Dynamics of RecA filaments on single-stranded DNA

RecA, the key protein in homologous recombination, performs its actions as a helical filament on single-stranded DNA (ssDNA). ATP hydrolysis makes the RecA–ssDNA filament dynamic and is essential for successful recombination. RecA has been studied extensively by single-molecule techniques on double-stranded DNA (dsDNA). Here we directly probe the structure and kinetics of RecA interaction with its biologically most relevant substrate, long ssDNA molecules. We find that RecA ATPase activity is required for the formation of long continuous filaments on ssDNA. These filaments both nucleate and extend with a multimeric unit as indicated by the Hill coefficient of 5.4 for filament nucleation. Disassembly rates of RecA from ssDNA decrease with applied stretching force, corresponding to a mechanism where protein-induced stretching of the ssDNA aids in the disassembly. Finally, we show that RecA–ssDNA filaments can reversibly interconvert between an extended, ATP-bound, and a compressed, ADP-bound state. Taken together, our results demonstrate that ATP hydrolysis has a major influence on the structure and state of RecA filaments on ssDNA.     

Morphology and sedimentology of (clustered) cold-water coral mounds at the south Rockall Trough margins,NE Atlantic Ocean

Cold-water coral mounds on both margins of the Rockall Trough (NE Atlantic Ocean) have a strongly different morphology. Single, isolated mounds occur on the SE margin and are mainly found on the upper slope between 900 and 650 m water depth, while large mound clusters are found on the SW margin in water depths between 600 and 1,000 m, in a narrow zone almost parallel to the slope. Sedimentation rates on the mounds are higher than on the surrounding seabed as a result of baffling of biogenic carbonate debris and siliciclastic particles by the coral framework covering the mounds. This is confirmed by ²¹⁰Pb measurements. The individual coral growth rate can be three times higher then the vertical growth rate of the coral cover (±10 mm year⁻¹) which in turn is more than an order of magnitude higher then the present-day overall mound growth rate (±0.25 mm year⁻¹). The presence of extensive hardgrounds and firmgrounds and the three-dimensional coral framework are considered to be responsible for the stability of the relatively steep slopes of the mounds. High current velocities in the intramound areas result in local non-sedimentation and erosion, as is shown by the presence of IRD (ice-rafted debris) lag deposits on the seabed and moats around some of the mounds. The morphology and sedimentology of cold-water coral-covered (mainly Lophelia pertusa and Madrepora oculata) mounds on the southern Rockall Trough margins (NE Atlantic Ocean) is discussed and a model describing the development of these mounds is presented.     

Effects of a defective ERAD pathway on growth and heterologous protein production in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation.     

Functional characteristics of the high affinity IgG receptor, FcγRI

IgG FcRs are important mediators of immunity and play a key role during Ab-based immunotherapy. Within the leukocyte IgG receptor family, only FcγRI is capable of IgG binding with high affinity. FcγRI exists as a complex of a ligand binding α-chain and an FcR γ-chain. The receptors' α-chain can, furthermore, elicit several functions independent of the ITAM-bearing FcR γ-chain. Functional implications of high-affinity IgG binding and mechanisms underlying FcR γ-chain-independent signaling remain unclear to this day. In this paper, we provide an overview of past literature on FcγRI and address the implications of recently described interactions between cytosolic proteins and the FcγRI α-chain, as well as cytokine-enhanced FcγRI immune complex binding. Furthermore, an analysis of potential polymorphisms within the FCGR1A gene is provided.     

An oral health care guideline for institutionalised older people

Institutionalized older people are prone to oral health problems and their negative impact due to frailty, disabilities, multi-morbidity, and multiple medication use. Until recently, no evidence-based oral health care guideline for institutionalized older people has been available. For that reason, the Dutch Association of Nursing Home Physicians developed the Oral health care Guideline for Older people in Long-term care Institutions (OGOLI), meeting the requirements of the AGREE instrument for assessing a guideline's quality. This short report presents the keynotes and the content of the Oral health care Guideline. Most recommendations are based on expert opinions. Only 4 recommendations (education, pneumonia, use of an electric toothbrush, and fluoride rinsing in case of a sudden increase of oral plaque amount) are based on evidence level A2 conclusions. This emphasizes the need for further research on oral health of institutionalized older people.     

WISECONDOR: detection of fetal aberrations from shallow sequencing maternal plasma based on a within-sample comparison scheme

Genetic disorders can be detected by prenatal diagnosis using Chorionic Villus Sampling, but the 1:100 chance to result in miscarriage restricts the use to fetuses that are suspected to have an aberration. Detection of trisomy 21 cases noninvasively is now possible owing to the upswing of next-generation sequencing (NGS) because a small percentage of fetal DNA is present in maternal plasma. However, detecting other trisomies and smaller aberrations can only be realized using high-coverage NGS, making it too expensive for routine practice. We present a method, WISECONDOR (WIthin-SamplE COpy Number aberration DetectOR), which detects small aberrations using low-coverage NGS. The increased detection resolution was achieved by comparing read counts within the tested sample of each genomic region with regions on other chromosomes that behave similarly in control samples. This within-sample comparison avoids the need to re-sequence control samples. WISECONDOR correctly identified all T13, T18 and T21 cases while coverages were as low as 0.15–1.66. No false positives were identified. Moreover, WISECONDOR also identified smaller aberrations, down to 20 Mb, such as del(13)(q12.3q14.3), +i(12)(p10) and i(18)(q10). This shows that prevalent fetal copy number aberrations can be detected accurately and affordably by shallow sequencing maternal plasma. WISECONDOR is available at bioinformatics.tudelft.nl/wisecondor.     

