Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division.

Members of the crenarchaeal kingdom, such as Sulfolobus, divide by binary fission yet lack genes for the otherwise near-ubiquitous tubulin and actin superfamilies of cytoskeletal proteins. Recent work has established that Sulfolobus homologs of the eukaryotic ESCRT-III and Vps4 components of the ESCRT machinery play an important role in Sulfolobus cell division. In eukaryotes, several pathways recruit ESCRT-III proteins to their sites of action. However, the positioning determinants for archaeal ESCRT-III are not known. Here, we identify a protein, CdvA, that is responsible for recruiting Sulfolobus ESCRT-III to membranes. Overexpression of the isolated ESCRT-III domain that interacts with CdvA results in the generation of nucleoid-free cells. Furthermore, CdvA and ESCRT-III synergize to deform archaeal membranes in vitro. The structure of the CdvA/ESCRT-III interface gives insight into the evolution of the more complex and modular eukaryotic ESCRT complex.

Maternal plasma LPCAT 1 mRNA correlates with lamellar body count.

Human lysophosphatidylcholine acyltransferase 1 (hLPCAT1) is a protein which helps produce surfactant in the fetal lung. We previously reported that levels of cell-free fetal mRNA for hLPCAT1 in amniotic fluid are correlated with lamellar body count (LBC) (r2=0.93). This short communication demonstrates that fetal hLPCAT1 mRNA is also present in maternal blood. Its quantity also correlates with amniotic fluid LBC (r2=0.81). Research in maternal plasma hLPCAT1 may assist in understanding fetal and placental maturational processes.

Amniotic fluid LPCAT1 mRNA correlates with the lamellar body count.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is required in the biosynthesis of pulmonary surfactant. This short communication describes our assessment of LPCAT1 mRNA levels in human amniotic fluid. We found a direct correlation between LPCAT1 mRNA copies and the amniotic fluid lamellar body count (LBC). This finding corroborates an association between LPCAT1 and surfactant phospholipid biosynthesis in humans. It may provide a model for future research in perinatal medicine.

The formation and positioning of cilia in Ciona intestinalis embryos in relation to the generation and evolution of chordate left-right asymmetry.

In the early mouse embryo monocilia on the ventral node rotate to generate a leftward flow of fluid. This nodal flow is essential for the left-sided expression of nodal and pitx2, and for subsequent asymmetric organ patterning. Equivalent left fluid flow has been identified in other vertebrates, including Xenopus and zebrafish, indicating it is an ancient vertebrate mechanism. Asymmetric nodal and Pitx expression have also been identified in several invertebrates, including the vertebrates' nearest relatives, the urochordates. However whether cilia regulate this asymmetric gene expression remains unknown, and previous studies in urochordates have not identified any cilia prior to the larval stage, when asymmetry is already long established. Here we use Scanning and Transmission Electron Microscopy and immunofluorescence to investigate cilia in the urochordate Ciona intestinalis. We show that single cilia are transiently present on each ectoderm cell of the late neurula/early tailbud stage embryo, a time point just before onset of asymmetric nodal expression. Mapping the position of each cilium on these cells shows they are posteriorly positioned, something also described for mouse node cilia. The C. intestinalis cilia have a 9+0 ring ultrastructure, however we find no evidence of structures associated with motility such as dynein arms, radial spokes or nexin. Furthermore the 9+0 ring structure becomes disorganised immediately after the cilia have exited the cell, indicative of cilia which are not capable of motility. Our results indicate that although cilia are present prior to molecular asymmetries, they are not motile and hence cannot be operating in the same way as the flow-generating cilia of the vertebrate node. We conclude that the cilia may have a role in the development of C. intestinalis left-right asymmetry but that this would have to be in a sensory capacity, perhaps as mechanosensors as hypothesised in two-cilia physical models of vertebrate cilia-driven asymmetry.

Evolution of GOLDEN2-LIKE gene function in C(3) and C (4) plants.

