Single molecule sequencing-guided scaffolding and correction of draft assemblies

Background

Although single molecule sequencing is still improving, the lengths of the generated sequences are inevitably an advantage in genome assembly. Prior work that utilizes long reads to conduct genome assembly has mostly focused on correcting sequencing errors and improving contiguity of de novo assemblies.

Results

We propose a disassembling-reassembling approach for both correcting structural errors in the draft assembly and scaffolding a target assembly based on error-corrected single molecule sequences. To achieve this goal, we formulate a maximum alternating path cover problem. We prove that this problem is NP-hard, and solve it by a 2-approximation algorithm.

Conclusions

Our experimental results show that our approach can improve the structural correctness of target assemblies in the cost of some contiguity, even with smaller amounts of long reads. In addition, our reassembling process can also serve as a competitive scaffolder relative to well-established assembly benchmarks.

     

Angle- and gender-specific quadriceps femoris muscle recruitment and knee extensor torque

The objectives were to examine knee angle-, and gender-specific knee extensor torque output and quadriceps femoris (QF) muscle recruitment during maximal effort, voluntary contractions. Fourteen young adult men and 15 young adult women performed three isometric maximal voluntary contractions (MVC), in a random order, with the knee at 0 degrees (terminal extension), 10 degrees, 30 degrees, 50 degrees, 70 degrees, and 90 degrees flexion. Knee extensor peak torque (PT), and average torque (AT) were expressed in absolute (N m), relative (N m kg(-1)) and allometric-modeled (N m kg(-n)) units. Vastus medialis (VM), vastus lateralis (VL), and rectus femoris (RF) muscle EMG signals were full-wave rectified and integrated over the middle 3 s of each contraction, averaged over the three trials at each knee angle, and normalized to the activity recorded at 0 degrees. Muscle recruitment efficiency was calculated as the ratio of the normalized EMG of each muscle to the allometric-modeled average torque (normalized to the values at 0 degrees flexion), and expressed as a percent. Men generated significantly greater knee extensor PT and AT than women in absolute, relative and allometric-modeled units. Absolute and relative PT and AT were significantly highest at 70 degrees, while allometric-modeled values were observed to increase significantly across knee joint angles 10-90 degrees. VM EMG was significantly greater than the VL and RF muscles across all angles, and followed a similar pattern to absolute knee extensor torque. Recruitment efficiency improved across knee joint angles 10-90 degrees and was highest for the VL muscle. VM recruitment efficiency improved more than the VL and RF muscles across 70-90 degrees flexion. The findings demonstrate angle-, and gender-specific responses of knee extensor torque to maximal-effort contractions, while superficial QF muscle recruitment was most efficient at 90 degrees, and less dependent on gender.     

A plant-specific subclass of C-terminal kinesins contains a conserved a-type cyclin-dependent kinase site implicated in folding and dimerization

Cyclin-dependent kinases (CDKs) control cell cycle progression through timely coordinated phosphorylation events. Two kinesin-like proteins that interact with CDKA;1 were identified and designated KCA1 and KCA2. They are 81% identical and have a similar three-partite domain organization. The N-terminal domain contains an ATP and microtubule-binding site typical for kinesin motors. A green fluorescent protein (GFP) fusion of the N-terminal domain of KCA1 decorated microtubules in Bright Yellow-2 cells, demonstrating microtubule-binding activity. During cytokinesis the full-length GFP-fusion protein accumulated at the midline of young and mature expanding phragmoplasts. Two-hybrid analysis and coimmunoprecipitation experiments showed that coiled-coil structures of the central stalk were responsible for homo- and heterodimerization of KCA1 and KCA2. By western-blot analysis, high molecular mass KCA molecules were detected in extracts from Bright Yellow-2 cells overproducing the full-length GFP fusion. Treatment of these cultures with the phosphatase inhibitor vanadate caused an accumulation of these KCA molecules. In addition to dimerization, interactions within the C-terminally located tail domain were revealed, indicating that the tail could fold onto itself. The tail domains of KCA1 and KCA2 contained two adjacent putative CDKA;1 phosphorylation sites, one of which is conserved in KCA homologs from other plant species. Site-directed mutagenesis of the conserved phosphorylation sites in KCA1 resulted in a reduced binding with CDKA;1 and abolished intramolecular tail interactions. The data show that phosphorylation of the CDKA;1 site provokes a conformational change in the structure of KCA with implications in folding and dimerization.     

