Interacting protein partners of Arabidopsis RNA-binding protein AtRBP45b.

RNA binding proteins, important players in post-transcriptional gene regulation, usually exist in ribonuclear complexes. However, even in model systems like Arabidopsis characterisation of RBP associated proteins is limited. In this study, we investigated the interacting proteins of the Arabidopsis AtRBP45b, which is involved in stress signalling. In vivo localisation of AtRBP45b was conducted using 35S-GFP. FLAG-tagged AtRBP45b under control of the 35S promoter in the Atrbp45b-1 mutant background was used to pull down AtRBP45b interacting proteins. Yeast two-hybrid analysis, fluorescence energy resonance transfer assays were used to confirm the veracity of the AtRBP45b interacting proteins. In planta GFP-tagging indicated AtRBP45b is localised to the nucleus and the cytosol. AtRBP45b protein has a N-terminal proline-rich region and a C-terminal glutamine-rich domain that are usually involved in protein-protein interactions. Co-immunoprecipitation followed by mass spectrometry-based protein sequencing led to identification of 30 proteins that interacted with AtRBP45b. Using information from interactome databases (BIOGRID, INTACT and STRING), pull-down assays and localisation data, 12 putative interacting proteins were selected for yeast two-hybrid analysis. Cap-binding protein (CBP20, At5g44200) and polyA-binding protein (PAB8, At1g49760) were shown to interact with AtRBP45b. Based on its interacting partners we speculate that AtRBP45b may play an important role in RNA metabolism, especially in aspects related to mRNA stability and translation initiation during stress conditions in plants.

Transferrin liposomes of docetaxel for brain-targeted cancer applications: formulation and brain theranostics.

Diagnosis and therapy of brain cancer was often limited due to low permeability of delivery materials across the blood-brain barrier (BBB) and their poor penetration into the brain tissue. This study explored the possibility of utilizing theranostic d-alpha-tocopheryl polyethylene glycol 1000 succinate mono-ester (TPGS) liposomes as nanocarriers for minimally invasive brain-targeted imaging and therapy (brain theranostics). The aim of this work was to formulate transferrin conjugated TPGS coated theranostic liposomes, which contain both docetaxel and quantum dots (QDs) for imaging and therapy of brain cancer. The theranostic liposomes with and without transferrin decoration were prepared and characterized for their particle size, polydispersity, morphology, drug encapsulation efficiency, in-vitro release study and brain theranostics. The particle sizes of the non-targeted and targeted theranostic liposomes were found below 200 nm. Nearly, 71% of drug encapsulation efficiency was achieved with liposomes. The drug release from transferrin conjugated theranostic liposomes was sustained for more than 72 h with 70% of drug release. The in-vivo results indicated that transferrin receptor-targeted theranostic liposomes could be a promising carrier for brain theranostics due to nano-sized delivery and its permeability which provided an improved and prolonged brain targeting of docetaxel and QDs in comparison to the non-targeted preparations.

Transferrin receptor-targeted vitamin E TPGS micelles for brain cancer therapy: preparation, characterization and brain distribution in rats.

The effective treatment of brain cancer is hindered by the poor transport across the blood-brain barrier (BBB) and the low penetration across the blood-tumor barrier (BTB). The objective of this work was to formulate transferrin-conjugated docetaxel (DTX)-loaded d-alpha-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or TPGS) micelles for targeted brain cancer therapy. The micelles with and without transferrin conjugation were prepared by the solvent casting method and characterized for their particle size, polydispersity, drug encapsulation efficiency, drug loading, in vitro release study and brain distribution study. Particle sizes of prepared micelles were determined at 25 °C by dynamic light scattering technique. The external surface morphology was determined by transmission electron microscopy analysis and atomic force microscopy. The encapsulation efficiency was determined by spectrophotometery. In vitro release studies of micelles and control formulations were carried out by dialysis bag diffusion method. The particle sizes of the non-targeted and targeted micelles were <20 nm. About 85% of drug encapsulation efficiency was achieved with micelles. The drug release from transferrin-conjugated micelles was sustained for >24 h with 50% of drug release. The in vivo results indicated that transferrin-targeted TPGS micelles could be a promising carrier for brain targeting due to nano-sized drug delivery, solubility enhancement and permeability which provided an improved and prolonged brain targeting of DTX in comparison to the non-targeted micelles and marketed formulation.

Copolymers of poly(lactic acid) and D-α-tocopheryl polyethylene glycol 1000 succinate-based nanomedicines: versatile multifunctional platforms for cancer diagnosis and therapy.

