In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends ...against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.
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•In T. thermophilus, small DNAs from the terminus of replication guide Argonaute•When gyrase is inhibited, TtAgo allows T. thermophilus to complete DNA replication•Without functional gyrase and TtAgo, T. thermophilus forms filaments•Acquisition of small DNA guides requires TtAgo endonucleolytic activity
Argonaute proteins use short nucleic acids as guides. In eukaryotes, these include siRNAs, miRNAs, and piRNAs, whose functions in fighting viruses, regulating gene expression, and silencing transposons have been studied extensively. The functions of Argonaute proteins in bacteria are less well understood. Jolly et al. report that, in the eubacterium Thermus thermophilus, Argonaute derives its DNA guides from the terminus of replication and participates in decatenating circular chromosomes, ensuring successful cell division.
The properties of the human immunodeficiency virus (HIV) pose serious difficulties for the development of an effective prophylactic vaccine. Here we describe the construction and characterization of ...recombinant (r), replication-competent forms of rhesus monkey rhadinovirus (RRV), a gamma-2 herpesvirus, containing a near-full-length (nfl) genome of the simian immunodeficiency virus (SIV). A 306-nucleotide deletion in the pol gene rendered this nfl genome replication-incompetent as a consequence of deletion of the active site of the essential reverse transcriptase enzyme. Three variations were constructed to drive expression of the SIV proteins: one with SIV's own promoter region, one with a cytomegalovirus (cmv) immediate-early promoter/enhancer region, and one with an RRV dual promoter (p26 plus PAN). Following infection of rhesus fibroblasts in culture with these rRRV vectors, synthesis of the early protein Nef and the late structural proteins Gag and Env could be demonstrated. Expression levels of the SIV proteins were highest with the rRRV-SIVcmv-nfl construct. Electron microscopic examination of rhesus fibroblasts infected with rRRV-SIVcmv-nfl revealed numerous budding and mature SIV particles and these infected cells released impressive levels of p27 Gag protein (>150 ng/ml) into the cell-free supernatant. The released SIV particles were shown to be incompetent for replication. Monkeys inoculated with rRRV-SIVcmv-nfl became persistently infected, made readily-detectable antibodies against SIV, and developed T-cell responses against all nine SIV gene products. Thus, rRRV expressing a near-full-length SIV genome mimics live-attenuated strains of SIV in several important respects: the infection is persistent; >95% of the SIV proteome is naturally expressed; SIV particles are formed; and CD8+ T-cell responses are maintained indefinitely in an effector-differentiated state. Although the magnitude of anti-SIV immune responses in monkeys infected with rRRV-SIVcmv-nfl falls short of what is seen with live-attenuated SIV infection, further experimentation seems warranted.
Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as ...PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD.
A probiotic goats’ milk yoghurt was developed containing Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. and using polymerized whey protein (PWP, 0.4%) and pectin (0.3%) as ...gelling agents. The yoghurt was analyzed for chemical composition, mould and yeast counts, changes in pH, titratable acidity, and viscosity, and probiotic survivability during storage at 4 °C. There was no significant difference in viscosity between weeks; however, changes in titratable acidity and pH showed significant differences between weeks during storage. L. casei and Bifidobacterium spp. remained viable with populations above 106 cfu g−1 during storage, but there were no viable counts of L. acidophilus by the fourth week. Scanning electron microscopy of goats’ milk yoghurt revealed that PWP interacted with casein micelles to form a comprehensive network in the yoghurt gel. Results indicated that PWP may be a novel protein-based thickening agent for improving the consistency of goats’ milk yoghurt and other products.
Dynamin-related protein 1 (Drp1) is the key regulator of mitochondrial fission. We and others have reported a strong correlation between enhanced Drp1 activity and impaired skeletal muscle insulin ...sensitivity. This study aimed to determine whether Drp1 directly regulates skeletal muscle insulin sensitivity and whole-body glucose homeostasis.
We employed tamoxifen-inducible skeletal muscle-specific heterozygous Drp1 knockout mice (mDrp1+/−). Male mDrp1+/− and wildtype (WT) mice were fed with either a high-fat diet (HFD) or low-fat diet (LFD) for four weeks, followed by tamoxifen injections for five consecutive days, and remained on their respective diet for another four weeks. In addition, we used primary human skeletal muscle cells (HSkMC) from lean, insulin-sensitive, and severely obese, insulin-resistant humans and transfected the cells with either a Drp1 shRNA (shDrp1) or scramble shRNA construct. Skeletal muscle and whole-body insulin sensitivity, skeletal muscle insulin signaling, mitochondrial network morphology, respiration, and H2O2 production were measured.
