Functional screening for cellulolytic activity in a metagenomic fosmid library of microorganisms associated with coral.

Cellulases are enzymes that degrade cellulosic materials. Cellulose is the most abundant renewable carbon resource on Earth, and cellulases are used in various industrial sectors. Although cellulases are obtained from a variety of sources, this is the first description of cellulolytic activity isolated from a coral metagenomic library. A metagenomic fosmid library of microorganisms associated with the coral Siderastrea stellata, comprising 3552 clones, was screened for cellulolytic activity; this allows access to non-cultivable microorganisms by exploiting the full biotechnological potential. Clones were grown on LB agar plates supplemented with 0.5% carboxymethylcellulose and cellulase positive clones revealed by staining with Congo red. Using this approach, six positive clones with cellulolytic activity were identified. The enzymatic index (EI) of the positive clones was calculated by the ratio between the hydrolysis zone diameter and colony diameter. All positive clones had an EI greater than 1.5. Digestion of the DNA isolated from the six positive clones, using the HindIII restriction endonuclease, revealed different restriction patterns in each clone, indicating that the DNA of each clone is different. There is a growing interest for new cellulolytic enzymes in various industry sectors. Here, we present the initial selection of potential clones for cellulose degradation that could be targets for future studies of enzymatic characterization.

Long-term results of treatment of neovascular age-related macular degeneration using antiangiogenic drugs: A review of the literature.

Age-related macular degeneration (AMD) is one of the main causes of visual acuity (VA) loss in people over 50 years of age worldwide, with neovascular AMD (nAMD) accounting for 80% of cases of severe vision loss due to this disease. Anti-vascular endothelial growth factor (anti-VEGF) drugs have been used for the treatment of this disease for more than a decade, changing drastically the visual prognosis of these patients. However, initial studies reporting data on outcomes were short term. Currently, there are different series published on the long-term results of AMD after treatment with anti-VEGF, and the aim of this review is to synthesize these results. The mean follow-up of the included studies was 8.2 years (range 5-12 years). The mean initial VA was 55.3 letters in the Early Treatment Diabetic Retinopathy Study (ETDRS) (range 45.6-65) and the mean final VA was 50.1 letters (range 33.0-64.3), with a mean loss of 5.2 letters. At the end of follow-up, 29.4% of the patients maintained a VA > 70 letters. The 67.9% of patients remained stable at the end of follow-up (< 15 letter loss), with a severe loss (≥ 15 letters) of 30.1%. Fibrosis and atrophy were the main causes of long-term VA loss, occurring at the end of follow-up in 52.5% and 60.5%, respectively.

Update on the utility of en face optical coherence tomography in the assessment of vitreoretinal diseases.

En face optical coherence tomography (EF-OCT) is a rapid, non-invasive, high-resolution imaging technique that has evolved in recent years to be a routine examination for the assessment and follow-up of various vitreoretinal diseases. With the introduction of swept-source OCT (SS-OCT), which can achieve up to 100,000 A-scans per second and better-quality imaging of deeper structures using a longer wavelength (1050nm), EF-OCT reconstruction can produce high-resolution frontal images of the retina and choroid (C-Scans) that give an overview of disease extent. These images allow a more accurate study of vitreoretinal interface pathologies such as epiretinal membranes, macular holes, and vitreomacular traction. They also provide key information in the study of various retinal vascular diseases and the differential diagnosis of cystic macular edema. EF-OCT provides valuable information about the severity of vitreoretinal interface alterations and precisely assesses the choriocapillaris and choroidal vasculature in pachychoroid disorders. Finally, this technique provides valuable information about atrophic and neovascular age-related macular degeneration and various uveitic entities. This review aims to describe the current clinical applications of EF-OCT in various vitreoretinal diseases as well as the latest findings and future perspectives.

Pancreatic islet-acinar cell interaction: amylase messenger RNA levels ar determined by insulin.

Pancreatic amylase messenger RNA progressively decreases in rats rendered diabetic with streptozotocin. Insulin reverses this effect, inducing a selective decrease in amylase messenger RNA in the pancreas. Parotid amylase messenger RNA is not significantly affected by either diabetes or insulin.

