Kinesin-II is required for axonal transport of choline acetyltransferase in Drosophila.

KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport.     

Sequence Conservation in Plasmodium falciparum α-Helical Coiled Coil Domains Proposed for Vaccine Development

Background

The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to α-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative α-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB.

Methodology/Principal Findings

14 α-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 α-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative α-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for α-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved α-helical coiled coil motifs.

Conclusion/Significance

We conclude that the selection of α-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation.     

Comparison of devices used for stent-assisted coiling of intracranial aneurysms

Introduction

Two self-expandable stents, the Neuroform and the Enterprise stent, are widely used for stent-assisted coiling (SAC) of complex shaped intracranial aneurysms. However, comparative knowledge about technical feasibility, peri- and post-procedural morbidity and mortality, packing densities as well as follow-up data is limited.

Material and Methods

We conducted a retrospective study to investigate differences in aneurysms stented with the Enterprise or Neuroform stents. Angiographic follow-up (mean 19.42 months) was available in 72.6% (61/84) of aneurysms treated with stent-assisted coiling. We further sought to compare stent-assisted coiling to a matched patient population with aneurysms treated by conventional coil embolization.

Results

The stenting success rate of the Enterprise was higher compared to the Neuroform stent (46/48 and 42/51, respectively). In 5 of 9 cases in which the Neuroform stent was not navigable to the landing zone, we successfully deployed an Enterprise stent instead. Eventually, 42 aneurysms were coiled after stenting in each group. We observed no significant differences in peri-procedural complication rate, post-procedural hospital stay, packing density, recurrence rate or number of in-stent stenosis. Strikingly, 36.1% of followed aneurysms in the SAC group showed progressive occlusion on angiographic follow-up imaging. The packing density was significantly higher in aneurysms treated by SAC as compared to conventionally coiled aneurysms, while recanalization rate was significantly lower in the SAC group.

Conclusion

The procedural success rate is higher using the Enterprise, but otherwise both stents exhibited similar characteristics. Lower recurrence frequency and complication rates comparable to conventional coil embolization emphasize the importance of stent-assisted coiling in the treatment of complex aneurysms. Progressive occlusion on angiographic follow-up was a distinct and frequent observation in the SAC group and may in part be due to flow diversion.     

Tissue Factor Pathway Inhibitor 2 Is Found in Skin and Its C-Terminal Region Encodes for Antibacterial Activity

Background

Tissue factor pathway inhibitor 2 (TFPI-2) is a matrix-associated serine protease inhibitor with an enigmatic function in vivo. Here, we describe that TFPI-2 is present in fibrin of wounds and also expressed in skin, where it is up-regulated upon wounding.

Methodology and Principal Findings

Neutrophil elastase cleaved TFPI-2, and a C-terminal fragment was found to bind to bacteria. Similarly, a prototypic peptide representing this C-terminal part, EDC34, bound to bacteria and bacterial lipopolysaccharide, and induced bacterial permeabilization. The peptide also induced leakage in artificial liposomes, and displayed a random coil conformation upon interactions with liposomes as well as lipopolysaccharide. EDC34 was antibacterial against both Gram-negative and Gram-positive bacteria in physiological buffer conditions.

Conclusions/Significance

The results demonstrate that the C-terminus of TFPI-2 encodes for antimicrobial activity, and may be released during wounding.     

Spontaneous Bleeding in Pancreatitis Treated by Transcatheter Arterial Coil Embolization: A Retrospective Study

Background/Objectives

A rare, but life-threatening complication in pancreatitis is a spontaneous bleeding from intestinal vessels with or without previous formation of (pseudo-) aneurysms. And yet, the optimal diagnostic and therapeutic strategies remain unclear.

Methods

We performed a retrospective analysis of all patients with pancreatitis and intraabdominal bleeding at a German tertiary referral center between January 2002 and December 2012.

Results

Bleeding occurred in <1% (14/3,421) of patients with pancreatitis. Most involved vessels were arteria lienalis, arteria gastroduodenalis, and arteria pancreaticoduodenalis. All bleedings could be stopped by transcatheter arterial coil embolization. Recurrent bleeding after coil embolization occurred in 2/14 (14%) patients.