INTRODUCTION: The major drawbacks associated with most of the anti-cancer drugs are their potential adverse effects. Distribution of these drugs throughout the body causes untoward adverse effects and less accumulation of drug at the site of tumors also causes decrease in therapeutic efficacy. Targeted nanomedicines are the emerging systems to improve the targetability of drug to the tumor site and to reduce the toxicity with maximum efficacy. Copolymers of poly-lactic acid (PLA) and D-α-tocopheryl polyethylene glycol 1000 succinate (Vitamin-E TPGS or TPGS) are innovative materials being actively investigated for the fabrication of non-targeted and targeted nanomedicines for diagnosis and therapy of cancer. AREAS COVERED: In this review, different nanomedicines of copolymers such as poly-lactic acid - polyoxyethylene sorbitan monooleate (PLA - Tween® 80), poly-lactic acid - poly-ethyleneglycol (PLA-PEG), poly-lactic acid-D-α-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS) and TPGS-based nanomedicines (i.e., TPGS emulsified polymeric nanoparticles, TPGS prodrugs, TPGS liposomes, and TPGS micelles) for the diagnosis and therapy of cancer have been discussed. EXPERT OPINION: PLA, PLA-Tween® 80, PLA-PEG, PLA-TPGS, and TPGS are the promising polymeric biomaterials well studied as cancer nanomedicines. These biomaterials have proved that they could be applied in the fabrication of multifunctional nanomedicines for the future needs in simultaneous diagnosis of cancer as well as targeted chemotherapy.

Varicella zoster virus vasculopathy: analysis of virus-infected arteries.

OBJECTIVE: Varicella zoster virus (VZV) is an under-recognized yet treatable cause of stroke. No animal model exists for stroke caused by VZV infection of cerebral arteries. Thus, we analyzed cerebral and temporal arteries from 3 patients with VZV vasculopathy to identify features that will help in diagnosis and lead to a better understanding of VZV-induced vascular remodeling. METHODS: Normal and VZV-infected cerebral and temporal arteries were examined histologically and by immunohistochemistry using antibodies directed against VZV, endothelium, and smooth muscle actin and myosin. RESULTS: All VZV-infected arteries contained 1) a disrupted internal elastic lamina; 2) a hyperplastic intima composed of cells expressing α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-myosin) but not endothelial cells expressing CD31; and 3) decreased medial smooth muscle cells. The location of VZV antigen, degree of neointimal thickening, and disruption of the media were related to the duration of disease. CONCLUSIONS: The presence of VZV primarily in the adventitia early in infection and in the media and intima later supports the notion that after reactivation from ganglia, VZV spreads transaxonally to the arterial adventitia followed by transmural spread of virus. Disruption of the internal elastic lamina, progressive intimal thickening with cells expressing α-SMA and SM-MHC, and decreased smooth muscle cells in the media are characteristic features of VZV vasculopathy. Stroke in VZV vasculopathy may result from changes in arterial caliber and contractility produced in part by abnormal accumulation of smooth muscle cells and myofibroblasts in thickened neointima and disruption of the media.

Review: The neurobiology of varicella zoster virus infection.

Varicella zoster virus (VZV) is a neurotropic herpesvirus that infects nearly all humans. Primary infection usually causes chickenpox (varicella), after which virus becomes latent in cranial nerve ganglia, dorsal root ganglia and autonomic ganglia along the entire neuraxis. Although VZV cannot be isolated from human ganglia, nucleic acid hybridization and, later, polymerase chain reaction proved that VZV is latent in ganglia. Declining VZV-specific host immunity decades after primary infection allows virus to reactivate spontaneously, resulting in shingles (zoster) characterized by pain and rash restricted to one to three dermatomes. Multiple other serious neurological and ocular disorders also result from VZV reactivation. This review summarizes the current state of knowledge of the clinical and pathological complications of neurological and ocular disease produced by VZV reactivation, molecular aspects of VZV latency, VZV virology and VZV-specific immunity, the role of apoptosis in VZV-induced cell death and the development of an animal model provided by simian varicella virus infection of monkeys.

Virus vasculopathy and stroke: an under-recognized cause and treatment target.