A pair of GOLDEN2-LIKE transcription factors is required for normal chloroplast development in land plant species that encompass the range from bryophytes to angiosperms. In the C(4) plant maize, compartmentalized function of the two GLK genes in bundle sheath and mesophyll cells regulates dimorphic chloroplast differentiation, whereas in the C(3) plants Physcomitrella patens and Arabidopsis thaliana the genes act redundantly in all photosynthetic cells. To assess whether the cell-specific function of GLK genes is unique to maize, we analyzed gene expression patterns in the C(4) monocot Sorghum bicolor and C(4) eudicot Cleome gynandra. Compartmentalized expression was observed in S. bicolor, consistent with the development of dimorphic chloroplasts in this species, but not in C. gynandra where bundle sheath and mesophyll chloroplasts are morphologically similar. The generation of single and double mutants demonstrated that GLK genes function redundantly in rice, as in other C(3) plants, despite the fact that GLK gene duplication in monocots preceded the speciation of rice, maize and sorghum. Together with phylogenetic analyses of GLK gene sequences, these data have allowed speculation on the evolutionary trajectory of GLK function. Based on current evidence, most species that retain single GLK genes belong to orders that contain only C(3) species. We therefore propose that the ancestral state is a single GLK gene, and hypothesize that GLK gene duplication enabled sub-functionalization, which in turn enabled cell-specific function in C(4) plants with dimorphic chloroplasts. In this scenario, GLK gene duplication preconditioned the evolution of C(4) physiology that is associated with chloroplast dimorphism.

Basal body movements orchestrate membrane organelle division and cell morphogenesis in Trypanosoma brucei.

The defined shape and single-copy organelles of Trypanosoma brucei mean that it provides an excellent model in which to study how duplication and segregation of organelles is interfaced with morphogenesis of overall cell shape and form. The centriole or basal body of eukaryotic cells is often seen to be at the centre of such processes. We have used a combination of electron microscopy and electron tomography techniques to provide a detailed three-dimensional view of duplication of the basal body in trypanosomes. We show that the basal body duplication and maturation cycle exerts an influence on the intimately associated flagellar pocket membrane system that is the portal for secretion and uptake from this cell. At the start of the cell cycle, a probasal body is positioned anterior to the basal body of the existing flagellum. At the G1-S transition, the probasal body matures, elongates and invades the pre-existing flagellar pocket to form the new flagellar axoneme. The new basal body undergoes a spectacular anti-clockwise rotation around the old flagellum, while its short new axoneme is associated with the pre-existing flagellar pocket. This rotation and subsequent posterior movements results in division of the flagellar pocket and ultimately sets parameters for subsequent daughter cell morphogenesis.

Flagellar motility is required for the viability of the bloodstream trypanosome.

The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures. How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.

Beyond 9+0: noncanonical axoneme structures characterize sensory cilia from protists to humans.

The intracellular amastigote stages of parasites such as Leishmania are often referred to as aflagellate. They do, however, possess a short axoneme of cryptic function. Here, our examination of the structure of this axoneme leads to a testable hypothesis of its role in the cell biology of pathogenicity. We show a striking similarity between the microtubule axoneme structure of the Leishmania mexicana parasite infecting a macrophage and vertebrate primary cilia. In both, the 9-fold microtubule doublet symmetry is broken by the incursion of one or more microtubule doublets into the axoneme core, giving rise to an architecture that we term here the 9v (variable) axoneme. Three-dimensional reconstructions revealed that no particular doublet initiated the symmetry break, and moreover it often involved 2 doublets. The tip of the L. mexicana flagellum was frequently intimately associated with the macrophage vacuole membrane. We propose that the main function of the amastigote flagellum is to act as a sensory organelle with important functions in host-parasite interactions and signaling in the intracellular stage of the L. mexicana life cycle.

Peptide-major histocompatibility complex dimensions control proximal kinase-phosphatase balance during T cell activation.

T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.

Perturbation of phosphatidylethanolamine synthesis affects mitochondrial morphology and cell-cycle progression in procyclic-form Trypanosoma brucei.

