carbohydrate supply influences positively carotenoid accumulation in fruits. We propose that early carbon starvation irreversibly promotes carotenoid accumulation.     

A horse's eye view: size and shape discrimination compared with other mammals

Mammals have adapted to a variety of natural environments from underwater to aerial and these different adaptations have affected their specific perceptive and cognitive abilities. This study used a computer-controlled touchscreen system to examine the visual discrimination abilities of horses, particularly regarding size and shape, and compared the results with those from chimpanzee, human and dolphin studies. Horses were able to discriminate a difference of 14% in circle size but showed worse discrimination thresholds than chimpanzees and humans; these differences cannot be explained by visual acuity. Furthermore, the present findings indicate that all species use length cues rather than area cues to discriminate size. In terms of shape discrimination, horses exhibited perceptual similarities among shapes with curvatures, vertical/horizontal lines and diagonal lines, and the relative contributions of each feature to perceptual similarity in horses differed from those for chimpanzees, humans and dolphins. Horses pay more attention to local components than to global shapes.     

Live-cell imaging reveals a stable cohesin-chromatin interaction after but not before DNA replication

Cohesin is a multisubunit protein complex that links sister chromatids from replication until segregation. The lack of obvious cohesin-targeting-specific sequences on DNA, as well as cohesin's molecular arrangement as a large ring, has led to the working hypothesis that cohesin acts as a direct topological linker. To preserve the identity of sister chromatids, such a linkage would need to stably persist throughout the entire S and G2 phases of the cell cycle. Unexpectedly, cohesin binds chromatin already in telophase, and a large fraction dissociates from chromosomes during prophase in a phosphorylation-dependent manner, whereas initiation of anaphase requires proteolytic cleavage of only a small fraction of cohesin. These observations raised the question of how and when cohesin interacts with chromatin during the cell cycle. Here, we report a cell-cycle dependence in the stability of cohesin binding to chromatin. Using photobleaching and quantitative live-cell imaging, we identified several cohesin pools with different chromatin binding stabilities. Although all chromatin bound cohesin dissociated after a mean residence time of less than 25 min before replication, about one-third of cohesin was bound much more stably after S phase and persisted until metaphase, consistent with long-lived links mediating cohesion between sister chromatids.