A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

The acoustic startle response is an evolutionarily conserved avoidance behavior. Disruptions in startle behavior, particularly startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. We uncovered mutations in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 and Dolk play critical roles in the spinal cord to regulate movement magnitude during the startle response and spontaneous swim movements. Moreover, we show that Kv1.1 protein is mislocalized in dolk mutants, suggesting they act in a common genetic pathway. Combined, our results identify a diverse set of eight genes, all associated with human disorders, that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway.     

Synthetic Aurones: New Features for Schistosoma mansoni Therapy

In this work, two synthetic aurones revealed moderate schistosomicidal potential in in vitro and in vivo assays. Aurones ( 1 ) and ( 2 ) promoted changes in tegument integrity and motor activity, leading to death of adult Schistosoma mansoni worms in in vitro assays. When administered orally (two doses of 50 mg/kg) in experimentally infected animals, synthetic aurones ( 1 ) and ( 2 ) promoted reductions of 56.20 % and 57.61 % of the parasite load and stimulated the displacement towards the liver of the remaining adult worms. The oogram analysis revealed that the treatment with both aurones interferes with the egg development kinetics in the intestinal tissue. Seeking an action target for compounds ( 1 ) and ( 2 ), the connection with NTPDases enzymes, recognized as important therapeutic targets for S. mansoni, was evaluated. Molecular docking studies have shown promising results. The dataset reveals the anthelmintic character of these compounds, which can be used in the development of new therapies for schistosomiasis.     

Post-Glacial Climate–Fire Interactions Control Tree Composition of Mesic Temperate Forests in Eastern North America

Stand-scale gap-phase dynamics is generally viewed as the main driver of development in mesic deciduous forests of the temperate biome. Soil charcoal of temperate forests in eastern North America are unnoticed in most surveys, thus explaining why fire is undervalued as a driver of forest succession. The extent to which gap-phase, fire, or other processes are responsible for the regeneration and maintenance of mesic deciduous forests is unknown because paleoecological evidence is lacking. We tested the fire-driven succession hypothesis on the development of this major forest type. Based on charcoal ¹⁴C dates of two sites, 44 and 55 fires occurred since early Holocene, with a mean interval of 170 to 215 years. The vegetation of both sites followed comparable post-glacial trajectories consisting of three distinct periods. Conifers dominated the two first periods during 5200–6000 years and were replaced by hardwoods–conifers over the last 3500 years. The first period was represented by boreal conifers, whereas the second period, dominated by white pine (Pinus strobus) forests, persisted during 3000–4300 years. The third period marked the development of hardwood (sugar maple, Acer saccharum) forests. Fires occurred continuously on the sites since early Holocene likely under dry conditions during the conifer periods and cooler and moister conditions during the hardwood–conifer period. Recurrent fires appear with climate as key drivers of the long-term dynamics of several temperate forests in eastern North America. Similar studies on other temperate forests should be pursued to test the hypothesis of climate–fire interactions influencing tree composition change.

     

Azole-resistant Aspergillus fumigatus is highly prevalent in the environment of Vietnam,with marked variability by land use type

Azole-resistant environmental Aspergillus fumigatus presents a threat to public health but the extent of this threat in Southeast Asia is poorly described. We conducted environmental surveillance in the Mekong Delta region of Vietnam, collecting air and ground samples across key land-use types, and determined antifungal susceptibilities of Aspergillus section Fumigati (ASF) isolates and azole concentrations in soils. Of 119 ASF isolates, 55% were resistant (or non-wild type) to itraconazole, 65% to posaconazole and 50% to voriconazole. Azole resistance was more frequent in A. fumigatus sensu stricto isolates (95%) than other ASF species (32%). Resistant isolates and agricultural azole residues were overrepresented in samples from cultivated land. cyp51A gene sequence analysis showed 38/56 resistant A. fumigatus sensu stricto isolates carried known resistance mutations, with TR₃₄/L98H most frequent (34/38).     

Arginine substitution by alanine at the P1 position increases the selectivity of CmPI-II,a non-classical Kazal inhibitor

CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while improving/maintaining SUBTA and elastases inhibition. Thus, an alanine mutant of this position (rCmPI-II R12A) was obtained by site-directed mutagenesis. The gene cmpiR12A was expressed in P. pastoris KM71H yeast. The recombinant protein (rCmPI-II R12A) was purified by the combination of two ionic exchange chromatography (1:cationic, 2 anionic) followed by and size exclusion chromatography. The N-terminal sequence obtained as well as the experimental molecular weight allowed verifying the identity of the recombinant protein, while the correct folding was confirmed by CD experiments. rCmPI-II R12A shows a slightly increase in potency against SUBTA and elastases. The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition, confirming the relevance of an arginine residue at P1 site in CmPI-II for trypsin inhibition and leading to a molecule with more potentialities in biomedicine.     