Partial deletion of the Drp1 gene in skeletal muscle led to improved whole-body glucose tolerance and insulin sensitivity (P < 0.05) in diet-induced obese, insulin-resistant mice but not in lean mice. Analyses of mitochondrial structure and function revealed that the partial deletion of the Drp1 gene restored mitochondrial dynamics, improved mitochondrial morphology, and reduced mitochondrial Complex I- and II-derived H2O2 (P < 0.05) under the condition of diet-induced obesity. In addition, partial deletion of Drp1 in skeletal muscle resulted in elevated circulating FGF21 (P < 0.05) and in a trend towards increase of FGF21 expression in skeletal muscle tissue (P = 0.095). In primary myotubes derived from severely obese, insulin-resistant humans, ShRNA-induced-knockdown of Drp1 resulted in enhanced insulin signaling, insulin-stimulated glucose uptake and reduced cellular reactive oxygen species (ROS) content compared to the shScramble-treated myotubes from the same donors (P < 0.05).
These data demonstrate that partial loss of skeletal muscle-specific Drp1 expression is sufficient to improve whole-body glucose homeostasis and insulin sensitivity under obese, insulin-resistant conditions, which may be, at least in part, due to reduced mitochondrial H2O2 production. In addition, our findings revealed divergent effects of Drp1 on whole-body metabolism under lean healthy or obese insulin-resistant conditions in mice.
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•Partial loss of skeletal muscle Drp1 enhances insulin sensitivity in obese mice.•Partial loss of skeletal muscle Drp1 reduces mitochondrial H2O2 production under obese conditions.•Skeletal muscle Drp1 has divergent impacts on insulin sensitivity under lean and obese conditions.
Intrinsically disordered cytoplasmic domains of T cell receptor (TCR) signaling subunits including ζ
cyt and CD3ε
cyt all contain one or more copies of an immunoreceptor tyrosine-based activation ...motif (ITAM), tyrosine residues of which are phosphorylated upon receptor triggering. Membrane binding-induced helical folding of ζ
cyt and CD3ε
cyt ITAMs is thought to control TCR activation. However, the question whether or not lipid binding of ζ
cyt and CD3ε
cyt is necessarily accompanied by a folding transition of ITAMs remains open. In this study, we investigate whether the membrane binding mechanisms of ζ
cyt and CD3ε
cyt depend on the membrane model used. Circular dichroic and fluorescence data indicate that binding of ζ
cyt and CD3ε
cyt to detergent micelles and unstable vesicles is accompanied by a disorder-to-order transition, whereas upon binding to stable vesicles these proteins remain unfolded. Using electron microscopy and dynamic light scattering, we show that upon protein binding, unstable vesicles fuse and rupture. In contrast, stable vesicles remain intact under these conditions. This suggests different membrane binding modes for ζ
cyt and CD3ε
cyt depending on the bilayer stability: (1) coupled binding and folding, and (2) binding without folding. These findings explain the long-standing puzzle in the literature and highlight the importance of the choice of an appropriate membrane model for protein–lipid interactions studies.
Dendritic cells (DCs) regulate both innate and adaptive immune responses. In this article, we exploit the unique avascularity of the cornea to examine a role for local or very early infiltrating DCs ...in regulating the migration of blood-derived innate immune cells toward HSV-1 lesions. A single systemic diphtheria toxin treatment 2 d before HSV-1 corneal infection transiently depleted CD11c(+) DCs from both the cornea and lymphoid organs of CD11c-DTR bone marrow chimeric mice for up to 24 h postinfection. Transient DC depletion significantly delayed HSV-1 clearance from the cornea through 6 d postinfection. No further compromise of viral clearance was observed when DCs were continuously depleted throughout the first week of infection. DC depletion did not influence extravasation of NK cells, inflammatory monocytes, or neutrophils into the peripheral cornea, but it did significantly reduce migration of NK cells and inflammatory monocytes, but not neutrophils, toward the HSV-1 lesion in the central cornea. Depletion of NK cells resulted in similar loss of viral control to transient DC ablation. Our findings demonstrate that resident corneal DCs and/or those that infiltrate the cornea during the first 24 h after HSV-1 infection contribute to the migration of NK cells and inflammatory monocytes into the central cornea, and are consistent with a role for NK cells and possibly inflammatory monocytes, but not polymorphonuclear neutrophils, in clearing HSV-1 from the infected cornea.