While arteriosclerotic disease and hypertension, with or without diabetes, are the most common causes of stroke, viruses may also produce transient ischemic attacks and stroke. The three most-well studied viruses in this respect are varicella zoster virus (VZV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV), all of which are potentially treatable with antiviral agents. Productive VZV infection in cerebral arteries after reactivation (zoster) or primary infection (varicella) has been documented as a cause of ischemic and hemorrhagic stroke, aneurysms with subarachnoid and intracerebral hemorrhage, arterial ectasia and as a co-factor in cerebral arterial dissection. CMV has been suggested to play a role in the pathogenesis of arteriosclerotic plaques in cerebral arteries. HIV patients have a small but definite increased incidence of stroke which may be due to either HIV infection or opportunistic VZV infection in these immunocompromised individuals. Importantly, many described cases of vasculopathy in HIV-infected patients were not studied for the presence of anti-VZV IgG antibody in CSF, a sensitive indicator of VZV vasculopathy. Unlike the well-documented role of VZV in vasculopathy, evidence for a causal link between HIV or CMV and stroke remains indirect and awaits further studies demonstrating productive HIV and CMV infection of cerebral arteries in stroke patients. Nonetheless, all three viruses have been implicated in stroke and should be considered in clinical diagnoses.

Psychogenic non-epileptic seizures, anticipatory grief and limited access to medical care.

Psychogenic non-epileptic seizures (PNES) in the context of meningoencephalitis can occur with sufficient stressors. Video-EEG is a critical diagnostic tool in such complex cases. Once the diagnosis of PNES is confirmed by video-EEG, psychiatric consultation with psychotherapeutic intervention to address underlying psychopathology and specific stressors that led to these conversion symptoms is required. Clinicians need to understand the importance of cultural themes, including the economics of healthcare, and anticipatory grief as causative stressors in pseudoseizures.

The varicella zoster virus vasculopathies: clinical, CSF, imaging, and virologic features.

BACKGROUND: Varicella zoster virus (VZV) vasculopathy produces stroke secondary to viral infection of cerebral arteries. Not all patients have rash before cerebral ischemia or stroke. Furthermore, other vasculitides produce similar clinical features and comparable imaging, angiographic, and CSF abnormalities. METHODS: We review our 23 published cases and 7 unpublished cases of VZV vasculopathy. All CSFs were tested for VZV DNA by PCR and anti-VZV IgG antibody and were positive for either or both. RESULTS: Among 30 patients, rash occurred in 19 (63%), CSF pleocytosis in 20 (67%), and imaging abnormalities in 29 (97%). Angiography in 23 patients revealed abnormalities in 16 (70%). Large and small arteries were involved in 15 (50%), small arteries in 11 (37%), and large arteries in only 4 (13%) of 30 patients. Average time from rash to neurologic symptoms and signs was 4.1 months, and from neurologic symptoms and signs to CSF virologic analysis was 4.2 months. CSF of 9 (30%) patients contained VZV DNA while 28 (93%) had anti-VZV IgG antibody in CSF; in each of these patients, reduced serum/CSF ratio of VZV IgG confirmed intrathecal synthesis. CONCLUSIONS: Rash or CSF pleocytosis is not required to diagnose varicella zoster virus (VZV) vasculopathy, whereas MRI/CT abnormalities are seen in almost all patients. Most patients had mixed large and small artery involvement. Detection of anti-VZV IgG antibody in CSF was a more sensitive indicator of VZV vasculopathy than detection of VZV DNA (p < 0.001). Determination of optimal antiviral treatment and benefit of concurrent steroid therapy awaits studies with larger case numbers.

The value of detecting anti-VZV IgG antibody in CSF to diagnose VZV vasculopathy.

BACKGROUND: Factors that may obscure the diagnosis of varicella zoster virus (VZV) vasculopathy include the absence of rash before TIAs or stroke as well as similar clinical features and imaging, angiographic, and CSF abnormalities to those of other vasculopathies. Diagnosis relies on virologic confirmation that detects VZV DNA, anti-VZV IgG antibody, or both in the CSF. METHODS: We reviewed our current 14 cases of patients diagnosed with VZV vasculopathy based on combined clinical, imaging, angiographic, or CSF abnormalities. All CSFs must have been tested for VZV DNA by PCR and for anti-VZV IgG antibody by enzyme immunoassay and found to be positive for either or both. Of the 14 subjects, 8 had a history of recent zoster, whereas 6 had no history of zoster rash before developing vasculopathy. RESULTS: All 14 subjects (100%) had anti-VZV IgG antibody in their CSF, whereas only 4 (28%) had VZV DNA. The detection of anti-VZV IgG antibody in CSF was a more sensitive indicator of VZV vasculopathy than detection of VZV DNA (p < 0.001). CONCLUSIONS: In varicella zoster virus (VZV) vasculopathy, the diagnostic value of detecting anti-VZV IgG antibody in CSF is greater than that of detecting VZV DNA. Although a positive PCR for VZV DNA in CSF is helpful, a negative PCR does not exclude the diagnosis of VZV vasculopathy. Only when the CSF is negative for both VZV DNA and anti-VZV IgG antibody can the diagnosis of VZV vasculopathy be excluded.

