Systemic Inflammation and Normocytic Anemia in DOCK11 Deficiency.

BACKGROUND: Increasing evidence links genetic defects affecting actin-regulatory proteins to diseases with severe autoimmunity and autoinflammation, yet the underlying molecular mechanisms are poorly understood. Dedicator of cytokinesis 11 (DOCK11) activates the small Rho guanosine triphosphatase (GTPase) cell division cycle 42 (CDC42), a central regulator of actin cytoskeleton dynamics. The role of DOCK11 in human immune-cell function and disease remains unknown. METHODS: We conducted genetic, immunologic, and molecular assays in four patients from four unrelated families who presented with infections, early-onset severe immune dysregulation, normocytic anemia of variable severity associated with anisopoikilocytosis, and developmental delay. Functional assays were performed in patient-derived cells, as well as in mouse and zebrafish models. RESULTS: We identified rare, X-linked germline mutations in DOCK11 in the patients, leading to a loss of protein expression in two patients and impaired CDC42 activation in all four patients. Patient-derived T cells did not form filopodia and showed abnormal migration. In addition, the patient-derived T cells, as well as the T cells from Dock11-knockout mice, showed overt activation and production of proinflammatory cytokines that were associated with an increased degree of nuclear translocation of nuclear factor of activated T cell 1 (NFATc1). Anemia and aberrant erythrocyte morphologic features were recapitulated in a newly generated dock11-knockout zebrafish model, and anemia was amenable to rescue on ectopic expression of constitutively active CDC42. CONCLUSIONS: Germline hemizygous loss-of-function mutations affecting the actin regulator DOCK11 were shown to cause a previously unknown inborn error of hematopoiesis and immunity characterized by severe immune dysregulation and systemic inflammation, recurrent infections, and anemia. (Funded by the European Research Council and others.).

Diphtheria toxin-mediated transposon-driven poly (A)-trapping efficiently disrupts transcriptionally silent genes in embryonic stem cells.

Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein-coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)-mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin-resistance gene of the vector. We found that a double-DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.

Collection of homozygous mutant mouse embryonic stem cells arising from autodiploidization during haploid gene trap mutagenesis.

Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.

Foot pressure-based analysis of gait while using a smartphone.

BACKGROUND: The number of incidents related to walking while using smartphones is rising. However, it is not clear how smartphone usage might affect a gait pattern in terms of the foot pressure, and this may address the mechanism leading to incidents while using smartphones. RESEARCH QUESTION: How do the characteristics of walking while using a smartphone affect foot pressure patterns? METHODS: In this cross-sectional study, we recruited 40 healthy young participants and investigated the walking speed, step length, coefficient of variance of the walking cycle (CV), anteroposterior length of the center of pressure (COP) trajectory (%Long), partial foot pressure ratios (% partial foot pressure [%PFP]), and COP existence time (COPexT) under the following four conditions: normal walking, screen gazing, while using social networking services (SNS), and while using a cognitive application. Parameters were compared among the four conditions using a repeated-measures ANOVA. Further, according to the presence or absence of an incident history (e.g. stumbles, collisions), participants were divided into either the incident or non-incident group. Parameters were compared between the two groups using a two-way repeated-measures ANOVA. RESULTS: Under the SNS and cognitive application conditions, the walking speed, step length, %Long, %PFP, and COPexT in the heel were significantly lower, and the CV and %PFP in the metatarsal region were higher than those under normal walking or screen gazing. %PFP in the heel and metatarsal regions showed a significant group-by-condition interaction; the incident group had lower %PFP in the heel region and higher %PFP in the metatarsal region than the non-incident group. SIGNIFICANCE: These findings indicate a trend of loading more pressure on the forefoot than on the heel. This pattern was markedly evident in individuals with a history of incidents related to the smartphone usage and may be one of the factors causing stumbles and collisions.

Nontuberculous mycobacteriosis oligoarthritis of the right hand misdiagnosed as rheumatoid arthritis: A case report.

Infection with Mycobacterium marinum has several different clinical presentations. Most commonly, it appears as a solitary papulonodular lesion on an extremity. A rare presentation of osteoarticular M. marinum involving multiple small joints and tenosynovitis of the hand, which was misdiagnosed as rheumatoid arthritis, is reported. The patient was initially treated for seronegative rheumatoid arthritis but failed to respond to methotrexate. Magnetic resonance imaging showed arthritis and tenosynovitis. Subsequently, synovial biopsy led to histological and microbiological diagnosis. Antimycobacterial treatment should be started promptly in such cases. The combined use of rifampicin, ethambutol, and clarithromycin appears to be effective, and debridement is indicated in patients with deep-seated infections.

Information quantity for secondary structure propensities of protein subsequences in the Protein Data Bank.

Elucidating the principles of sequence-structure relationships of proteins is a long-standing issue in biology. The nature of a short segment of a protein is determined by both the subsequence of the segment itself and its environment. For example, a type of subsequence, the so-called chameleon sequences, can form different secondary structures depending on its environments. Chameleon sequences are considered to have a weak tendency to form a specific structure. Although many chameleon sequences have been identified, they are only a small part of all possible subsequences in the proteome. The strength of the tendency to take a specific structure for each subsequence has not been fully quantified. In this study, we comprehensively analyzed subsequences consisting of four to nine amino acid residues, or N-gram (4≤N≤9), observed in non-redundant sequences in the Protein Data Bank (PDB). Tendencies to form a specific structure in terms of the secondary structure and accessible surface area are quantified as information quantities for each N-gram. Although the majority of observed subsequences have low information quantity due to lack of samples in the current PDB, thousands of N-grams with strong tendencies, including known structural motifs, were found. In addition, machine learning partially predicted the tendency of unknown N-grams, and thus, this technique helps to extract knowledge from the limited number of samples in the PDB.