Competitive binding of MatP and topoisomerase IV to the MukB hinge domain

Structural Maintenance of Chromosomes (SMC) complexes have ubiquitous roles in compacting DNA linearly, thereby promoting chromosome organization-segregation. Interaction between the Escherichia coli SMC complex, MukBEF, and matS-bound MatP in the chromosome replication termination region, ter, results in depletion of MukBEF from ter, a process essential for efficient daughter chromosome individualisation and for preferential association of MukBEF with the replication origin region. Chromosome-associated MukBEF complexes also interact with topoisomerase IV (ParC2E2), so that their chromosome distribution mirrors that of MukBEF. We demonstrate that MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding, which occurs with the same dimer to dimer stoichiometry. Furthermore, we show that matS DNA competes with the MukB hinge for MatP binding. Cells expressing MukBEF complexes that are mutated at the ParC/MatP binding interface are impaired in ParC binding and have a mild defect in MukBEF function. The data highlight competitive binding as a means of globally regulating MukBEF-topoisomerase IV activity in space and time.

Case Report: A Novel Mutation in the Mitochondrial MT-ND5 Gene Is Associated With Leber Hereditary Optic Neuropathy (LHON)

Leber hereditary optic neuropathy (LHON) is a mitochondrial disease causing severe bilateral visual loss, typically in young adults. The disorder is commonly caused by one of three primary point mutations in mitochondrial DNA, but a number of other rare mutations causing or associated with the clinical syndrome of LHON have been reported. The mutations in LHON are almost exclusively located in genes encoding subunits of complex I in the mitochondrial respiratory chain. Here we report two patients, a mother and her son, with the typical LHON phenotype. Genetic investigations for the three common mutations were negative, instead we found a new and previously unreported mutation in mitochondrial DNA. This homoplasmic mutation, m.13345G>A, is located in the MT-ND5 gene, encoding a core subunit in complex I in the mitochondrial respiratory chain. Investigation of the patients mitochondrial respiratory chain in muscle found a mild defect in the combined activity of complex I+III. In the literature six other mutations in the MT-ND5 gene have been associated with LHON and by this report a new putative mutation in the MT-ND5 can be added.

Nonspecific DNA binding by P1 ParA determines the distribution of plasmid partition and repressor activities

The faithful segregation, or “partition,” of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parAR351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a “super-repressor.” In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.

Three-Dimensional Holey-Graphene Architectures for Highly Sensitive Enzymatic Electrochemical Determination of Hydrogen Peroxide

Three-dimensional (3D) graphene with high specific surface area, excellent conductivity and designed porosity is essential for many practical applications. Herein, holey graphene oxide with nano pores was facilely prepared via a convenient mild defect-etching reaction and then fabricated to 3D nanostructures via a reduction method. Based on the 3D architectures, a novel enzymatic hydrogen peroxide sensor was successfully fabricated. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were used to characterize the 3D holey graphene oxide architectures (3DHGO). Cyclic voltammetry (CV) was used to evaluate the electrochemical performance of 3DHGO at glassy carbon electrode (GCE). Excellent electrocatalytic activity to the reduction of H2O2 was observed, and a linear range of 5.0×10-8~5.0×10-5 M with a detection limit of 3.8×10-9 M was obtained. These results indicated that 3DHGO have potential as electrochemical biosensors.

CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine remodeling

ABSTRACTRegulation of axon guidance and pruning of inappropriate synapses by class 3 semaphorins is key to development of neural circuits. Collapsin response mediator protein 2 (CRMP2) has been shown to regulate axon guidance by mediating Semaphorin 3A (Sema3A) signaling and its dysfunction has been linked to schizophrenia, however, nothing is known about its role in the synapse pruning. Here, using newly generated crmp2−/− mice we demonstrate that while CRMP2 has only a moderate effect on Sema3A-dependent axon guidance in vivo, it is essential for Sema3F-dependent axon pruning and dendritic spine remodeling. We first demonstrate that CRMP2 deficiency interferes with Sema3A signaling in compartmentalized neuron cultures and leads to a mild defect in axon guidance in peripheral nerves and corpus callosum. Strikingly, we show that crmp2−/− mice display more prominent defects in dendritic spine pruning and stereotyped axon pruning in hippocampus and visual cortex consistent with impaired Sema3F signaling and with autism spectrum disorder (ASD)-rather than schizophrenia-like phenotype. Indeed, we demonstrate that CRMP2 mediates Sema3F-induced axon retraction in primary neurons and that crmp2−/− mice display early postnatal behavioral changes linked to ASD. In conclusion, we demonstrate that CRMP2 is an essential mediator of Sema3F-dependent synapse pruning and its dysfunction in early postnatal stages shares histological and behavioral features of ASD.