Presence of VZV and HSV-1 DNA in human nodose and celiac ganglia.

Polymerase chain reaction (PCR) revealed herpes simplex virus (HSV) and varicella zoster virus (VZV) DNA in human nodose and celiac ganglia. This is the first detection of VZV DNA in ganglia of the human autonomic nervous system. The ability of reactivated VZV to produce serious, sometimes fatal neurological disease in the absence of rash, raises the possibility that VZV reactivation from autonomic ganglia might be involved in visceral disease.

An animal model of varicella virus infection.

Varicella-zoster virus (VZV) causes chickenpox in children; establishes latency in cranial nerve, dorsal root, and autonomic ganglia; and reactivates decades later to produce zoster. VZV produces disease only in humans. Although attempts to produce disease and study VZV latency in experimentally infected animals have resulted in virus in trigeminal or dorsal root ganglia, no clinical signs of infection or reactivation developed. In contrast, simian varicella virus (SVV) produces a naturally occurring exanthematous disease in non-human primates that mimics human varicella. Experimental inoculation of non-human primates causes similar, if not identical, clinical and pathological changes observed in monkeys naturally infected with SVV. Like VZV, SVV becomes latent in ganglia and reactivates, often with entire body rash. SVV and VZV encode antigenically related polypeptides. Both virus genomes have been sequenced and shown to be colinear, sharing up to 75% DNA homology. During latency, an SVV homolog of one of the five VZV genes transcribed in latently infected human ganglia has been detected in monkey ganglia. Preliminary studies in which monkeys were inoculated intratracheally with SVV revealed the presence of viral DNA and RNA in multiple tissues, including blood mononuclear cells, months after experimental infection. These findings differed from the expected restricted localization of the virus DNA to ganglia only and the expected limited viral gene expression, and probably reflect the high virus load delivered intratracheally compared to natural SVV infection in monkeys. Nevertheless, clinical, pathological, and molecular similarities between SVV and VZV indicate that SVV infection in non-human primates has considerable potential as an animal model for human varicella.

Acute, chronic, and recurrent varicella zoster virus neuropathy without zoster rash.

The authors report three patients with acute, chronic, and recurrent neuropathy associated with varicella zoster virus (VZV) infection but without zoster rash. CSF of all three patients contained VZV immunoglobulin G antibody, but not herpes simplex virus. In two patients, serum/CSF ratios of VZV immunoglobulin G were reduced compared to normal ratios for immunoglobulin G and albumin, and one patient also had VZV immunoglobulin M in CSF. All three patients received antiviral therapy and improved. The diagnosis of nervous system infection by VZV may be confirmed by the presence of antibody to VZV in CSF even without detectable VZV DNA.

Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays.

We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced-PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced-PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for approximately 1,000 stress-induced and -repressed Arabidopsis genes.

The DNA sequence of the simian varicella virus genome.

In nonhuman primates, simian varicella virus (SVV) causes a natural disease which is clinically similar to human varicella-zoster virus (VZV) infections. The SVV and VZV genomes are similar in size and structure and share extensive DNA homology. This report presents the complete DNA sequence of the SVV genome. SVV DNA is 124,138 bp in size, 746 bp shorter than VZV DNA, and 40.4% G + C. The viral genome includes a 104,104-bp unique long component bracketed by 8-bp inverted repeat sequences and a short component composed of a 4904-bp unique short region bracketed by 7557-bp inverted repeat sequences. A total of 69 distinct SVV open reading frames (ORFs) were identified, including three that are duplicated within the inverted repeats of the short component. Each of the SVV ORFs shares extensive homology to a corresponding VZV gene. The only major difference between SVV and VZV DNA occurs at the leftward terminus. SVV lacks a VZV ORF 2 homolog. In addition, SVV encodes an 882-bp ORF A that is absent in VZV, but has homology to the SVV and VZV ORF 4. The results of this study confirm the relatedness of SVV and VZV and provide further support for simian varicella as a model to investigate VZV pathogenesis and latency.

Simian varicella virus infects ganglia before rash in experimentally infected monkeys.

