Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria

Planarians have been a classic model system for the study of regeneration, tissue homeostasis, and stem cell biology for over a century, but they have not historically been accessible to extensive genetic manipulation. Here we utilize RNA-mediated genetic interference (RNAi) to introduce large-scale gene inhibition studies to the classic planarian system. 1065 genes were screened. Phenotypes associated with the RNAi of 240 genes identify many specific defects in the process of regeneration and define the major categories of defects planarians display following gene perturbations. We assessed the effects of inhibiting genes with RNAi on tissue homeostasis in intact animals and stem cell (neoblast) proliferation in amputated animals identifying candidate stem cell, regeneration, and homeostasis regulators. Our study demonstrates the great potential of RNAi for the systematic exploration of gene function in understudied organisms and establishes planarians as a powerful model for the molecular genetic study of stem cells, regeneration, and tissue homeostasis.     

Killed Saccharomyces cerevisiae Protects Against Lethal Challenge of Cryptococcus grubii

Heat-killed Saccharomyces cerevisiae (HKY) vaccination protects mice against aspergillosis, coccidioidomycosis, mucormycosis, or candidiasis. We studied HKY protection against murine cryptococcosis. Once weekly subcutaneous HKY doses (S, 6 × 10⁷; 2S, 1.2 × 10⁸; 3S, 2.4 × 10⁸) began 28 (×3), 35 (×4), or 42 (×6) days prior to intravenous Cryptococcus grubii infection. Survival through 28 days, and CFU in the organs of survivors, were compared to saline-vaccinated controls. In the initial experiment, S, S×4, or 2S reduced brain CFU; liver or spleen CFU was reduced by S×4 or 2S. In a more lethal second experiment, 2S×6, 2S, or 3S×4 improved survival, and HKY regimens reduced CFU in the brain, liver, or spleen, with 2S×6, 2S, or 3S×4 most efficacious. Dose size appears more important than the number of doses: Regimens >S were superior, and 2S and 2S×6 were equivalent. 2S and 3S were equivalent, suggesting doses >2S do not provide additional protection. HKY protects against Cryptococcus, supporting components of HKY as a basis for the development of a panfungal vaccine.     

Calcium signaling in Parkinson’s disease

Calcium (Ca²⁺) is an almost universal second messenger that regulates important activities of all eukaryotic cells. It is of critical importance to neurons, which have developed extensive and intricate pathways to couple the Ca²⁺ signal to their biochemical machinery. In particular, Ca²⁺ participates in the transmission of the depolarizing signal and contributes to synaptic activity. During aging and in neurodegenerative disease processes, the ability of neurons to maintain an adequate energy level can be compromised, thus impacting on Ca²⁺ homeostasis. In Parkinson’s disease (PD), many signs of neurodegeneration result from compromised mitochondrial function attributable to specific effects of toxins on the mitochondrial respiratory chain and/or to genetic mutations. Despite these effects being present in almost all cell types, a distinguishing feature of PD is the extreme selectivity of cell loss, which is restricted to the dopaminergic neurons in the ventral portion of the substantia nigra pars compacta. Many hypotheses have been proposed to explain such selectivity, but only recently it has been convincingly shown that the innate autonomous activity of these neurons, which is sustained by their specific Cav1.3 L-type channel pore-forming subunit, is responsible for the generation of basal metabolic stress that, under physiological conditions, is compensated by mitochondrial buffering. However, when mitochondria function becomes even partially compromised (because of aging, exposure to environmental factors or genetic mutations), the metabolic stress overwhelms the protective mechanisms, and the process of neurodegeneration is engaged. The characteristics of Ca²⁺ handling in neurons of the substantia nigra pars compacta and the possible involvement of PD-related proteins in the control of Ca²⁺ homeostasis will be discussed in this review.     

Effects of seed burial on germination,protein mobilisation and seedling survival in Dodonaea viscosa

Ecological restoration of disturbed areas requires substantial knowledge of the germination of native plants and the creation of novel methods to increase seedling establishment in the field. We studied the effects of soil matrix priming on the germination of Dodonaea viscosa seeds, which exhibit physical dormancy. To this end, we buried both pre‐scarified (in H₂SO₄, 3 min) and non‐pre‐scarified seeds in the Parque Ecológico de la Ciudad de México. After seeds were unearthed, they were post‐scarified for 0, 2, 6 and 10 min and their germination percentages compared to the germination of a control batch of laboratory‐stored seeds. For both control and unearthed seeds, the protein pattern was determined in the enriched storage protein fraction in SDS‐PAGE gels stained with Coomassie blue. Percentage germination increased as the scarification time increased. Pre‐scarification significantly increased percentage germination of post‐scarified seeds in relation to the control and non‐pre‐scarified seeds. In seeds unearthed from the forest site, the buried pre‐scarified seeds had relatively high percentage germination, even in the absence of post‐scarification treatment. A 48‐kDa protein was not found in unearthed, pre‐scarified seeds nor in the control germinated seeds, indicating that mobilisation of this protein occurred during soil priming. Burying seeds for a short period, including the beginning of the rainy season, promoted natural priming, which increased protein mobilisation. Functionally, priming effects were reflected in high percentage seedling survival in both the shade house and the field. Seed burial also reduced the requirement for acidic post‐scarification.     