Conclusions

In cases of intraabdominal hemorrhage in patients with pancreatitis, transcatheter arterial coil embolization should be considered as the first interventional procedure.     

Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport

This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled- coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.     

The Pentameric Channel of COMPcc in Complex with Different Fatty Acids

Background

COMPcc forms a pentameric left-handed coiled coil that is known to bind hydrophilic signaling molecules such as vitamin D₃, and vitamin A.

Principal Findings

In an integrated approach we reveal the unique binding properties of COMPcc for saturated and unsaturated fatty acids. Our observations suggest that residues Met33 (gating pore), Thr40/Asn41 (water chamber) and Gln54 (electrostatic trap) are key elements for the binding of fatty acids by COMPcc. In addition, this work characterizes the binding of various fatty acids to COMPcc using fluorescence spectroscopy. Our findings reveal a binding trend within the hydrophobic channel of COMPcc, namely, that is driven by length of the methylene tail and incorporation of unsaturation.

Conclusion/Significance

The unique binding properties imply that COMPcc may be involved in signalling functions in which hydrophilic ligands are involved. The pentameric channel is a unique carrier for lipophilic compounds. This opens the exciting possibility that COMPcc could be developed as a targeted drug delivery system.     

Expression Levels of a Kinesin-13 Microtubule Depolymerase Modulates the Effectiveness of Anti-Microtubule Agents

Background

Chemotheraputic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs. In this report, we test the hypothesis that the cellular levels of microtubule depolymerases, in this case kinesin-13 s, modulate the effectiveness of the microtubule disrupting drug colchicine.

Methodology/Principal Findings

We used a combination of RNA interference (RNAi), high-throughput microscopy, and time-lapse video microscopy in Drosophila S2 cells to identify a specific MAP, kinesin-like protein 10A (KLP10A), that contributes to the efficacy of the anti-microtubule drug colchicine. KLP10A is an essential microtubule depolymerase throughout the cell cycle. We find that depletion of KLP10A in S2 cells confers resistance to colchicine-induced microtubule depolymerization to a much greater extent than depletion of several other destabilizing MAPs. Using image-based assays, we determined that control cells retained 58% (±2%SEM) of microtubule polymer when after treatment with 2 µM colchicine for 1 hour, while cells depleted of KLP10A by RNAi retained 74% (±1%SEM). Likewise, overexpression of KLP10A-GFP results in increased susceptibility to microtubule depolymerization by colchicine.

Conclusions/Significance

Our results demonstrate that the efficacy of microtubule destabilization by a pharmacological agent is dependent upon the cellular expression of a microtubule depolymerase. These findings suggest that expression levels of Kif2A, the human kinesin-13 family member, may be an attractive biomarker to assess the effectiveness of anti-microtubule chemotherapies. Knowledge of how MAP expression levels affect the action of anti-microtubule drugs may prove useful for evaluating possible modes of cancer treatment.     

Current status of IMRT in head and neck cancer

Background

IMRT provides highly conformal dose distributions creating non uniform spatial intensity using different segments in the beam.

Material & Methods and Results

Different retrospective studies have shown a high capability of IMRT to treat tumours close to the base of skull. Prospective studies have shown a decrease in xerostomia compared with conventional 3D conformal treatment (3DCRT). Modulation of intensity is performed by the movement of the multileaf collimator (MLC) that can deliver the radiation in different ways, such as static field segments, dynamic field segments and rotational delivery (arc therapy and tomotherapy). There are slight differences among the different techniques in terms of homogeneity, dose conformity and treatment delivery time.

Conclusions

The best method to deliver IMRT will depend on multiple factors such as deliverability, practicality, user training and plan quality.     

In silico characterization of a novel pathogenic deletion mutation identified in XPA gene in a Pakistani family with severe xeroderma pigmentosum

Background

Xeroderma Pigmentosum (XP) is a rare skin disorder characterized by skin hypersensitivity to sunlight and abnormal pigmentation. The aim of this study was to investigate the genetic cause of a severe XP phenotype in a consanguineous Pakistani family and in silico characterization of any identified disease-associated mutation.