Rhizobium nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs

Application of Nod factors to growing, responsive root hairs of the bean Phaseolus vulgaris induces marked changes in both the intracellular cytosolic free calcium (Ca2+) and in the influx of extracellular [Ca2+]. The intracellular [Ca2+], which has been measured by ratiometric imaging in cells microinjected with fura-2-dextran (70 kDa), elevates within 5 min from approximately 400 nM to 1500 nM in localised zones in the root hair apex. Of particular note is the observation that the elevated regions of [Ca2+] appear to shift position during short time intervals. Increases in and fluctuations of the intracellular [Ca2+] are also observed in the perinuclear region after 10-15 min treatment with Nod factors. The extracellular Ca2+ flux, detected with the non-invasive, calcium specific vibrating electrode, is inwardly directed and also increases quickly in response to Nod factors from 13 pmol cm-2 s-1 to 28 pmol cm-2 s-1. Chitin-oligomers, which are structurally similar but biologically inactive when compared to the active Nod factors, fail to elicit changes in either intracellular or extracellular Ca2+. The similar timing and location of the intracellular elevations and the increased extracellular influx provide support for the idea that Ca2+ participates in secretion and cell wall remodelling, which occur in anticipation of root hair deformation and curling.

Rearrangement of actin microfilaments in plant root hairs responding to rhizobium etli nodulation signals

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.

Constant subcutaneous infusion of rhIL-11 in mice: efficient delivery enhances biological activity.

Previous studies have shown that subcutaneous (SC) bolus administration of recombinant human interleukin-11 (rhIL-11) stimulates megakaryocytopoiesis and increases peripheral platelet counts in naive mice. This study was designed to determine whether administration of rhIL-11 by constant SC infusion altered either the magnitude of the nature of the hematologic response. Female C57BL/6 mice were implanted subcutaneously with 7-day Alzet mini-osmotic pumps containing either rhIL-11 with 0.5% homologous mouse serum (delivery rate of 250 microg/kg/d) or vehicle alone. Mice were sacrificed on days 3, 7, 10, and 13 after pump implantation, and the hematopoietic response was compared to mice receiving an equivalent dose of rhIL-11 administered by SC injection (250 microg/kg/d, 7 days) or vehicle controls. Subcutaneous injection of rhIL-11 resulted in a significant increase in peripheral platelet counts with a maximum platelet increase of 44% over controls observed on day 7 of the study. Platelet counts subsequently declined (24% by day 10) returning to control values by day 13. The increase in peripheral platelet counts was accompanied by an increase in reticulated platelets on day 7 and a shift to higher ploidy bone marrow megakaryocytes on days 3 and 7. Compared to SC injection, both the magnitude and duration of the platelet increase were significantly enhanced following continuous SC infusion of rhIL-11. Maximum platelet counts were detected on day 10 (115% above vehicle controls), and platelets remained significantly elevated on day 13 (84%), 6 days after rhIL-11 administration had stopped. Consistent with the platelet response, the modal ploidy of bone marrow megakaryocytes was shifted from 16N to 32N on days 3 and 7, with increases in 32N megakaryocytes still apparent on days 10 and 13. There was also a significant increase in reticulated platelets detected in the peripheral blood on days 3, 7, and 10 compared to mice administered rhIL-11 by SC injection, The changes in reticulated platelets and bone marrow megakaryocyte ploidy are consistent with the increased and prolonged platelet response following SC infusion of rhIL-11. In addition to the effects observed on peripheral platelet counts, constant SC infusion of rhIL-11 dramatically enhanced splenic hematopoietic activity, increasing spleen weight and cellularity as well as splenic megakaryocyte, erythroid, granulocyte, and macrophage progenitors compared to mice receiving rhIL-11 by SC injection.

Actin monoubiquitylation is induced in plants in response to pathogens and symbionts.