The Lack of Side Effects of an Ineffective Treatment Facilitates the Development of a Belief in Its Effectiveness

Some alternative medicines enjoy widespread use, and in certain situations are preferred over conventional, validated treatments in spite of the fact that they fail to prove effective when tested scientifically. We propose that the causal illusion, a basic cognitive bias, underlies the belief in the effectiveness of bogus treatments. Therefore, the variables that modulate the former might affect the latter. For example, it is well known that the illusion is boosted when a potential cause occurs with high probability. In this study, we examined the effect of this variable in a fictitious medical scenario. First, we showed that people used a fictitious medicine (i.e., a potential cause of remission) more often when they thought it caused no side effects. Second, the more often they used the medicine, the more likely they were to develop an illusory belief in its effectiveness, despite the fact that it was actually useless. This behavior may be parallel to actual pseudomedicine usage; that because a treatment is thought to be harmless, it is used with high frequency, hence the overestimation of its effectiveness in treating diseases with a high rate of spontaneous relief. This study helps shed light on the motivations spurring the widespread preference of pseudomedicines over scientific medicines. This is a valuable first step toward the development of scientifically validated strategies to counteract the impact of pseudomedicine on society.     

Measuring Software Timing Errors in the Presentation of Visual Stimuli in Cognitive Neuroscience Experiments

Because of the features provided by an abundance of specialized experimental software packages, personal computers have become prominent and powerful tools in cognitive research. Most of these programs have mechanisms to control the precision and accuracy with which visual stimuli are presented as well as the response times. However, external factors, often related to the technology used to display the visual information, can have a noticeable impact on the actual performance and may be easily overlooked by researchers. The aim of this study is to measure the precision and accuracy of the timing mechanisms of some of the most popular software packages used in a typical laboratory scenario in order to assess whether presentation times configured by researchers do not differ from measured times more than what is expected due to the hardware limitations. Despite the apparent precision and accuracy of the results, important issues related to timing setups in the presentation of visual stimuli were found, and they should be taken into account by researchers in their experiments.     

Regional and segmental differences in the embryonic expression of a putative leech Hox gene,Lox2, by central neurons immunoreactive to FMRFamide-like neuropeptides

We performed immunofluorescence experiments using a rat polyclonal antibody on formaldehyde-fixed whole-mount embryos to characterize the expression of a putative leech Hox gene, Lox2, during embryonic development. The main goal was to determine whether the differentiation of subsets of FMRFamide-like immunoreactive (FLI) neurons coincide with the expression domain of Lox2. The earliest expression of Lox2 was detected in relatively large, prominent nuclei in the posterior region at embryonic day 4, a very early stage. Lox2 expression was also detected in subsets of central neurons (neurons located in the CNS) located in midbody ganglia 6 (M6)–M21. In addition, Lox2 was expressed by a number of segment-specific and segmentally repeated central FLI neurons. Lox2-positive FLI neurons of interest included some of those previously identified: the rostral most ventral (RMV) neurons, the circular ventral (CV) neurons, and cell 261. The paired RMVs, which are located in all midbody ganglia, expressed Lox2 only in M7–M19. The CV neurons, specialized motor neurons that innervate the circular ventral muscles of the body wall, expressed Lox2 in M7–M19. The putative cell 261 expressed Lox2 in M7–M12, where Lox1 is also expressed. FMRFamide staining in putative segmental homologs of cell 261 was not detected in other segmental ganglia. Our results suggest a role for Lox2 in very early embryonic development (before the formation of the CNS), and in the differentiation of segmentally repeated and region-specific FLI neurons.     

Agrobacterium-mediated transformation of African violet

A method for genetic transformation of Saintpaulia ionantha by co-cultivation of in vitro-grown leaves and petioles with Agrobacterium tumefaciens is described. Two bacterial strains, EHA105 and A281 both harbouring the binary plasmid pKIWI105 carrying the genes uidA and nptII, were used in the experiments. Regenerants were not obtained using the disarmed strain EHA105. The oncogenic strain A281 resulted in efficient transient and stable expression of the transferred traits for petiole explants only. After transformation and regeneration, the integration of the transgenes in the plant genome was confirmed by PCR analysis and Southern hybridization.     

Mapping quantitative trait loci for root development under hypoxia conditions in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Key message

Greatest potential, QTLs for hypoxia and waterlogging tolerance in soybean roots were detected using a new phenotypic evaluation method.

Abstract

Waterlogging is a major environmental stress limiting soybean yield in wet parts of the world. Root development is an important indicator of hypoxia tolerance in soybean. However, little is known about the genetic control of root development under hypoxia. This study was conducted to identify quantitative trait loci (QTLs) responsible for root development under hypoxia. Recombinant inbred lines (RILs) developed from a cross between a hypoxia-sensitive cultivar, Tachinagaha, and a tolerant landrace, Iyodaizu, were used. Seedlings were subjected to hypoxia, and root development was evaluated with the value change in root traits between after and before treatments. We found 230 polymorphic markers spanning 2519.2 cM distributed on all 20 chromosomes (Chrs.). Using these, we found 11 QTLs for root length (RL), root length development (RLD), root surface area (RSA), root surface area development (RSAD), root diameter (RD), and change in average root diameter (CARD) on Chrs. 11, 12, 13 and 14, and 7 QTLs for hypoxia tolerance of these root traits. These included QTLs for RLD and RSAD between markers Satt052 and Satt302 on Chr. 12, which are important markers of hypoxia tolerance in soybean; those QTLs were stable between 2 years. To validate the QTLs, we developed a near-isogenic line with the QTL region derived from Iyodaizu. The line performed well under both hypoxia and waterlogging, suggesting that the region contains one or more genes with large effects on root development. These findings may be useful for fine mapping and positional cloning of gene responsible for root development under hypoxia.