Results

The XP complementation group was assigned by genotyping of family for known XP loci. Genotyping data mapped the family to complementation group A locus, involving XPA gene. Mutation analysis of the candidate XP gene by DNA sequencing revealed a novel deletion mutation (c.654del A) in exon 5 of XPA gene. The c.654del A, causes frameshift, which pre-maturely terminates protein and result into a truncated product of 222 amino acid (aa) residues instead of 273 (p.Lys218AsnfsX5). In silico tools were applied to study the likelihood of changes in structural motifs and thus interaction of mutated protein with binding partners. In silico analysis of mutant protein sequence, predicted to affect the aa residue which attains coiled coil structure. The coiled coil structure has an important role in key cellular interactions, especially with DNA damage-binding protein 2 (DDB2), which has important role in DDB-mediated nucleotide excision repair (NER) system.

Conclusions

Our findings support the fact of genetic and clinical heterogeneity in XP. The study also predicts the critical role of DDB2 binding region of XPA protein in NER pathway and opens an avenue for further research to study the functional role of the mutated protein domain.     

Sensitization prevalence, antibody cross-reactivity and immunogenic peptide profile of Api g 2, the non-specific lipid transfer protein 1 of celery

Background

Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model.

Methodology

786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs.

Results

IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients'' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides.

Conclusions

Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.     

Effect of Atomoxetine on Hyperactivity in an Animal Model of Attention-Deficit/Hyperactivity Disorder (ADHD)

Background

Hyperactivity related behaviors as well as inattention and impulsivity are regarded as the nuclear symptoms of attention-deficit/hyperactivity disorder (ADHD).

Purpose

To investigate the therapeutic effects of atomoxetine on the motor activity in relation to the expression of the dopamine (DA) D2 receptor based on the hypothesis that DA system hypofunction causes ADHD symptoms, which would correlate with extensive D2 receptor overproduction and a lack of DA synthesis in specific brain regions: prefrontal cortex (PFC), striatum, and hypothalamus.

Methods

Young male spontaneously hypertensive rats (SHR), animal models of ADHD, were randomly divided into four groups according to the daily dosage of atomoxetine and treated for 21 consecutive days. The animals were assessed using an open-field test, and the DA D2 receptor expression was examined.

Results

The motor activity improved continuously in the group treated with atomoxetine at a dose of 1 mg/Kg/day than in the groups treated with atomoxetine at a dose of 0.25 mg/Kg/day or 0.5 mg/Kg/day. With respect to DA D₂ receptor immunohistochemistry, we observed significantly increased DA D₂ receptor expression in the PFC, striatum, and hypothalamus of the SHRs as compared to the WKY rats. Treatment with atomoxetine significantly decreased DA D₂ expression in the PFC, striatum, and hypothalamus of the SHRs, in a dose-dependent manner.

Conclusion

Hyperactivity in young SHRs can be improved by treatment with atomoxetine via the DA D2 pathway.     

Phosphodiesterase 6 subunits are expressed and altered in idiopathic pulmonary fibrosis

Background

Idiopathic Pulmonary Fibrosis (IPF) is an unresolved clinical issue. Phosphodiesterases (PDEs) are known therapeutic targets for various proliferative lung diseases. Lung PDE6 expression and function has received little or no attention. The present study aimed to characterize (i) PDE6 subunits expression in human lung, (ii) PDE6 subunits expression and alteration in IPF and (iii) functionality of the specific PDE6D subunit in alveolar epithelial cells (AECs).

Methodology/Principal Findings

PDE6 subunits expression in transplant donor (n = 6) and IPF (n = 6) lungs was demonstrated by real-time quantitative (q)RT-PCR and immunoblotting analysis. PDE6D mRNA and protein levels and PDE6G/H protein levels were significantly down-regulated in the IPF lungs. Immunohistochemical analysis showed alveolar epithelial localization of the PDE6 subunits. This was confirmed by qRT-PCR from human primary alveolar type (AT)II cells, demonstrating the down-regulation pattern of PDE6D in IPF-derived ATII cells. In vitro, PDE6D protein depletion was provoked by transforming growth factor (TGF)-β1 in A549 AECs. PDE6D siRNA-mediated knockdown and an ectopic expression of PDE6D modified the proliferation rate of A549 AECs. These effects were mediated by increased intracellular cGMP levels and decreased ERK phosphorylation.