Most dramatic examples of actin reorganization have been described during host-microbe interactions. Plasticity of actin is, in part, due to posttranslational modifications such as phosphorylation or ubiquitylation. Here, we show for the first time that actins found in root nodules of Phaseolus vulgaris are modified transiently during nodule development by monoubiquitylation. This finding was extended to root nodules of other legumes and to other plants infected with mycorrhiza or plant pathogens such as members of the genera Pseudomonas and Phytophthora. However, neither viral infections nor diverse stressful conditions (heat shock, wounding, or osmotic stress) induced this response. Additionally, this phenomenon was mimicked by the addition of a yeast elicitor or H2O2 to Phaseolus vulgaris suspension culture cells. This modification seems to provide increased stability of the microfilaments to proteolytic degradation and seems to be found in fractions in which the actin cytoskeleton is associated with membranes. All together, these data suggest that actin monoubiquitylation may be considered an effector mechanism of a general plant response against microbes.

Sulfation of nod factors via nodHPQ is nodD independent in Rhizobium tropici CIAT899.

A cosmid from the Rhizobium tropici CIAT899 symbiotic plasmid, containing most of the nodulation genes described in this strain, has been isolated. Although this cosmid does not carry a nodD gene, it confers ability to heterologous Rhizobium spp. to nodulate R. tropici hosts (Phaseolus vulgaris, Macroptilium atropurpureum, and Leucaena leucocephala). The observed phenotype is due to constitutive expression of the nodABCSUIJ operon, which has lost its regulatory region and is expressed from a promoter present in the cloning vector. Thin-layer chromatography (TLC) analysis of the Nod factors produced by this construction shows that it is still capable of synthesizing sulfated compounds, suggesting that the nodHPQ genes are organized as an operon that is transcribed in a nodD-independent manner and is not regulated by flavonoids.

Characterization of Rhizobium tropici CIAT899 nodulation factors: the role of nodH and nodPQ genes in their sulfation.

We have purified and characterized the nodulation factors produced by Rhizobium tropici CIAT899. This strain produces a large variety of nodulation factors, these being a mixture of sulfated or nonsulfated penta- or tetra-chito-oligosaccharides to which any of six different fatty acyl moieties may be attached to nitrogen of the nonreducing terminal residue. In this mixture we have also found methylated or nonmethylated lipo-chitin oligosaccharides. Here we describe a novel lipo-chitin-oligosaccharide consisting of a linear backbone of 4 N-acetylglucosamine residues and one mannose that is the reducing-terminal residue and bearing a C18:1 fatty acyl moiety on the nonreducing terminal residue. In addition, we have identified, cloned, and sequenced R. tropici nodH and nodPQ genes, generated mutations in the nodH and nodQ genes, and tested the mutant strains for nodulation in Phaseolus and Leucaena plants. Our results indicate that the sulfate group present in wild-type Nod factors plays a major role in nodulation of Leucaena plants by strain CIAT899 of R. tropici.

A rapid-cycling bipolar patient treated with long nights, bedrest, and light.

BACKGROUND: Stabilization of rapid-cycling bipolar disorder is extremely difficult. METHODS: A refractory bipolar I rapid-cycling patient on valproate was treated with long "nights" (extended sleep in darkness) and daytime light therapy. RESULTS: Rapid cycling immediately stopped on initiation of a 10 hour dark/rest period. This was extended to 14 hours (plus a self-selected 1 hour midday nap) without problems. Depression gradually improved when midday light therapy was added; near-euthymia was attained after light therapy was shifted to the morning. CONCLUSIONS: Nonpharmacological chronobiological treatments may be a means to interrupt rapid cycling.

The role of the nodI and nodJ genes in the transport of Nod metabolites in Rhizobium etli.

A kinetic analysis of secretion of lipo-chitin oligosaccharides (LCO) produced by Rhizobium etli (Re) wild-type (wt) strain and derivatives carrying disrupted nodI or nodJ genes was performed. LCO were detected in the growth media of the wt strain as early as 1 h after nod gene induction. In contrast, strains carrying nodI or nodJ mutations secreted less LCO, and accumulated LCO metabolites intracellularly after 4 h of induction. These Re mutants presented a delayed nodulation phenotype and a reduction in the maximum number of nodules formed in Phaseolus vulgaris roots.

Nucleotide sequence of the Rhizobium etli nodS gene.