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are the two major constituents of eukaryotic cell membranes. In the protist Trypanosoma brucei, PE and PC are synthesized exclusively via the Kennedy pathway. To determine which organelles or processes are most sensitive to a disruption of normal phospholipid levels, the cellular consequences of a decrease in the levels of PE or PC, respectively, were studied following RNAi knock-down of four enzymes of the Kennedy pathway. RNAi against ethanolamine-phosphate cytidylyltransferase (ET) disrupted mitochondrial morphology and ultrastructure. Electron microscopy revealed alterations of inner mitochondrial membrane morphology, defined by a loss of disk-like cristae. Despite the structural changes in the mitochondrion, the cells maintained oxidative phosphorylation. Our results indicate that the inner membrane morphology of T. brucei procyclic forms is highly sensitive to a decrease of PE levels, as a change in the ultrastructure of the mitochondrion is the earliest phenotype observed after RNAi knock-down of ET. Interference with phospholipid synthesis also impaired normal cell-cycle progression. ET RNAi led to an accumulation of multinucleate cells. In contrast, RNAi against choline-/ethanolamine phosphotransferase, which affected PC as well as PE levels, caused a cell division phenotype characterized by non-division of the nucleus and production of zoids.

Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon.

The functional properties of tendon require an extracellular matrix (ECM) rich in elongated collagen fibrils in parallel register. We sought to understand how embryonic fibroblasts elaborate this exquisite arrangement of fibrils. We show that procollagen processing and collagen fibrillogenesis are initiated in Golgi to plasma membrane carriers (GPCs). These carriers and their cargo of 28-nm-diam fibrils are targeted to previously unidentified plasma membrane (PM) protrusions (here designated "fibripositors") that are parallel to the tendon axis and project into parallel channels between cells. The base of the fibripositor lumen (buried several microns within the cell) is a nucleation site of collagen fibrillogenesis. The tip of the fibripositor is the site of fibril deposition to the ECM. Fibripositors are absent at postnatal stages when fibrils increase in diameter by accretion of extracellular collagen, thereby maintaining parallelism of the tendon. Thus, we show that the parallelism of tendon is determined by the late secretory pathway and interaction of adjacent PMs to form extracellular channels.

Risk Factors for Infection After Intramedullary Nailing of Open Tibial Shaft Fractures in Low- and Middle-Income Countries.

OBJECTIVES: (1) To determine the infection rate after fixation of open tibial shaft fractures using the Surgical Implant Generation Network (SIGN) intramedullary nail in low- and middle-income countries (LMICs) and (2) to identify risk factors for infection. DESIGN: Prospective cohort study using an international online database. SETTING: Multiple hospitals in LMICs worldwide. PATIENTS/PARTICIPANTS: A total of 1061 open tibia fractures treated with the SIGN nail in LMICs between March 2000 and February 2013. INTERVENTION: Intravenous antibiotic administration, surgical debridement, and definitive intramedullary nailing within 14 days of injury. MAIN OUTCOME MEASUREMENTS: Deep or superficial infection at follow-up, implant breakage/loosening, angular deformity >10 degrees, repeat surgery, radiographic union, weight bearing, and ability to kneel. RESULTS: The overall infection rate was 11.9%. Infection rates by the Gustilo and Anderson classification were type 1: 5.1%, type II: 12.6%, type IIIa: 12.5%, type IIIb: 29.1%, and type IIIc: 16.7% (P = 0.001 between groups). Patients who developed infection had a longer mean time from injury to definitive surgery (4.7 vs. 3.9 days, P = 0.03) and from injury to wound closure (13.7 vs. 3.6 days, P < 0.001). Distal fractures had a higher infection rate than midshaft fractures (13.3% vs. 8.2%, P = 0.03). Infection rates were not associated with time from injury to initial debridement, time from injury to initial antibiotic administration, or total duration of antibiotics. CONCLUSIONS: Open tibia fractures can be managed effectively using the SIGN intramedullary nail in LMICs with an overall infection rate of 11.9%. Risk factors for infection identified include more severe soft-tissue injury, delayed nailing, delayed wound closure, and distal fracture location. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Anti-biotin antibodies offer superior organelle-specific labelling of mitochondria over avidin or streptavidin.