Conclusions/Significance

Collectively, we report previously unrecognized PDE6 expression in human lungs, significant alterations of the PDE6D and PDE6G/H subunits in IPF lungs and characterize the functional role of PDE6D in AEC proliferation.     

The Video Head Impulse Test (vHIT) Detects Vertical Semicircular Canal Dysfunction

Background

The video head impulse test (vHIT) is a useful clinical tool to detect semicircular canal dysfunction. However vHIT has hitherto been limited to measurement of horizontal canals, while scleral search coils have been the only accepted method to measure head impulses in vertical canals. The goal of this study was to determine whether vHIT can detect vertical semicircular canal dysfunction as identified by scleral search coil recordings.

Methods

Small unpredictable head rotations were delivered by hand diagonally in the plane of the vertical semicircular canals while gaze was directed along the same plane. The planes were oriented along the left-anterior-right-posterior (LARP) canals and right-anterior-left-posterior (RALP) canals. Eye movements were recorded simultaneously in 2D with vHIT (250 Hz) and in 3D with search coils (1000 Hz). Twelve patients with unilateral, bilateral and individual semicircular canal dysfunction were tested and compared to seven normal subjects.

Results

Simultaneous video and search coil recordings were closely comparable. Mean VOR gain difference measured with vHIT and search coils was 0.05 (SD = 0.14) for the LARP plane and −0.04 (SD = 0.14) for the RALP plane. The coefficient of determination R² was 0.98 for the LARP plane and 0.98 for the RALP plane and the results of the two methods were not significantly different. vHIT and search coil measures displayed comparable patterns of covert and overt catch-up saccades.

Conclusions

vHIT detects dysfunction of individual vertical semicircular canals in vestibular patients as accurately as scleral search coils. Unlike search coils, vHIT is non-invasive, easy to use and hence practical in clinics.     

P68 RNA helicase (DDX5) alters activity of cis- and trans-acting factors of the alternative splicing of H-Ras

Background

H-Ras pre-mRNA undergoes an alternative splicing process to render two proteins, namely p21 H-Ras and p19 H-Ras, due to either the exclusion or inclusion of the alternative intron D exon (IDX), respectively. p68 RNA helicase (p68) is known to reduce IDX inclusion.

Principal Findings

Here we show that p68 unwinds the stem-loop IDX-rasISS1 structure and prevents binding of hnRNP H to IDX-rasISS1. We also found that p68 alters the dynamic localization of SC35, a splicing factor that promotes IDX inclusion. The knockdown of hnRNP A1, FUS/TLS and hnRNP H resulted in upregulation of the expression of the gene encoding the SC35-binding protein, SFRS2IP. Finally, FUS/TLS was observed to upregulate p19 expression and to stimulate IDX inclusion, and in vivo RNAi-mediated depletion of hnRNP H decreased p19 H-Ras abundance.

Significance

Taken together, p68 is shown to be an essential player in the regulation of H-Ras expression as well as in a vital transduction signal pathway tied to cell proliferation and many cancer processes.     

Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice

Background

Phosphoprotein phosphatase 2A (PP2A), a major serine-threonine protein phosphatase in eukaryotes, is an oligomeric protein comprised of structural (A) and catalytic (C) subunits to which a variable regulatory subunit (B) can associate. The C subunit contains a methyl ester post-translational modification on its C-terminal leucine residue, which is removed by a specific methylesterase (PME-1). Methylesterification is thought to control the binding of different B subunits to AC dimers, but little is known about its physiological significance in vivo.

