mEosFP-based green-to-red photoconvertible subcellular probes for plants.

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.

Effect of Silicon Nutrition on Oxidative Stress Induced by Phytophthora melonis Infection in Cucumber.

The effect of silicon nutrition on root rot of cucumber caused by Phytophthora melonis was studied in a greenhouse experiment. Two cucumber cultivars (Cucumis sativus 'Dominus' and 'Super Dominus') fertilized with three concentrations of Si (0.0, 1.0, and 1.7 mM Si in the form of sodium silicate) were not inoculated or were inoculated with P. melonis. The P. melonis isolate significantly decreased root dry weights, although the magnitude of growth reduction varied with cultivar and Si concentration. Silicon nutrition at either concentration significantly reduced disease severity relative to the treatment that received no silicon. There was a significant negative correlation between the extent of root rot caused by P. melonis and the extent of electrolyte leakage of roots. Roots infected with P. melonis had greater root catalase (CAT) and ascorbate peroxidase (APX) activities. There was a positive correlation between silicon concentrations and CAT and APX activities in plants inoculated with P. melonis. Silicon improved activity of antioxidant enzymes, resulting in enhanced crop resistance to oxidative stress induced by P. melonis infection and improved cucumber growth.

Case report: Xanthogranulomatous pyelonephritis masquerading as cystic renal cell carcinoma.

Background: Xanthogranulomatous pyelonephritis (XGP) is a rare chronic bacterial inflammation of the renal parenchyma and is often a diagnostic dilemma.Case Presentation: We present a challenging case of a patient with XGP. Initially thought to have had renal cell cancer she was treated accordingly with a partial nephrectomy. However, on the final pathology, she was found to have XGP and required further antibiotic therapy and referral to the infectious disease service.Discussion: Management of XGP and diagnostic pitfalls are discussed.Conclusion: XGP is a diagnostic and therapeutic dilemma. Partial Nephrectomy may be appropriate in management of XGP in select cases.

The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases.

BLM and WRN, the products of the Bloom's and Werner's syndrome genes, are members of the RecQ family of DNA helicases. Although both have been shown previously to unwind simple, partial duplex DNA substrates with 3'-->5' polarity, little is known about the structural features of DNA that determine the substrate specificities of these enzymes. We have compared the substrate specificities of the BLM and WRN proteins using a variety of partial duplex DNA molecules, which are based upon a common core nucleotide sequence. We show that neither BLM nor WRN is capable of unwinding duplex DNA from a blunt-ended terminus or from an internal nick. However, both enzymes efficiently unwind the same blunt-ended duplex containing a centrally located 12 nt single-stranded 'bubble', as well as a synthetic X-structure (a model for the Holliday junction recombination intermediate) in which each 'arm' of the 4-way junction is blunt-ended. Surprisingly, a 3'-tailed duplex, a standard substrate for 3'-->5' helicases, is unwound much less efficiently by BLM and WRN than are the bubble and X-structure substrates. These data show conclusively that a single-stranded 3'-tail is not a structural requirement for unwinding of standard B-form DNA by these helicases. BLM and WRN also both unwind a variety of different forms of G-quadruplex DNA, a structure that can form at guanine-rich sequences present at several genomic loci. Our data indicate that BLM and WRN are atypical helicases that are highly DNA structure specific and have similar substrate specificities. We interpret these data in the light of the genomic instability and hyper-recombination characteristics of cells from individuals with Bloom's or Werner's syndrome.

Waiting room educational media effect on preinjection anxiety for initial intravitreal injections.

OBJECTIVE: To ascertain the effectiveness of an educational web page in reducing anxiety associated with initial intravitreal anti-vascular endothelial growth factor injections. DESIGN: Single-centred, observation-enriched, randomized controlled trial. PARTICIPANTS: Ninety-six patients receiving intravitreal injections at the Hamilton Regional Eye Institute. METHODS: Patients aged 18 years or older scheduled to receive their first intravitreal injection were randomized to either view an educational web page pertaining to the injection procedure or wait 30 minutes. Both groups then completed the State-Trait Anxiety Inventory (STAI). A third cohort of patients who previously had intravitreal injections waited 30 minutes before completing the STAI. The difference between STAI anxiety scores across cohorts 1 through 3 was assessed using analysis of variance and independent t tests where applicable. RESULTS: Ninety-six patients completed the STAI questionnaires, of which 55 (57.3%) were female and 86 (89.6%) were Caucasian. The mean age of participants was 68.5 ± 14.2 years, 72.7 ± 12.9 years, and 70.4 ± 11.7 years for control, intervention, and treatment-experienced cohorts, respectively. The mean STAI score was 40.3 ± 12.0 for the control cohort, 39.3 ± 11.1 for the intervention cohort, and 30.2 ± 9.9 for the treatment-experienced cohort. No significant difference in STAI scores was observed between intervention and control cohorts (p = 0.716). The effect size between treatment-naïve and treatment-experienced cohorts was high, ranging from 0.862 and 0.919, and the mean difference in STAI scores was significant. CONCLUSIONS: Compared to treatment-experienced patients, treatment-naïve patients are more anxious. Electronic educational information about the intravitreal injection process may be ineffective at reducing procedure-induced anxiety.