HSV-specific CD8(+) T cells provide constant immunosurveillance of HSV-1 latently infected neurons in sensory ganglia, and their functional properties are influenced by the presence of latent virus. ...In this study, we show that ganglionic HSV-specific CD8(+) T cells exhibit a higher functional avidity (ability to respond to low epitope density) than their counterparts in noninfected lungs, satisfying a need for memory effector cells that can respond to low densities of viral epitopes on latently infected neurons. We further show that lack of CD4(+) T cell help during priming leads to a transient inability to control latent virus, which was associated with a PD-1/PD-L1 mediated reduced functional avidity of ganglionic HSV-specific CD8(+) T cells. CD4(+) T cells are not needed to maintain CD8(+) T cell memory through 34 d after infection, nor do they have a direct involvement in the maintenance of HSV-1 latency.
Vascular remodeling in response to implantation of a tissue engineering scaffold such as a flow diverter (FD) leads to the cure of intracranial aneurysms. We hypothesize that the vascular response is ...dependent on FD design, and CD34+ progenitor cells play an important role in the endothelialization of the implant.
Sixteen rabbit aneurysms were randomly treated with two different single-layer braided FDs made of cobalt-chrome alloys. The FD-48 and FD-72 devices had 48 and 72 wires, respectively. Aneurysm occlusion rate was assessed during the final digital subtraction angiogram at 10, 20, 30, and 60 days (n=2 per device per time point). Implanted vessels were analyzed with scanning electron microscopy for tissue coverage, endothelialization, and immuno-gold labeling for CD34+ cells.
Complete aneurysm occlusion rates were similar between the devices; however, complete or near complete occlusion was more frequently observed in aneurysms with neck ≤4.2 mm (p=0.008). Total tissue coverage at 10 days over the surface of the FD-48 and FD-72 devices was 56.4±11.6% and 76.6±3.6%, respectively. Endothelial cell growth over the surface was time-dependent for the FD-72 device (Spearman's r=0.86, p=0.013) but not for the FD-48 device (Spearman's r=-0.59, p=0.094). The endothelialization score was marginally correlated with the distance from the aneurysm neck for the FD-48 device (Spearman's r=1, p=0.083) but not for the FD-72 device (Spearman's r=0.8, p=0.33). CD34+ cells were present along the entirety of both devices at all time points.
This study gives preliminary evidence that temporal and spatial endothelialization is dependent on FD design. Circulating CD34+ progenitor cells contribute to endothelialization throughout the healing process.
Intracranial in-stent hyperplasia is a stroke-associated complication that requires routine surveillance.
To compare the results of in vivo experiments to determine the accuracy and precision of ...in-stent hyperplasia measurements obtained with modified C-arm contrast-enhanced, cone-beam CT (CE-CBCT) imaging with those obtained by 'gold standard' histomorphometry. Additionally, to carry out clinical analyses comparing this CE-CBCT protocol with digital subtraction angiography (DSA).
A non-binned CE-CBCT protocol (VasoCT) was used that acquires x-ray images with a small field-of-view and applies a full-scale reconstruction algorithm providing high-resolution three-dimensional (3D) imaging with 100 µm isotropic voxels. In an vivo porcine model, VasoCT cross-sectional area measurements were compared with gold standard vessel histology. VasoCT and DSA were used to calculate in-stent stenosis in 23 imaging studies.
Porcine VasoCT cross-sectional stent, lumen, and in-stent hyperplasia areas strongly correlated with histological measurements (r(2)=0.97, 0.93, 0.90; slope=1.14, 1.07, and 0.76, respectively; p<0.0001). Clinical VasoCT percentage stenosis correlated well with DSA percentage stenosis (r(2)=0.84; slope=0.76), and the two techniques were free of consistent bias (Bland-Altman, bias=3.29%; 95% CI -14.75% to 21.33%). An illustrative clinical case demonstrated the advantages of VasoCT, including 3D capability and non-invasive IV contrast administration, for detection of in-stent hyperplasia.
C-arm VasoCT is a high-resolution 3D capable imaging technique that has been validated in an animal model for measurement of in-stent tissue growth. Successful clinical implementation of the protocol was performed in a small case series.
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