Monkeys experimentally infected with simian varicella virus (SVV) develop rash 10-14 days later. However, the route and the time of ganglionic infection are unknown. Using PCR, we analyzed DNA extracted from tissues of 13 monkeys 5 to 60 days after either intratracheal or intravenous inoculation with SVV. SVV DNA was detected in ganglia from four of five monkeys sacrificed 6 to 7 days after intratracheal inoculation. Further, analysis of ganglia from monkeys sacrificed at 10 days revealed that intravenous inoculation produced a higher proportion of SVV DNA-positive ganglia (63%) than that after intratracheal inoculation (13%), pointing to the role of hematogenous spread in ganglionic infection. Like other organs, monkey ganglia become infected with SVV before the appearance of rash.

Sequence analysis of the leftward end of simian varicella virus (EcoRI-I fragment) reveals the presence of an 8-bp repeat flanking the unique long segment and an 881-bp open-reading frame that is absent in the varicella zoster virus genome.

Simian varicella virus (SVV) causes varicella (chickenpox) in nonhuman primates, becomes latent in cranial and dorsal root ganglia, and reactivates to produce zoster (shingles). Because the clinical and molecular features of SVV closely resemble those of varicella zoster virus (VZV) infection of humans, SVV infection of primates has served as an experimental model of VZV pathogenesis and latency. The SVV genome has been completely mapped, but attempts to clone the 3600-bp EcoRI fragment located at the leftward end of the virus genome have hitherto been unsuccessful. Herein, we report the cloning and the complete nucleotide sequence of this region. Comparison of the SVV and VZV sequences in this region revealed an 8-bp inverted repeat sequence flanking the unique long segment of the SVV genome; an 879-bp open-reading frame (ORF) A in SVV that is absent in VZV but has 42% amino acid identity to SVV ORF 4 and 49% to VZV ORF 4; a 342-bp ORF B in SVV with 35% amino acid identity to a 387-bp ORF located to the left of ORF 1 on the VZV genome; and a 303-bp ORF in SVV with 27% amino acid identity to VZV ORF 1. No homologue of VZV ORF 2 was detected. Transcripts specific for ORFs A and B were present in SVV-infected cells in culture and in acutely infected monkey ganglia. Overall, there are more than 2000 bp of DNA in the SVV genome that are absent in the VZV genome.

Solidification and stabilization of asbestos waste from an automobile brake manufacturing facility using cement.

Currently, the generated brake lining waste dust, which contains asbestos as its major component, is disposed of into a secure landfill without any additional treatment. As an alternative to this, solidification/stabilization (S/S) disposal of the dust was investigated using Portland cement alone and Portland cement mixed with activated carbon (AC), as the binders. Toxicity Characteristics Leaching Procedure (TCLP) results on the solidified matrix showed that cement was able to immobilize the heavy metals, Ba, Zn, Cr, Pb, Cu and Fe, to within the limits set by the US EPA for TCLP. Addition of AC to the cement reduced the leaching of heavy metals by an additional 4-24% compared to cement alone. The pH of the TCLP leachate extracted from virgin cement, and from dust treated with cement with or without AC was found to increase to 10.9-12.5 as opposed to an initial value of 4.93 for the TCLP extract for the untreated dust. Results of ANS 16.1 (modified) leach protocol revealed that Ba in cement-treated samples showed the highest leach rate, followed by Zn, Pb, Cr, Cu and Fe. The leach rate of heavy metals decreased with progress in time. Cement mixed with AC exhibited similar leach characteristics, however, the leach rate was lower. The linear relationship between the cumulative fraction leached (CFL) and the square root of leaching time in all cement-based samples indicate that a diffusional process is the controlling transport mechanism for the leaching of the heavy metals. The obtained Leachability Indices (L(i)) of 7.6-9.1 and 8.3-9.5 for cement and cement with AC, respectively, were low but exceeded the guidance value of 6, which clearly indicates that all the heavy metals studied are retained well within solid matrices. Cement-based S/S hardening times increased from 30 to 96 h as the dust content increased from 40 to 70 wt.%. The resulting solid matrices exhibited a compressive strength ranging from 1 to 12 MPa, which was well above the specified limit of 414 kPa for such matrices. An economic analysis indicates that the disposal costs for the dust in the only available secure landfill would increase by 40.3% if one were to go for the cement S/S option. Addition of AC to the cement would escalate this by an additional 43.8%. Although the S/S of brake lining dust using cement effectively immobilized the heavy metals of concern, cost considerations may hinder the commercial adaptation of this technique for waste disposal unless new regulatory demands are implemented.