A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.     

Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca²⁺ permeant cation channels (MscCa) and large conductance (200 pS) Ca²⁺-sensitive K⁺ (BK_Ca2+) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK_Ca2+ channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca²⁺-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca²⁺] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.     

Glasgow Coma Scale and Outcomes after Structural Traumatic Head Injury in Early Childhood

Objective

To assess the association of the Glasgow Coma Scale (GCS) with radiological evidence of head injury (the Abbreviated Injury Scale for the head region, AIS-HR) in young children hospitalized with traumatic head injury (THI), and the predictive value of GCS and AIS-HR scores for long-term impairment.

Methods

Our study involved a 10-year retrospective review of a database encompassing all patients admitted to Starship Children’s Hospital (Auckland, New Zealand, 2000–2010) with THI.

Results

We studied 619 children aged <5 years at the time of THI, with long-term outcome data available for 161 subjects. Both GCS and AIS-HR scores were predictive of length of intensive care unit and hospital stay (all p<0.001). GCS was correlated with AIS-HR (ρ=-0.46; p<0.001), although mild GCS scores (13–15) commonly under-estimated the severity of radiological injury: 42% of children with mild GCS scores had serious–critical THI (AIS-HR 3–5). Increasingly severe GCS or AIS-HR scores were both associated with a greater likelihood of long-term impairment (neurological disability, residual problems, and educational support). However, long-term impairment was also relatively common in children with mild GCS scores paired with structural THI more severe than a simple linear skull fracture.

Conclusion

Severe GCS scores will identify most cases of severe radiological injury in early childhood, and are good predictors of poor long-term outcome. However, young children admitted to hospital with structural THI and mild GCS scores have an appreciable risk of long-term disability, and also warrant long-term follow-up.     

Sex and differentiation: population genetic divergence and sexual dimorphism in Mexican goodeid fish

Genetic differentiation arises due to the interaction between natural and sexual selection, migration and genetic drift. A potential role of sexual selection in speciation has received much interest, although comparative studies are inconsistent in finding supporting evidence. A poorly tested prediction is that species subject to a higher intensity of sexual selection should show greater genetic differentiation amongst populations because females from these populations should be more choosy in mate choice. The Goodeinae is a group of endemic Mexican fishes in which female choice has driven some species to be morphologically sexually dimorphic, whereas others are relatively monomorphic. Here, we measured population divergence, using microsatellite loci, within four goodeid species which show contrasting levels of sexual dimorphism. We found higher levels of differentiation between populations of the more dimorphic species, implying less gene flow between populations. We also found evidence of higher levels of genetic differences between the sexes within populations of the dimorphic species, consistent with greater dispersal in males. Adjusted for geographic distance, the mean F(ST) for the dimorphic species is 0.25 compared with 0.16 for the less dimorphic species. We conclude that population differentiation is accelerated in more sexually dimorphic species, and that comparative phylogeography may provide a more powerful approach to detecting processes, such as an influence of sexual selection on differentiation, than broad-scale comparative studies.     

Multi-segment foot kinematics and ground reaction forces during gait of individuals with plantar fasciitis

Background

Clinically, plantar fasciitis (PF) is believed to be a result and/or prolonged by overpronation and excessive loading, but there is little biomechanical data to support this assertion. The purpose of this study was to determine the differences between healthy individuals and those with PF in (1) rearfoot motion, (2) medial forefoot motion, (3) first metatarsal phalangeal joint (FMPJ) motion, and (4) ground reaction forces (GRF).

Methods

We recruited healthy (n=22) and chronic PF individuals (n=22, symptomatic over three months) of similar age, height, weight, and foot shape (p>0.05). Retro-reflective skin markers were fixed according to a multi-segment foot and shank model. Ground reaction forces and three dimensional kinematics of the shank, rearfoot, medial forefoot, and hallux segment were captured as individuals walked at 1.35 ms⁻¹.

Results

Despite similarities in foot anthropometrics, when compared to healthy individuals, individuals with PF exhibited significantly (p<0.05) (1) greater total rearfoot eversion, (2) greater forefoot plantar flexion at initial contact, (3) greater total sagittal plane forefoot motion, (4) greater maximum FMPJ dorsiflexion, and (5) decreased vertical GRF during propulsion.

Conclusion

These data suggest that compared to healthy individuals, individuals with PF exhibit significant differences in foot kinematics and kinetics. Consistent with the theoretical injury mechanisms of PF, we found these individuals to have greater total rearfoot eversion and peak FMPJ dorsiflexion, which may put undue loads on the plantar fascia. Meanwhile, increased medial forefoot plantar flexion at initial contact and decreased propulsive GRF are suggestive of compensatory responses, perhaps to manage pain.     

Orthoses posted in both the forefoot and rearfoot reduce moments and angular impulses on lower extremity joints during walking

The purpose of the present study was to determine the effects of orthoses designed to support the forefoot and rearfoot on the kinematics and kinetics of the lower extremity joints during walking. Fifteen participants volunteered for this study. Kinematic and kinetic variables during overground walking were compared with the participants wearing sandals without orthoses or sandals with orthoses. Orthoses increased knee internal abduction moment during late stance and knee abduction angular impulse, and reduced the medial ground reaction force during late stance, adduction free moment, forefoot eversion angle, ankle inversion moment and angular impulse, hip adduction angle, hip abduction moment, and hip external rotation moment and angular impulse (p<0.05). Orthoses decreased the torsional forces on the lower extremity and reduced the loading at the hip during walking. These findings combined with our previous studies and those of others suggest that forefoot abnormalities are critically important in influencing lower extremity kinematics and kinetics, and may underlie some non-traumatic lower extremity injuries.     

Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.