A number of endogenously biotinylated proteins are found in both cytosol and mitochondria of mammalian cells from many tissues, including liver, spleen, pancreas, kidney, and intestine. Therefore, caution should be taken when using the biotin detection system. Endogenous biotin can interfere with staining systems that employ the use of biotin-avidin- or biotin-streptavidin-based detection systems and may therefore result in high, non-specific background staining. Here, we show that this endogenous biotin reactivity can be deliberately exploited and used as a specific mitochondrial marker in both light and electron microscopy as well as for identifying mitochondrial fractions on Western blot.

Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria.

Multiple copies of the mitochondrial genome in eukaryotic cells are organized into protein-DNA complexes called nucleoids. Mitochondrial genome repair mechanisms have been reported, but they are less well characterized than their nuclear counterparts. To expand our knowledge of mitochondrial genome maintenance, we have studied the localization of the BRCA1 protein, known to be involved in nuclear repair pathways. Our confocal and immunoelectron microscopy results show that BRCA1 is present in mitochondria of several human cancer cell lines and in primary breast and nasal epithelial cells. BRCA1 localization in mitochondria frequently overlapped that of nucleoids. Small interfering RNA-mediated knockdown of BRCA1 in human cancer cells (confirmed by Western blot) results in decreased nuclear, cytoplasmic, and mitochondrial staining after immunofluorescence microscopy, establishing the specificity of the BRCA1 immunolabeling. Furthermore, using cell fractionation, dephosphorylation, and enzyme protection experiments, we show that a 220-kDa phosphorylated isoform of BRCA1 is enriched in mitochondrial and nuclear fractions but reduced in cytoplasmic subcellular fractions. Submitochondrial fractionation confirmed the presence of BRCA1 protein in isolated mitoplasts. Because phosphorylation of BRCA1 and subsequent changes in subcellular localization are known to follow DNA damage, our data support a universal role for BRCA1 in the maintenance of genome integrity in both mitochondria and nucleus.

The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules.

BACKGROUND: Impairment of cilia and flagella function underlies a growing number of human genetic diseases. Mutations in hydin in hy3 mice cause lethal communicating hydrocephalus with early onset. Hydin was recently identified as an axonemal protein; however, its function is as yet unknown. RESULTS: Here we use RNAi in Trypanosoma brucei to address this issue and demonstrate that loss of Hydin causes slow growth and a loss of cell motility. We show that two separate defects in newly-formed flagellar central pair microtubules underlie the loss of cell motility. At early time-points after RNAi induction, the central pair becomes mispositioned, while at later time points the central pair is lost. While the basal body is unaffected, both defects originate at the basal plate, reflecting a role for TbHydin throughout the length of the central pair. CONCLUSION: Our data provide the first evidence of Hydin's role within the trypanosome axoneme, and reveal central pair anomalies and thus impairment of ependymal ciliary motility as the likely cause of the hydrocephalus observed in the hy3 mouse.

High cell surface expression of CD4 allows distinction of CD4(+)CD25(+) antigen-specific effector T cells from CD4(+)CD25(+) regulatory T cells in murine experimental autoimmune encephalomyelitis.

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up-regulate cell surface expression of CD4. The CD4(high) sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25(+) sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses.

T-Cells Specific for a Self-Peptide of ApoB-100 Exacerbate Aortic Atheroma in Murine Atherosclerosis.

On the basis of mouse I-Ab-binding motifs, two sequences of the murine apolipoprotein B-100 (mApoB-100), mApoB-1003501-3515 (designated P3) and mApoB-100978-992 (designated P6), were found to be immunogenic. In this report, we show that P6 is also atherogenic. Immunization of Apoe-/- mice fed a high-fat diet (HFD) with P6 resulted in enhanced development of aortic atheroma as compared to control mice immunized with an irrelevant peptide MOG35-55 or with complete Freund's adjuvant alone. Adoptive transfer of lymph node cells from P6-immunized donor mice to recipients fed an HFD caused exacerbated aortic atheromas, correlating P6-primed cells with disease development. Finally, P6-specific T cell clones were generated and adoptive transfer of T cell clones into recipients fed an HFD led to significant increase in aortic plaque coverage when compared to control animals receiving a MOG35-55-specific T cell line. Recipient mice not fed an HFD, however, did not exhibit such enhancement, indicating that an inflammatory environment facilitated the atherogenic activity of P6-specific T cells. That P6 is identical to or cross-reacts with a naturally processed peptide of ApoB-100 is evidenced by the ability of P6 to stimulate the proliferation of T cells in the lymph node of mice primed by full-length human ApoB-100. By identifying an atherogenic T cell epitope of ApoB-100 and establishing specific T cell clones, our studies open up new and hitherto unavailable avenues to study the nature of atherogenic T cells and their functions in the atherosclerotic disease process.