Molecular dissection of plant cytokinesis and phragmoplast structure: a survey of GFP-tagged proteins

To identify molecular players implicated in cytokinesis and division plane determination, the Arabidopsis thaliana genome was explored for potential cytokinesis genes. More than 100 open reading frames were selected based on similarity to yeast and animal cytokinesis genes, cytoskeleton and polarity genes, and Nicotiana tabacum genes showing cell cycle-controlled expression. The subcellular localization of these proteins was determined by means of GFP tagging in tobacco Bright Yellow-2 cells and Arabidopsis plants. Detailed confocal microscopy identified 15 proteins targeted to distinct regions of the phragmoplast and the cell plate. EB1- and MAP65-like proteins were associated with the plus-end, the minus-end, or along the entire length of microtubules. The actin-binding protein myosin, the kinase Aurora, and a novel cell cycle protein designated T22, accumulated preferentially at the midline. EB1 and Aurora, in addition to other regulatory proteins (homologs of Mob1, Sid1, and Sid2), were targeted to the nucleus, suggesting that this organelle operates as a coordinating hub for cytokinesis.     

Identification of integrin beta subunit mutations that alter heterodimer function in situ

We conducted a genetic screen for mutations in myospheroid, the gene encoding the Drosophila betaPS integrin subunit, and identified point mutants in all of the structural domains of the protein. Surprisingly, we find that mutations in very strongly conserved residues will often allow sufficient integrin function to support the development of adult animals, including mutations in the ADMIDAS site and in a cytoplasmic NPXY motif. Many mutations in the I-like domain reduce integrin expression specifically when betaPS is combined with activating alphaPS2 cytoplasmic mutations, indicating that integrins in the extended conformation are unstable relative to the inactive, bent heterodimers. Interestingly, the screen has identified alleles that show gain-of-function characteristics in cell culture, but have negative effects on animal development or viability. This is illustrated by the allele mys(b58); available structural models suggest that the molecular lesion of mys(b58), V409>D, should promote the "open" conformation of the beta subunit I-like domain. This expectation is supported by the finding that alphaPS2betaPS (V409>D) promotes adhesion and spreading of S2 cells more effectively than does wild-type alphaPS2betaPS, even when betaPS is paired with alphaPS2 containing activating cytoplasmic mutations. Finally, comparisons with the sequence of human beta8 suggest that evolution has targeted the "mys(b58)" residue as a means of affecting integrin activity.     

The effects of rest interval length on ratings of perceived exertion during dynamic knee extension exercise

The objective of this study was to examine the effects of rest interval length on perceived exertion and during 3 sets of 10 inertial knee extension repetitions. Thirty healthy men (n = 15) and women (n = 15) volunteers were randomly assigned to 1 of 3 groups (1-, 2-, or 3-minute rest interval length) following the establishment of each subject's 1 repetition maximum (1RM) for inertial knee extension exercise. Subjects in each group performed 3 sets of 10 repetitions at 70% of a theoretical 10RM (based on each subject's 1RM), with a 1-, 2-, or 3-minute rest interval between each set. Perceived exertion was recorded, via the Borg category-ratio scale, from each subject after each repetition of each set. The results demonstrated no significant rest interval length effect on perceived exertion across the 3 sets of 10 repetitions. The results revealed a significantly higher perceived exertion value following the first repetition in set 3 as compared to sets 2 and 1 in all groups. The increase in perceived exertion within each set, as described by the slope, was found to be significantly lowest in set 1, as compared to sets 2 and 3. The major findings of this study demonstrate that perceived exertion significantly increases in a similar manner across 3 sets of 10 knee extension repetitions, despite rest interval lengths of 1-3 minutes.     

Chloroplast protein import: solve the GTPase riddle for entry

The fidelity of the numerous intracellular protein-trafficking pathways to different organelles is dictated by the interactions between the intrinsic targeting signals of substrate proteins and specific receptors that deliver the substrate to the proper organelle. Recent studies of protein targeting to chloroplasts suggest a novel mechanism in which GTP-dependent substrate recognition is coupled to a GTP-driven motor that initiates the translocation of proteins into the organelle.