A new unique form of microRNA from human heart,microRNA-499c,promotes myofibril formation and rescues cardiac development in mutant axolotl embryos

Background

A recessive mutation “c” in the Mexican axolotl, Ambystoma mexicanum, results in the failure of normal heart development. In homozygous recessive embryos, the hearts do not have organized myofibrils and fail to beat. In our previous studies, we identified a noncoding Myofibril-Inducing RNA (MIR) from axolotls which promotes myofibril formation and rescues heart development.

Results

We randomly cloned RNAs from fetal human heart. RNA from clone #291 promoted myofibril formation and induced heart development of mutant axolotls in organ culture. This RNA induced expression of cardiac markers in mutant hearts: tropomyosin, troponin and α-syntrophin. This cloned RNA matches in partial sequence alignment to human microRNA-499a and b, although it differs in length. We have concluded that this cloned RNA is unique in its length, but is still related to the microRNA-499 family. We have named this unique RNA, microRNA-499c. Thus, we will refer to this RNA derived from clone #291 as microRNA-499c throughout the rest of the paper.

Conclusions

This new form, microRNA-499c, plays an important role in cardiac development.     

A Developmental Framework for Complex Plasmodesmata Formation Revealed by Large-Scale Imaging of the Arabidopsis Leaf Epidermis

Plasmodesmata (PD) form tubular connections that function as intercellular communication channels. They are essential for transporting nutrients and for coordinating development. During cytokinesis, simple PDs are inserted into the developing cell plate, while during wall extension, more complex (branched) forms of PD are laid down. We show that complex PDs are derived from existing simple PDs in a pattern that is accelerated when leaves undergo the sink–source transition. Complex PDs are inserted initially at the three-way junctions between epidermal cells but develop most rapidly in the anisocytic complexes around stomata. For a quantitative analysis of complex PD formation, we established a high-throughput imaging platform and constructed PDQUANT, a custom algorithm that detected cell boundaries and PD numbers in different wall faces. For anticlinal walls, the number of complex PDs increased with increasing cell size, while for periclinal walls, the number of PDs decreased. Complex PD insertion was accelerated by up to threefold in response to salicylic acid treatment and challenges with mannitol. In a single 30-min run, we could derive data for up to 11k PDs from 3k epidermal cells. This facile approach opens the door to a large-scale analysis of the endogenous and exogenous factors that influence PD formation.     

The Use of Ice to Enrich the Environment of Pigs Housed Indoors

Pigs used in research are often housed in barren environments. The effects of ice as a simple enrichment tool for newly weaned pigs were investigated. Four replicates of 120 pigs were separated into 3 groups. One group was given free access to blocks of ice (ice group), another group had access to Classic Kong toys (Kong group), and a 3rd group did not receive any enrichment (control group). The behavior of each group was observed every 5 min from 08:00 hr to 12:00 hr during 4 consecutive days. Pigs were motivated to explore the ice blocks (4.85% ± 1.34) over the Classic Kong toys (2.03% ± 0.59). No differences in other behaviors were found between treatments. Ice is an effective and easy-to-replace enrichment device. Blocks of ice can be used as enrichment devices for pigs housed in research facilities.     

Dog Obesity: Can Dog Caregivers' (Owners') Feeding and Exercise Intentions and Behaviors Be Predicted From Attitudes?

Dog obesity is a common nutritional disorder affecting up to 40% of the companion animal (pet) dog population in Australia and other developed nations. A clear understanding of factors determining relevant caregiver (owner) behaviors underpins effective treatment for this disorder. The theory of planned behavior can be used to understand factors contributing to human behavior. This article describes research informed by this theory. The research examined relationships between owners' behavioral beliefs and barriers, normative beliefs and perceptions of control, owners' feeding and exercise behaviors toward their dogs, and the body condition scores (BCSs) of dogs. The study recruited a sample of 182 dog and owner dyads. The researcher independently assessed BCSs. Owners completed a questionnaire measuring relevant feeding and exercise beliefs and behaviors. This revealed significant correlations between many psychological variables and BCSs and between psychological variables and specific owner behaviors: for example, the relationship of low levels of intentions to feed appropriately to ambivalent beliefs toward feeding appropriately and low perceived control. Careful consideration of the specific variables identified will permit the development of more effective interventions.