The Bradyrhizobium Sp. LmicA16 Type VI Secretion System Is Required for Efficient Nodulation of Lupinus Spp.

Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and Lupinus angustifolius. A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild-type (wt) strain and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26-kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidity than the wt strain in free-living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free-living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain.

RNA sequencing and analysis of three Lupinus nodulomes provide new insights into specific host-symbiont relationships with compatible and incompatible Bradyrhizobium strains.

Nitrogen fixation in the legume root-nodule symbiosis has a critical importance in natural and agricultural ecosystems and depends on the proper choice of the symbiotic partners. However, the genetic determinism of symbiotic specificity remains unclear. To study this process, we inoculated three Lupinus species (L. albus, L. luteus, L. mariae-josephae), belonging to the under-investigated tribe of Genistoids, with two Bradyrhizobium strains (B. japonicum, B. valentinum) presenting contrasted degrees of symbiotic specificity depending on the host. We produced the first transcriptomes (RNA-Seq) from lupine nodules in a context of symbiotic specificity. For each lupine species, we compared gene expression between functional and non-functional interactions and determined differentially expressed (DE) genes. This revealed that L. luteus and L. mariae-josephae (nodulated by only one of the Bradyrhizobium strains) specific nodulomes were richest in DE genes than L. albus (nodulation with both microsymbionts, but non-functional with B. valentinum) and share a higher number of these genes between them than with L. albus. In addition, a functional analysis of DE genes highlighted the central role of the genetic pathways controlling infection and nodule organogenesis, hormones, secondary, carbon and nitrogen metabolisms, as well as the implication of plant defence in response to compatible or incompatible Bradyrhizobium strains.

Functional and expression analysis of the metal-inducible dmeRF system from Rhizobium leguminosarum bv. viciae.

A gene encoding a homolog to the cation diffusion facilitator protein DmeF from Cupriavidus metallidurans has been identified in the genome of Rhizobium leguminosarum UPM791. The R. leguminosarum dmeF gene is located downstream of an open reading frame (designated dmeR) encoding a protein homologous to the nickel- and cobalt-responsive transcriptional regulator RcnR from Escherichia coli. Analysis of gene expression showed that the R. leguminosarum dmeRF genes are organized as a transcriptional unit whose expression is strongly induced by nickel and cobalt ions, likely by alleviating the repressor activity of DmeR on dmeRF transcription. An R. leguminosarum dmeRF mutant strain displayed increased sensitivity to Co(II) and Ni(II), whereas no alterations of its resistance to Cd(II), Cu(II), or Zn(II) were observed. A decrease of symbiotic performance was observed when pea plants inoculated with an R. leguminosarum dmeRF deletion mutant strain were grown in the presence of high concentrations of nickel and cobalt. The same mutant induced significantly lower activity levels of NiFe hydrogenase in microaerobic cultures. These results indicate that the R. leguminosarum DmeRF system is a metal-responsive efflux mechanism acting as a key element for metal homeostasis in R. leguminosarum under free-living and symbiotic conditions. The presence of similar dmeRF gene clusters in other Rhizobiaceae suggests that the dmeRF system is a conserved mechanism for metal tolerance in legume endosymbiotic bacteria.

Genetic diversity of indigenous rhizobial symbionts of the Lupinus mariae-josephae endemism from alkaline-limed soils within its area of distribution in Eastern Spain.

The genomic diversity of a collection of 103 indigenous rhizobia isolates from Lupinus mariae-josephae (Lmj), a recently described Lupinus species endemic to alkaline-limed soils from a restricted habitat in Eastern Spain, was investigated by molecular methods. Isolates were obtained from soils of four geographic locations in the Valencia province that harbored the known Lmj plant populations. Using an M13 RAPD fingerprinting technique, 19 distinct RAPD profiles were identified. Phylogenetic analysis based on 16S rDNA and the housekeeping genes glnII, recA and atpD showed a high diversity of native Bradyrhizobium strains that were able to establish symbiosis with Lmj. All the strains grouped in a clade unrelated to strains of the B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. The phylogenetic tree based on concatenated glnII, recA and atpD gene sequences grouped the Lmj isolates in six different operational taxonomic units (OTUs) at the 93% similarity level. These OTUs were not associated to any specific geographical location, and their observed divergence predicted the existence of different Bradyrhizobium genomic species. In contrast, phylogenetic analysis of symbiotic genes based on nodC and nodA gene sequences, defined only two distinct clusters among the Lmj strains. These two Lmj nod gene types were largely distinct from nod genes of bradyrhizobia nodulating other Old World lupine species. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host.