Ozurdex in age-related macular degeneration as adjunct to ranibizumab (The OARA Study).

OBJECTIVE: To evaluate the utility of dexamethasone intravitreal implant (DXI; Ozurdex; Allergan, Irvine, Calif.) in combination with ranibizumab (Lucentis; Novartis Pharma AG, Basel, Switzerland) versus ranibizumab monotherapy on visual acuity (VA) and anatomical outcomes in a neovascular age-related macular degeneration (nAMD) cohort. DESIGN: Multicentred, single-blinded, pilot randomized control trial. PARTICIPANTS: Ten patients 50 years or older with subfoveal choroidal neovascularization secondary to AMD were randomized to receive DXI in combination with ranibizumab (group 1) or ranibizumab alone (group 2) after a 3-month ranibizumab loading period. METHODS: Group 1 patients received 1 DXI after the loading phase with the option of retreatment at months 4 to 6. Ranibizumab was administered pro re nata for 6 months in both study arms. Mean VA and central macular thickness (CMT) reductions from baseline to study endpoint (9 months) were reported in addition to adverse event frequency across study cohorts. RESULTS: From baseline to the study endpoint, VA improved by 10.8 ± 13.2 Early Treatment of Diabetic Retinopathy Study letters in the control arm and 3.0 ± 10.5 letters in the intervention arm (p = 0.331). CMT decreased by 31.7% ± 17.5% and 13.3% ± 27.0% (p = 0.236) for the control and intervention cohorts, respectively. One patient developed intraocular pressure in excess of 30 mm Hg 3 months after DXI administration. CONCLUSIONS: For this nAMD population, no visual or anatomical benefits were observed when treating with DXI in adjunct to ranibizumab relative to ranibizumab monotherapy. DXI-related adverse events were consistent with those previously documented for dexamethasone.

Predictors of functional vision changes after cataract surgery: the PROVISION study.

OBJECTIVE: To ascertain whether time-to-treatment, sex, age, preoperative functional vision scores, education, and ocular comorbidities predict change in functional vision pre- to postoperatively in patients receiving cataract surgery. DESIGN: Prospective cohort study. PARTICIPANTS: Three hundred and forty-three cataract patients at the Hamilton Regional Eye Institute. METHODS: Participants 18 years or older scheduled to undergo cataract surgery completed the Catquest-9SF functional vision questionnaire on the day of their surgery and were mailed a survey 2-3 months postoperatively. Multivariate linear regression was used to determine the ability of predictors to explain variability in functional vision change between questionnaire administrations. RESULTS: One hundred and sixty-six patients completed both baseline and follow-up questionnaires. Mean age of the cohort was 73.8 ± 8.1 years. Most patients were female (59.6%), had cataract surgery performed for the first time (66.9%), and had spent a mean time of 20.3 ± 20.7 weeks waiting for surgery. Functional vision improved in 83.7% of patients. The mean baseline Catquest-9SF score was the only significant predictor of functional vision improvement (adjusted R(2) = 0.47; F1,159 = 144.6; p < 0.001). Controlling for other variables, functional vision improved by 0.74 logits when mean baseline survey score increased by 1 logit. CONCLUSIONS: In most patients, functional vision improved after cataract surgery. Mean baseline Catquest-9SF score was a moderate predictor of the observed improvement.

A case of familial third ventricular colloid cyst.

Colloid cyst of the third ventricle is a rare benign intracranial lesion, and familial cases are rarer still. They may be asymptomatic or present with symptoms of raised intracranial pressure, including sudden death. Surgical excision is curative. We report a 24 year old pregnant woman with familial colloid cyst, who presented with headaches and suffered a cardiorespiratory arrest. Early computed tomography scan of the brain is advised in patients with a family history of third ventricular colloid cyst presenting to the accident and emergency department with headache.

Tibial cortical lesions: a multimodality pictorial review.

Shin pain is a common complaint, particularly in young and active patients, with a wide range of potential diagnoses and resulting implications. We review the natural history and multimodality imaging findings of the more common causes of cortically-based tibial lesions, as well as the rarer pathologies less frequently encountered in a general radiology department.

Analysis of mutations in the tudor domain of the survival motor neuron protein SMN.

Autosomal recessive childhood onset spinal muscular atrophy (SMA) is a leading cause of infant mortality caused by mutations in the survival motor neuron (SMN) gene. The SMN protein is involved in RNA processing and is localised in structures called GEMs in the nucleus. Nothing is yet understood about why mutations in SMN gene result in the selective motor neuron loss observed in patients. The SMN protein domains conserved across several species may indicate functionally significant regions. Exon 3 of SMN contains homology to a tudor domain, where a Type I SMA patient has been reported to harbour a missense mutation. We have generated missense mutants in this region of SMN and have tested their ability to form GEMs when transfected into HeLa cells. Our results show such mutant SMN proteins still localise to GEMs. Furthermore, exon 7 deleted SMN protein appears to exert a dominant negative effect on localisation of endogenous SMN protein. However, exon 3 mutant protein and exon 5 deleted protein exert no such effect.