Adoptive transfer of myelin basic protein-induced experimental autoimmune encephalomyelitis between SJL and B10.S mice: correlation of priming milieus with susceptibility and resistance phenotypes.

To study the mechanisms of EAE resistance, we directly transfer MBP-primed EAE-susceptible SJL lymph node cells into EAE-resistant B10.S recipients and vice versa. These transfers were unsuccessful because of strong alloreactivity between the two strains. Neonatal tolerance to SJL antigens was induced in B10.S mice and in these hosts MBP-primed SJL lymph node cells readily induce development of adoptive EAE. Conversely, transfer of MBP-primed B10.S lymph node cells into EAE-susceptible (SJL x B10.S)F1 recipients failed to induce EAE. These results are consistent with the notion that the priming milieus in the donor mice affect the expression of susceptible and resistant phenotypes.

Retropubic Dilation With a Foley Catheter Balloon: A Novel Technique for Penile Prosthesis Reservoir Placement.

BACKGROUND: The 3-piece inflatable penile prosthesis was introduced in 1973 as a treatment for men with erectile dysfunction. Consisting of 2 corporal cylinders, 1 pump, and a fluid-filled reservoir, the prosthesis is placed by blunt dissection into the retropubic space. The dissection for the reservoir is performed blindly into a space juxtaposed with nerves, vessels, and the bladder. OBJECTIVE: To propose a novel approach for inflatable penile prosthesis reservoir placement involving gentle dilation of the retropubic space using a Foley catheter balloon. METHODS: Patient medical records from 1 surgeon were reviewed. Patients did not have a history of pelvic surgery or prostatectomy. Each implant was approached using a penoscrotal incision, and the retropubic space was dilated with a 30-mL Foley catheter balloon filled to 100-mL capacity before reservoir placement. The postoperative visits were examined for complications, including reservoir infection and herniation. A literature search of penile prosthesis reservoir placement technique and complications (eg, herniation, infection) of reservoir placement was also performed. RESULTS: Fifteen patient records were examined. The reservoir herniation rate was 0% and the infection rate was 7%. The average reservoir herniation rate is reported to be 1% to 3%, and the average infection rate is reported to be 1% to 5%. CONCLUSION: The use of a Foley catheter balloon is a safe, atraumatic, cost-effective, and easily performed method of dilating the retropubic space for subsequent inflatable penile prosthesis reservoir placement.

Evaluation of the King-Devick test as a concussion screening tool in high school football players.

OBJECTIVE: Concussion is the most common type of traumatic brain injury, and results from impact or impulsive forces to the head, neck or face. Due to the variability and subtlety of symptoms, concussions may go unrecognized or be ignored, especially with the pressure placed on athletes to return to competition. The King-Devick (KD) test, an oculomotor test originally designed for reading evaluation, was recently validated as a concussion screening tool in collegiate athletes. A prospective study was performed using high school football players in an attempt to study the KD as a concussion screening tool in this younger population. METHODS: 343 athletes from four local high school football teams were recruited to participate. These athletes were given baseline KD tests prior to competition. Individual demographic information was collected on the subjects. Standard team protocol was employed to determine if a concussion had occurred during competition. Immediately after diagnosis, the KD test was re-administered to the concussed athlete for comparison to baseline. Post-season testing was also performed in non-concussed individuals. RESULTS: Of the 343 athletes, nine were diagnosed with concussions. In all concussed players, cumulative read times for the KD test were significantly increased (p<0.001). Post-season testing of non-concussed athletes revealed minimal change in read times relative to baseline. Univariate analysis revealed that history of concussion was the only demographic factor predictive of concussion in this cohort. CONCLUSION: The KD test is an accurate and easily administered sideline screening tool for concussion in adolescent football players.