Anatomical and functional damage in experimental glaucoma.

Glaucoma is a progressive neurodegenerative disease caused by retinal ganglion cell (RGC) loss. One important risk factor for glaucoma is elevated intraocular pressure and thus many animal models are based on spontaneous or induced ocular hypertension (OHT). Using these models it has been shown that RGCs initially suffer an impairment of the active axonal transport that progresses to a lack of passive diffusion along the axon. This axonal damage eventually causes the death of the parent RGCs in pie-shaped sectors of the retina, but there is also diffuse RGC loss, without involving displaced amacrine cells. Recent data show that OHT results in a protracted insult to the inner and outer retina that causes functional alterations and ultimately, degeneration and death of cones.

Alanine aminotransferase levels are not significantly elevated in patients with HIV/HBV co-infection and lamivudine resistance.

In hepatitis B virus (HBV) monoinfection, alanine aminotransferase (ALT) levels are linearly correlated with HBV DNA levels and lamivudine resistance. In human immunodeficiency virus (HIV)/HBV co-infection, little is known about the association between ALT, HBV DNA, and lamivudine resistance. We assessed HBV DNA, lamivudine resistance and ALT levels in 45 time points in 11 patients with HIV/HBV co-infection during lamivudine-containing antiretroviral therapy. High HBV DNA levels (>10(6) copies/mL) and lamivudine resistance developed in 45% and 91% of patients, respectively. However, ALT levels were not elevated in the setting of high HBV DNA levels (mean ALT, 48 IU/mL) or lamivudine resistance (mean ALT, 44 IU/mL). HBV viraemia and lamivudine resistance during extended lamivudine-containing antiretroviral therapy are common in HIV/HBV co-infection, occurring in the absence of significant ALT elevations. In HIV/HBV co-infection, measurement of HBV DNA and HBV resistance mutations may identify HBV virological failure before biochemical changes and should be routinely used in the management of HIV/HBV co-infection.

Biodiversity of uptake hydrogenase systems from legume endosymbiotic bacteria.

Uptake hydrogenases in legume endosymbiotic bacteria recycle hydrogen produced during the nitrogen fixation process in legume nodules. Despite the described beneficial effect on plant productivity, the hydrogen oxidation capability is not widespread in the Rhizobiaceae family. Characterization of hydrogenase gene clusters in strains belonging to Rhizobium, Bradyrhizobium and Azorhizobium reveals a similar overall genetic organization along with important differences in gene regulation. In addition, phylogenetic analysis of hup genes indicates distinct evolutionary origins for hydrogenase genes in Rhizobia.

Genetics and biotechnology of the H(2)-uptake [NiFe] hydrogenase from Rhizobium leguminosarum bv. viciae, a legume endosymbiotic bacterium.

A limited number of strains belonging to several genera of Rhizobiaceae are capable of expressing a hydrogenase system that allows partial or full recycling of hydrogen evolved by nitrogenase, thus increasing the energy efficiency of the nitrogen fixation process. This review is focused on the genetics and biotechnology of the hydrogenase system from Rhizobium leguminosarum bv. viciae, a frequent inhabitant of European soils capable of establishing symbiotic association with peas, lentils, vetches and other legumes.

[Central retinal vein occlusion in a factor V leiden and G21210A prothrombin variant carrier]. / Obstrucción de la vena central de la retina en portador de factor V de Leiden y variante G20210A de la protrombina.

CASE REPORT: A fifty-five year old man complained of diminished visual acuity in his right eye and reported a deep venous thrombosis in his right leg five years ago. Examination showed a central retinal vein occlusion in the right eye. Mutations in the factor V gene and prothrombin gene were found in a thrombophilia study. The patient was anticoagulated and no laser photocoagulation was required. DISCUSSION: Various coagulation disorders induced by genetic mutations are often associated with an increased risk for retinal vein occlusion although there are no statistically significant associations reported in the literature.

Obstrucción de la vena central de la retina en portador de factor V de Leiden y variante G20210a de la protrombina / Central retinal vein occlusion in a factor V leiden and G21210a prothrombin variant carrier

Caso clínico: Varón de 55 años con déficit de agudeza visual en ojo derecho y un episodio de trombosis venosa profunda en la pierna derecha cinco años atrás. Presentaba una obstrucción de la vena central de la retina. El estudio de trombofilia mostró mutaciones en el gen del factor V (factor Leiden) y de la protrombina (G20210A).El paciente fue anticoagulado, no precisando fotocoagulación láser en su evolución. Discusión: Diversos trastornos de la coagulación originados por mutaciones genéticas se han relacionado con un riesgo elevado de oclusión venosa retiniana, aunque diversos estudios no han demostrado una asociación estadísticamente significativa (AU)

Comparison of quality of life, work productivity and medical resource utilization of peginterferon alpha 2a vs the combination of interferon alpha 2b plus ribavirin as initial treatment in patients with chronic hepatitis C.