Reducing transmitter release from nerve terminals influences motoneuron survival in developing rats.

Motoneurons in neonatal rats die following injury to the peripheral nerve. However, this vulnerability to nerve injury declines rapidly so that nerve injury at five days of age results in little if any motoneuron death. We have proposed that the role of the target during this critical period of development is to up-regulate the release of transmitter from developing motor nerve terminals. Here we show that reducing the release of acetylcholine from nerve terminals in neonatal rats can affect motoneuron maturation and survival. The soleus muscle in neonatal rats was treated with either magnesium or hemicholinium, and the number of motoneurons that survived was established 10 weeks later by retrograde labelling. Following treatment with magnesium, only 58.1% (+/-10.4 S.E.M., n=5) of the motoneurons in the soleus motor pool survived, although hemicholinium had no effect on motoneuron survival. However, those motoneurons that survived following treatment with either magnesium or hemicholinium did not develop normally since they remained susceptible to axotomy-induced cell death for longer than normal. In adult animals in which the sciatic nerve was crushed at five days of age following prior treatment with either magnesium or hemicholinium, only 27.6% (+/-6.2 S.E.M., n=5) and 44% (+/-6.1 S.E.M., n=4) of motoneurons in the sciatic motor pool survived, respectively, although no motoneurons died following injury alone or when injury was preceded by treatment with control implants containing NaCl. These results indicate that the release of acetylcholine from motor nerve terminals plays an important role in the development and survival of motoneurons.

DNA helicase deficiencies associated with cancer predisposition and premature ageing disorders.

Deficiency in a helicase of the RecQ family is found in at least three human genetic disorders associated with cancer predisposition and/or premature ageing. The RecQ helicases encoded by the BLM, WRN and RECQ4 genes are defective in Bloom's, Werner's and Rothmund-Thomson syndromes, respectively. Cells derived from individuals with these disorders in each case show inherent genomic instability. Recent studies have demonstrated direct interactions between these RecQ helicases and human nuclear proteins required for several aspects of chromosome maintenance, including p53, BRCA1, topoisomerase III, replication protein A and DNA polymerase delta. Here, we review this network of protein interactions, and the clues that they present regarding the potential roles of RecQ family members in DNA repair, replication and/or recombination pathways.

Characterization of a gene encoding survival motor neuron (SMN)-related protein, a constituent of the spliceosome complex.

Mutations in the gene encoding the Survival Motor Neuron (SMN) protein are responsible for autosomal recessive proximal spinal muscular atrophy (SMA). SMN orthologues have been identified in the nematode worm Caenorhabditis elegans and the yeast Schizosaccharomyces pombe but, to date, no human paralogues have been described. Here we describe identification and characterization of an SMN-related protein (SMNrp) gene that encodes a novel protein of 239 amino acids, which has recently been identified as a constituent of the spliceosome complex and designated SPF30. Significant similarity to the SMN protein is apparent only within a central region of SMNrp that represents a tudor domain. The SMNrp/SPF30 gene has been mapped to chromosome 10q23. It is differentially expressed, with abundant levels in skeletal muscle. An exclusively nuclear localization for SMNrp in cultured cells and muscle sections was revealed using GFP fusion constructs and thereafter confirmed with a polyclonal antibody raised against SMNrp. Overexpression of SMNrp as a fusion protein in HeLa cells in culture induced dose-dependent apoptosis with positive TUNEL staining. In addition to a possible role for this protein as a pro-apoptotic factor, SMN and its related protein share significant similarities in sequence and cellular function.

Direct interaction of Smn with dp103, a putative RNA helicase: a role for Smn in transcription regulation?

Spinal muscular atrophy is an autosomal recessive neurodegenerative disease of childhood, resulting from deletion or mutation of the survival motor neuron ( SMN ) gene on chromosome 5q13. SMN exists as part of a 300 kDa multi-protein complex, incorporating several proteins critically required in pre-mRNA splicing. Although SMN mutations render SMN defective in this role, the specific alpha-motor neuron degenerative phenotype seen in the disease remains unexplained. Here we demonstrate the isolation from mouse brain of the murine homologue of a recently identified novel RNA helicase of the DEAD box family, DP103, and its direct and specific binding of SMN. Previous work has shown that DP103 binds viral proteins known to interact with a cellular transcription factor to modulate gene expression. We suggest that the interaction between SMN and DP103 is further evidence for a role for SMN in transcriptional regulation and that SMN may be involved in the regulation of neuron-specific genes essential in neuronal development.

Role of the Bloom's syndrome helicase in maintenance of genome stability.

The RecQ family of DNA helicases has members in all organisms analysed. In humans, defects in three family members are associated with disease conditions: BLM is defective in Bloom's syndrome, WRN in Werner's syndrome and RTS in Rothmund-Thomson syndrome. In each case, cells from affected individuals show inherent genomic instability. The focus of our work is the Bloom's syndrome gene and its product, BLM. Here, we review the latest information concerning the roles of BLM in the maintenance of genome integrity.