The complete mitochondrial genome of Taeniogonalos taihorina (Bischoff) (Hymenoptera: Trigonalyidae) reveals a novel gene rearrangement pattern in the Hymenoptera

The family Trigonalyidae is considered to be one of the most basal lineages in the suborder Apocrita of Hymenoptera. Here, we determine the first complete mitochondrial genome of the Trigonalyidae, from the species Taeniogonalos taihorina (Bischoff, 1914). This mitochondrial genome is 15,927 bp long, with a high A + T-content of 84.60%. It contains all of the 37 typical animal mitochondrial genes and an A + T-rich region. The orders and directions of all genes are different from those of previously reported hymenopteran mitochondrial genomes. Eight tRNA genes, three protein-coding genes and the A + T-rich region were rearranged, with the dominant gene rearrangement events being translocation and local inversion. The arrangements of three tRNA clusters, trnY–trnM–trnI–trnQ, trnW–trnL2–trnC, and trnH–trnA–trnR–trnN–trnS–trnE–trnF, and the position of the cox1 gene, are novel to the Hymenoptera, even the insects. Six long intergenic spacers are present in the genome. The secondary structures of the RNA genes are normal, except for trnS2, in which the D-stem pairing is absent.     

Computational and experimental investigation of local stress fiber orientation in uniaxially and biaxially constrained microtissues

The orientation of cells and associated F-actin stress fibers is essential for proper tissue functioning. We have previously developed a computational model that qualitatively describes stress fiber orientation in response to a range of mechanical stimuli. In this paper, the aim is to quantitatively validate the model in a static, heterogeneous environment. The stress fiber orientation in uniaxially and biaxially constrained microscale tissues was investigated using a recently developed experimental system. Computed and experimental stress fiber orientations were compared, while accounting for changes in orientation with location in the tissue. This allowed for validation of the model, and additionally, it showed how sensitive the stress fiber orientation in the experimental system is to the location where it is measured, i.e., the heterogeneity of the stress fiber orientation. Computed and experimental stress fiber orientations showed good quantitative agreement in most regions. A strong local alignment near the locations where boundary conditions were enforced was observed for both uniaxially and biaxially constrained tissues. Excepting these regions, in biaxially constrained tissues, no preferred orientation was found and the distribution was independent of location. The stress fiber orientation in uniaxially constrained tissues was more heterogeneous, and stress fibers mainly oriented in the constrained direction or along the free edge. These results indicate that the stress fiber orientation in these constrained microtissues is mainly determined by the local mechanical environment, as hypothesized in our model, and also that the model is a valid tool to predict stress fiber orientation in heterogeneously loaded tissues.     

Computational model predicts cell orientation in response to a range of mechanical stimuli

To build anisotropic, mechanically functioning tissue, it is essential to understand how cells orient in response to mechanical stimuli. Therefore, a computational model was developed which predicts cell orientation, based on the actin stress fiber distribution inside the cell. In the model, the stress fiber distribution evolves dynamically according to the following: (1) Stress fibers contain polymerized actin. The total amount of depolymerized plus polymerized actin is constant. (2) Stress fibers apply tension to their environment. This active tension is maximal when strain rate and absolute strain are zero and reduces with increasing shortening rate and absolute strain. (3) A high active fiber stress in a direction leads to a large amount of fibers in this direction. (4) The cell is attached to a substrate; all fiber stresses are homogenized into a total cell stress, which is in equilibrium with substrate stress. This model predicts that on a substrate of anisotropic stiffness, fibers align in the stiffest direction. Under cyclic strain when the cellular environment is so stiff that no compaction occurs (1 MPa), the model predicts strain avoidance, which is more pronounced with increasing strain frequency or amplitude. Under cyclic strain when the cellular environment is so soft that cells can compact it (10 kPa), the model predicts a preference for the cyclically strained compared to the compacting direction. These model predictions all agree with experimental evidence. For the first time, a computational model predicts cell orientation in response to this range of mechanical stimuli using a single set of parameters.     

Divided we stand: splitting synthetic cells for their proliferation

With the recent dawn of synthetic biology, the old idea of man-made artificial life has gained renewed interest. In the context of a bottom-up approach, this entails the de novo construction of synthetic cells that can autonomously sustain themselves and proliferate. Reproduction of a synthetic cell involves the synthesis of its inner content, replication of its information module, and growth and division of its shell. Theoretical and experimental analysis of natural cells shows that, whereas the core synthesis machinery of the information module is highly conserved, a wide range of solutions have been realized in order to accomplish division. It is therefore to be expected that there are multiple ways to engineer division of synthetic cells. Here we survey the field and review potential routes that can be explored to accomplish the division of bottom-up designed synthetic cells. We cover a range of complexities from simple abiotic mechanisms involving splitting of lipid-membrane-encapsulated vesicles due to physical or chemical principles, to potential division mechanisms of synthetic cells that are based on prokaryotic division machineries.