The on-treatment impact of interferon-based therapies on quality of life (QOL), work productivity, and medical resource utilization has not been systematically studied. We evaluated the effects of treatment with peginterferon alpha (pegIFNalpha) 2a monotherapy and the combination of interferon alpha (IFNalpha) 2b plus ribavirin (RBV) on health-related QOL, work productivity and resource utilization. A total of 412 patients with hepatitis C infection were randomized to open-label treatment with either pegIFNalpha 2a (n = 206) or IFNalpha 2b/RBV (n = 206). PegIFNalpha 2a was administered subcutaneously at a dose of 180 microg once weekly for 48 weeks; and IFNalpha 2b/RBV at doses of 3 MU thrice weekly subcutaneously and 1000-1200 mg/day orally. Outcome measures included the SF-36 Health Survey Questionnaire and additional generic and specific scales. During treatment, for all SF-36 summary and Hepatitis Quality of Life Questionnaire (HQLQ)-specific scales, the pegIFNalpha 2a group experienced less impairment than did the IFNalpha 2b/RBV patients. The between-treatment differences were significant for many of the scores particularly in the first 24 weeks of treatment. Across all measures of work functioning and productivity at each visit, patients randomized to pegIFNalpha 2a treatment showed less impairment relative to the group treated with IFNalpha 2b/RBV. Hence treatment with pegIFNalpha 2a relative to IFNalpha 2b/RBV minimizes the adverse impact of therapy on health-related QOL. Patients randomized to pegIFNalpha 2a had improved work productivity, less activity impairment, decreased need for prescription drugs to treat adverse effects, and better adherence to therapy.

[Proliferative radiation retinopathy]. / Retinopatía proliferativa por radiación.

CASE REPORT: A thirty year-old-woman that had received radiotherapy three years before for a frontal glyoma consulted because of diminished visual acuity in her left eye. Examination showed a proliferative radiation retinopathy in the left eye and non-proliferative radiation retinopathy in the right eye that was confirmed by fluorescein angiography. The patient was treated with panretinal photocoagulation, and her visual acuity remained stable. DISCUSSION: Patients receiving cranial or neck radiotherapy should be followed for long periods of time because radiation retinopathy may appear many years after the treatment. Follow-up may permit early diagnosis of ischemic radiation retinopathy that can benefit from laser photocoagulation.

Retinopatía proliferativa por radiación / Proliferative radiation retinopathy

Caso Clínico: Mujer de treinta años, tratada con radioterapia por un glioma frontal tres años atrás, que refería déficit de agudeza visual en el ojo izquierdo (OI). Presentaba una retinopatía isquémica proliferante en OI y no proliferante en el derecho, lo cual fue confirmado mediante angiografía fluoresceínica. La paciente fue panfotocoagulada, quedando estabilizada la agudeza visual. Discusión: La retinopatía por radiación puede aparecer muchos años después de aplicada ésta, por lo que los pacientes que reciben radioterapia de cabeza y cuello deben ser controlados para detectarla precozmente ya que se pueden beneficiar del tratamiento con láser (AU)

Insulin secretory responses to rising and falling glucose concentrations are delayed in subjects with impaired glucose tolerance.

AIMS/HYPOTHESIS: We hypothesized that beta-cell responses to changes in glucose would not be normal in subjects with impaired glucose tolerance (IGT). METHODS: Three groups of 6 subjects were studied: normal weight with normal glucose tolerance (control subjects); obese with normal glucose tolerance (Obese-NGT); and obese with IGT (Obese-IGT). All subjects had a graded glucose infusion protocol to increase (step-up) and then decrease (step-down) plasma glucose. We obtained average insulin-secretion rates (ISR) over the glucose range common to all three groups during step-up and step-down phases, minimal model indices of beta-cell function (f(b), f(d), f(s), T(up), T(down) ), and insulin sensitivity (Si). RESULTS: ISR differed significantly between step-up and -down phases only in Obese-IGT individuals. Basal (f(b)) and stimulated (f(d), f(s)) beta-cell sensitivity to glucose were similar in the three groups. Delays between glucose stimulus and beta-cell response during both step-up (T(up)) and -down (T(down)) phases were higher in Obese-IGT compared to Controls and Obese-NGT individuals. The product ISR x Si (10(-5.)min(-2) x l) was lower in Obese-IGT compared to Controls, both during step-up (919 +/- 851 vs 3192 +/- 1185, p < 0.05) and step-down (1455 +/- 1203 vs 3625 +/- 691, p < 0.05) phases. Consistently, the product f(s) x Si (10(-14.)min(-2). pmol(-1) x l) was lower in Obese-IGT than in control subjects (27.6 +/- 25.4 vs 103.1 +/- 20.2, p < 0.05). CONCLUSION/INTERPRETATION: Subjects with IGT are not able to secrete insulin to compensate adequately for insulin resistance. They also show delays in the timing of the beta-cell response to glucose when glucose levels are either rising or falling.