Methodology/Principal Findings

Here, we show that targeted disruption of the PME-1 gene causes perinatal lethality in mice, a phenotype that correlates with a virtually complete loss of the demethylated form of PP2A in the nervous system and peripheral tissues. Interestingly, PP2A catalytic activity over a peptide substrate was dramatically reduced in PME-1(−/−) tissues, which also displayed alterations in phosphoproteome content.

Conclusions

These findings suggest a role for the demethylated form of PP2A in maintenance of enzyme function and phosphorylation networks in vivo.     

A Peek into Tropomyosin Binding and Unfolding on the Actin Filament

Background

Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin.

Principal Findings

Tropomyosin''s periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle α-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism).

Conclusions

This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.     

Comparison of the Association of Sac Growth and Coil Compaction with Recurrence in Coil Embolized Cerebral Aneurysms

Background and Purpose

In recurrent cerebral aneurysms treated by coil embolization, coil compaction is regarded as the presumptive mechanism. We test the hypothesis that aneurysm growth is the primary recurrence mechanism. We also test the hypothesis that the coil mass will translate a measurable extent when recurrence occurs.

Methods

An objective, quantitative image analysis protocol was developed to determine the volumes of aneurysms and coil masses during initial and follow-up visits from 3D rotational angiograms. The population consisted of 15 recurrence and 12 non-recurrence control aneurysms initially completely coiled at a single center. An investigator sensitivity study was performed to assess the objectivity of the methods. Paired Wilcoxon tests (p<0.05, one-tailed) were performed to assess for aneurysm and coil growth. The translation of the coil mass center at follow-up was computed. A Mann Whitney U-Test (p<0.05, one-tailed) was used to compare translation of coil mass centers between recurrence and control subjects.

Results

Image analysis protocol was found to be insensitive to the investigator. Aneurysm growth was evident in the recurrence cohort (p=0.003) but not the control (p=0.136). There was no evidence of coil compaction in either the recurrence or control cohorts (recurrence: p=0.339; control: p=0.429). The translation of the coil mass centers was found to be significantly larger in the recurrence cohort than the control cohort (p=0.047).

Conclusion

Aneurysm sac growth, not coil compaction, was the primary mechanism of recurrence following successful coil embolization. The coil mass likely translates to a measurable extent when recurrence occurs and has the potential to serve as a non-angiographic recurrence marker.     

Vitamin D Deficiency in Medical Patients at a Central Hospital in Malawi: A Comparison with TB Patients from a Previous Study

Objectives

To determine the prevalence of vitamin D deficiency (VDD) in adult medical, non-tuberculous (non-TB) patients. To investigate associations with VDD. To compare the results with a similar study in TB patients at the same hospital.

Design

Cross-sectional sample.

Setting

Central hospital in Malawi.

Participants

Adult non-TB patients (n = 157), inpatients and outpatients.

Outcome Measures

The primary outcome was the prevalence of VDD. Potentially causal associations sought included nutritional status, in/outpatient status, HIV status, anti-retroviral therapy (ART) and, by comparison with a previous study, a diagnosis of tuberculosis (TB).

Results

Hypovitaminosis D (≤75 nmol/L) occurred in 47.8% (75/157) of patients, 16.6% (26/157) of whom had VDD (≤50 nmol/L). None had severe VDD (≤25 nmol/L). VDD was found in 22.8% (23/101) of in-patients and 5.4% (3/56) of out-patients. In univariable analysis in-patient status, ART use and low dietary vitamin D were significant predictors of VDD. VDD was less prevalent than in previously studied TB patients in the same hospital (68/161 = 42%). In multivariate analysis of the combined data set from both studies, having TB (OR 3.61, 95%CI 2.02–6.43) and being an in-patient (OR 2.70, 95%CI 1.46–5.01) were significant independent predictors of VDD.

Conclusions

About half of adult medical patients without TB have suboptimal vitamin D status, which is more common in in-patients. VDD is much more common in TB patients than non-TB patients, even when other variables are controlled for, suggesting that vitamin D deficiency is associated with TB.     

Electron Tomography of Cryofixed,Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

Background

Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.

Methodology

We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the “target zone”, situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.

Conclusion

We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.