The complete nucleotide sequence of the nodS gene from the bean-nodulating Rhizobium etli, presumably encoding a methyltransferase, was determined. A phylogenetic analysis of five different NodS proteins from three genera of Gram- soil bacteria, Azorhizobium, Bradyrhizobium and Rhizobium, was performed.

Posttraumatic delayed sleep phase syndrome.

Circadian rhythm sleep disorders may occur after traumatic brain injury. We describe a 48-year-old man who presented with sleep onset insomnia and cognitive dysfunction after a car accident. A diagnosis of delayed sleep phase syndrome (DSPS) was confirmed by sleep logs and actigraphy, which revealed sleep onset in the early morning hours and awakening around noon.

Open-ring imaging sign: highly specific for atypical brain demyelination.

OBJECTIVE: To test the specificity for demyelination of a new neuroimaging sign: contrast enhancement shaped as an open ring or a crescent circumscribed to the white matter. BACKGROUND: Brain demyelination can cause ring enhancement mimicking neoplasm or infection on CT or MRI. METHODS: A MEDLINE search of pathology-proved demyelination yielded 32 illustrated cases of ring-enhancing lesions published between 1981 and 1995. Controls consisted of the same number of published images of neoplasms and infections, pathology proved, and matched by year of publication, and age and gender of the patient. Two neuroradiologists read the images twice independently 1 year apart. RESULTS: Interrater agreement was good (kappa = 0.64 and 0.66 for either reading). Test-retest reliability was high (kappa = 0.75 and 0.74 for either rater). The open-ring sign clearly distinguished demyelinating lesions from neoplasms and infections. For demyelination versus neoplasm or infection, the specificity of the reading by the first neuroradiologist was 93.8 (95% CI, 86 to 98), and that of the second was 84.4 (95% CI, 74 to 92). The likelihood ratio of demyelination versus neoplasm averaged 5.2, and versus infection, 17.2. That is, if the lesions had the same incidence in the population, in the presence of an open-ring sign demyelination would be five times more likely than neoplasm and 17 times more likely than infection. However, given the much higher incidence of neoplasms and infections, these lesions are still frequently responsible for open-ring enhancement. CONCLUSIONS: The open-ring sign is often present in large, contrast-enhancing demyelinating lesions and helps to differentiate them from neoplasms and infections.

Median and ulnar palm-wrist studies.

OBJECTIVES: Routine carpal tunnel electrodiagnosis frequently includes median (MPW) and ulnar (UPW) palm-wrist mixed nerve conduction latency determinations over 8 cm. Despite widespread use, normative palmar latency difference (PLD) and UPW values, and the relative utility of onset latency (OL) or peak latency (PL) measurements are controversial. The current study was conducted to determine normative values for these parameters. METHODS: MPW and UPW studies were performed unilaterally in 33 normal controls. The PLD-OL and PLD-PL were calculated. The mean, range, standard deviation, and upper limits of normal were determined. 74 hands (50 patients) with both clinical and electrophysiologic median neuropathy were also studied. RESULTS: The abnormal MPW and UPW cut-offs were both 1.8 ms (OL), and 2.3 ms (PL). The abnormal PLD cut-offs were 0.5 ms (OL and PL). Using either OL or PL, PLD parameters were similar within controls, and also within CTS patients. Using either OL or PL, UPW parameters were similar between controls and CTS patients. CONCLUSIONS: An abnormal PLD cut-off of 0.5 is recommended. This is slightly higher than some prior recommendations, however it should minimize the likelihood of false positive studies. Onset and peak latency measurements are likely to have similar clinical utility.

Biological nitrogen fixation and future challenges of agriculture. The endophytic connection.

Feeding the growing global population, anticipated to be 8 billion by the year 2020, is one of the most important recent challenges of agriculture. The increase in cereal grain yield, to cope with this demand, directly implies a dramatic increase in the use of nitrogen-based fertilizers and agrochemicals. Some of these intensive agricultural practices have progressive detrimental effects on the environment. This review is focused on some novel insights gained into the understanding of associative and symbiotic interactions of plants with nitrogen-fixing organisms that makes Biological Nitrogen Fixation (BNF) a viable answer to this compelling dilemma.