Engineering the Rhizobium leguminosarum bv. viciae hydrogenase system for expression in free-living microaerobic cells and increased symbiotic hydrogenase activity.

Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.

Neuroprotective effects of brimonidine against transient ischemia-induced retinal ganglion cell death: a dose response in vivo study.

The purpose of this study was to investigate the dose-response effects of topically administered brimonidine (BMD) on retinal ganglion cell (RGC) survival, short and long periods of time after transient retinal ischemia. In adult Sprague-Dawley rats, RGCs were retrogradely labeled with the fluorescent tracer fluorogold (FG) applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 90 min. One hr prior to retinal ischemia, two 5 microl drops of saline alone or saline containing 0.0001, 0.001, 0.01 or 0.1% BMD were instilled on the left eye. Rats were processed 7, 14 or 21 days later and densities of surviving RGCs were estimated by counting FG-labeled RGCs in 12 standard regions of each retina. The following have been found. (1) Seven days after 90 min of transient ischemia there is loss of approximately 46% of the RGC population. (2) topical pre-treatment with BMD prevents ischemia-induced RGC death in a dose-dependent manner. Administration of 0.0001% BMD resulted in the loss of approximately 37% of the RGC population and had no significant neuroprotective effects. Administration of higher concentrations of BMD (0.001 or 0.01%) resulted in the survival of 76 or 90%, respectively, of the RGC population, and 0.1% BMD fully prevented RGC death in the first 7 days after ischemia. (3) Between 7 and 21 days after ischemia there was an additional slow cell loss of approximately 25% of the RGC population. Pre-treatment with 0.1% BMD also reduced significantly this slow cell death. These results indicate that the neuroprotective effects of BMD, when administered topically, are dose-dependent and that the 0.1% concentration achieves optimal neuroprotective effects against the early loss of RGCs. Furthermore, this concentration is also effective to diminish the protracted loss of RGCs that occurs with time after transient ischemia.

Retinal ganglion cell death after acute retinal ischemia is an ongoing process whose severity and duration depends on the duration of the insult.

In adult Sprague-Dawley rats we have investigated retinal ganglion cell survival after transient intervals of retinal ischemia of 30, 45, 60, 90 or 120 min duration, induced by ligature of the ophthalmic vessels. Animals were killed 5, 7, 14, 21, 30, 60, 90 or 180 days later and densities of surviving retinal ganglion cells were estimated in retinal whole mounts by counting cells labelled with diAsp. This dye was applied, 3 days prior to death, to the ocular stump of the intraorbitally transected optic nerve. We found that retinal ganglion cell loss after retinal ischemia proceeds for different lengths of time. All the ischemic intervals induced loss of retinal ganglion cells whose severity and duration was related to the length of the ischemic interval. Following 30 or 45 min of ischemia, cell loss lasted 14 days and caused the death of 46 or 50%, respectively, of the population of retinal ganglion cells. Sixty, 90 or 120 min of retinal ischemia were followed by a period of cell loss that lasted up to 90 days and caused the death of 75%, 87% or 99%, respectively, of the population of retinal ganglion cells. We conclude that retinal ganglion cell loss after retinal ischemia is an ongoing process that may last up to 3 months after the injury and that its severity and duration are determined by the ischemic interval.

Brimonidine's neuroprotective effects against transient ischaemia-induced retinal ganglion cell death.