Differences in dental age of small gestational age children based on the severity of enamel dental defects

Small Gestational Age (SGA) is the term used for a new born baby with birth-severe below the 10th percentile on the intrauterine Lubchenco curve, due to Intra Uterine Growth Restriction (IUGR) which can cause defects of several organs such as deciduous teeth enamel. Dental age is the indicator for growth and development of the teeth that determine biological age which is more accurate than chronological age and could be determined by calcification or teeth eruption. The purpose of this study was to obtain dental age in SGA children based on the enamel defect severity level of deciduous teeth as compared to the chronological age. This study was a comparative analytic study with given sample size, conducted towards 18 SGA children aged 4-6 years old, and 21 children with Appropriate Gestational Age (AGA) aged 4-6 years old as the control group. Deciduous teeth enamel defect severity level determined by the developmental defect of enamel (DDE) score FDI modified. In order to determine the permanent teeth growth and development, the indicator was based on dental age using the panoramic radiograph with Dermijian method. The t-test was used to compare the permanent teeth dental age difference between SGA and AGA children as well as the permanent teeth dental age difference of SGA children based on the enamel defect severity level of deciduous teeth as seen in their chronological age. The result showed that there was a difference in the permanent teeth dental age between the SGA and AGA children at chronological ages of 4, ,5, and 6 years old (t count = 3,36; 2,35; and 3,49). Based on the average of permanent teeth dental age, the value of AGA children was higher than SGA children. Furthermore, it also showed that the severe score of EDS in the SGA children at the age of 4, 5, and 6 years old, their permanent teeth dental age was lower than the mild EDS (t count = 1,45). The conclusion of this study was the permanent teeth dental age in SGA children was lower than the AGA children as well as difference between the permanent teeth dental age in SGA children based on their deciduous teeth enamel defect severity, and the severe defect refered to slower permanent teeth dental age than the mild defect.

NLRP3 recruitment by NLRC4 during Salmonella infection

NLRC4 and NLRP3, of the NOD-like receptor (NLR) family of intracellular proteins, are expressed in innate immune cells and are thought to nucleate distinct inflammasome complexes that promote caspase-1 activation, secretion of the proinflammatory cytokines IL-1β and IL-18, and a form of cell death termed pyroptosis. We show that NLRP3 associates with NLRC4 in macrophages infected with Salmonella typhimurium or transfected with flagellin. The significance of the interaction between the NLRC4 NACHT domain and NLRP3 was revealed when Nlrc4S533A/S533A bone marrow–derived macrophages (BMDMs) expressing phosphorylation site mutant NLRC4 S533A had only a mild defect in caspase-1 activation when compared with NLRC4-deficient BMDMs. NLRC4 S533A activated caspase-1 by recruiting NLRP3 and its adaptor protein ASC. Thus, Nlrc4S533A/S533A Nlrp3−/− BMDMs more closely resembled Nlrc4−/− BMDMs in their response to S. typhimurium or flagellin. The interplay between NLRP3 and NLRC4 reveals an unexpected overlap between what had been considered distinct inflammasome scaffolds.

The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles

Listeria monocytogenesis an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on themlp-actA-plcBoperon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcBor Δmplstrains resulted in the formation of small infection foci. As expected, the ΔplcBstrain displayed a strong defect in vacuole escape. However, the Δmplstrain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmplstrain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmplstrain, the ΔactA207–238strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207–238strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution.

Cooperation of Nectin-1 and Nectin-3 Is Required for Maintenance of Epidermal Stratification and Proper Hair Shaft Formation in the Mouse

Nectins constitute a family of four cell adhesion molecules which are localized on cell membrane. Mutations in NECTIN-1 gene cause the human ectodermal dysplasia syndrome (CLPED1) manifesting severe defects in skin and its appendages. However, nectin-1 null mutant mice have only a mild defect in epidermal stratification suggesting compensation by other nectins. We have analysed the epidermal and hair phenotypes of nectin-1; nectin-3 compound mutants. Epidermis was fragile and displayed severe defects in stratification, hair follicles were hypoplastic, and hair shaft structure was abnormal. Immunohistochemical analysis revealed severe defects in cell-cell junctions including adherens and tight junctions as well as desmosomes. It is therefore likely that the phenotypes were caused by impaired cell adhesion. The expression patterns of nectin-1 and nectin-3 together with the phenotypes in compound mutants indicated that heterophilic interactions between the two nectins are required for proper formation of epidermis and hair in mice. The nectin-1; nectin-3 compound mutant mice partially reproduced the phenotype of human CLPED1 patients.