PURPOSE: Brimonidine is a lowering pressure agent currently used in glaucoma. This chronic degenerative condition is characterised by neuronal death, and an agent which offers neuroprotection may slow down or impede the progression of neuronal cell death. METHODS: The effects of brimonidine (BMD) on the short- and long-term survival of retinal ganglion cells (RGCs) after transient retinal ischaemia are reported here using a rat model. The fluorescent tracer Fluorogold (FG) was applied to both superior colliculi to retrogradely label RGCs. A ninety-minute period of ischaemia was induced and densities of surviving RGCs were estimated over time by counting FG-labelled RGCs in 12 standard regions of each retina. RESULTS: Seven days after inducing transient ischaemia, there was loss of approximately half of the RGC population. Topical pre-treatment with 0.1% or 0.5% BMD prevented ischaemia-induced RGC death. CONCLUSIONS: These results indicate that optimal neuroprotective effects against the early loss of RGCs are seen with 0.1% or 0.5% BMD. Ischaemia-induced RGC loss continued between day 7 and day 21 in the vehicle treated groups and amounted to approximately 25% of the RGC population. Topical pre-treatment with 0.1% or 0.5% BMD was also effective in reducing the slow loss of RGCs.

Neuroprotective effects of alpha(2)-selective adrenergic agonists against ischemia-induced retinal ganglion cell death.

PURPOSE: To investigate in adult rats the effects of two alpha(2)-selective adrenergic agonists (alpha(2)-SAs; AGN 191103 and AGN 190342) on retinal ganglion cell (RGC) survival after transient retinal ischemia. METHODS: RGCs were labeled with a Fluorogold (FG) tracer applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 60 or 90 minutes. In one group, a single dose of saline or one alpha(2)-SA was administered intraperitoneally (IP) or topically 1 hour before ischemia. In another group, a single dose of AGN 190342 was administered IP, 1, 2, 4, 24, or 72 hours after ischemia. Rats were processed 7, 14, or 21 days later. Densities of surviving RGCs were estimated by counting FG-labeled cells in 12 standard retinal areas. RESULTS: Seven days after 60 or 90 minutes of retinal ischemia, death had occurred in 36% or 47%, respectively, of the RGC population, and by 21 days the loss of RGCs amounted to 42% or 62%, respectively. Systemic pretreatment with an alpha(2)-SA resulted in enhanced survival of ischemic-injured RGCs. Topical pretreatment with an alpha(2)-SA prevented up to 100% of the ischemia-induced RGC loss. Pretreatment with an alpha(2)-SA abolished the secondary slow RGC loss that occurred between days 7 and 21 after ischemia. When administered shortly after ischemia (up to 2 hours) AGN 190342 rescued substantial proportions of RGCs destined to die and diminished slow RGC death. CONCLUSIONS: Pretreatment and early posttreatment with an alpha(2)-SA induces marked long-lasting neuroprotective in vivo protection against ischemia-induced cell death in RGCs.

Retinal ganglion cell death induced by retinal ischemia. neuroprotective effects of two alpha-2 agonists.

We have investigated in adult Sprague-Dawley rats the neuroprotective effects of two alpha-2-selective agonists [AGN 191,103 (AGN) and brimonidine tartrate (BMD)] on retinal ganglion cell (RGC) survival after transient retinal ischemia. RGCs were labelled with Fluorogold (FG) applied to both superior colliculi. Seven days later, 90 min of retinal ischemia were induced in the left eyes by ligature of the ophthalmic vessels (LOV). In one group of animals, vehicle or AGN (0.01 mg/kg) were administered systemically 1 hr before ischemia. In another group of animals, two 5 microl drops of vehicle, AGN (0.05%) or BMD (0.1%) were administered topically in the left eye 1 hr before ischemia. The animals were processed 7 or 21 days later. RGC survival was estimated by counting FG-labelled cells in 12 standard areas of each retina. In control retinas of systemically pretreated animals, mean densities of labelled RGCs were 2372 +/- 49 cells/mm(2) (mean +/- SEM; n = 6). In experimental retinas of systemically pretreated animals, mean RGC densities had decreased 7 days after ischemia to 53% (n = 6) or 81% (n = 6) of control in the groups treated with vehicle or AGN, respectively. Twenty-one days after ischemia, mean RGC densities had decreased to 38% (n = 6) or 79% (n = 6) of control in the groups treated with vehicle or AGN, respectively. In control retinas of topically pretreated animals, mean densities of labelled RGCs were 2208 +/- 29 cells/mm(2) (n = 6). In experimental retinas of topically pretreated animals, mean RGC densities had decreased 7 days after ischemia to 54% (n = 6), 95% (n = 6) or 96% (n = 6) of control in the groups treated with vehicle, AGN or BMD, respectively. These results indicate that pretreatment with a single systemic or topical dose of AGN or BMD can prevent completely the early rapid phase of RGC loss and abolish the delayed RGC loss observed after 90 min of retinal ischemia induced by ligature of the ophthalmic vessels.