Double blocking of carbon metabolism causes a large increase of Calvin-Benson cycle compounds in cyanobacteria.

Carbon-flow-regulator A (CfrA) adapts carbon flux to nitrogen conditions in nondiazotrophic cyanobacteria. Under nitrogen deficiency, CfrA leads to the storage of excess carbon, which cannot combine with nitrogen, mainly as glycogen. cfrA overexpression from the arsenite-inducible, nitrogen-independent ParsB promoter allows analysis of the metabolic effects of CfrA accumulation. Considering that the main consequence of cfrA overexpression is glycogen accumulation, we examined carbon distribution in response to cfrA expression in Synechocystis sp. PCC 6803 strains impaired in synthesizing this polymer. We carried out a comparative phenotypic analysis to evaluate cfrA overexpression in the wild-type strain and in a mutant of ADP-glucose pyrophosphorylase (ΔglgC), which is unable to synthesize glycogen. The accumulation of CfrA in the wild-type background caused a photosynthetic readjustment although growth was not affected. However, in a ΔglgC strain, growth decreased depending on CfrA accumulation and photosynthesis was severely affected. An elemental analysis of the H, C, and N content of cells revealed that cfrA expression in the wild-type caused an increase in the C/N ratio, due to decreased nitrogen assimilation. Metabolomic study indicated that these cells store sucrose and glycosylglycerol, in addition to the previously described glycogen accumulation. However, cells deficient in glycogen synthesis accumulated large amounts of Calvin-Benson cycle intermediates as cfrA was expressed. These cells also showed increased levels of some amino acids, mainly alanine, serine, valine, isoleucine, and leucine. The findings suggest that by controlling cfrA expression, in different conditions and strains, we could change the distribution of fixed carbon, with potential biotechnological benefits.

Stress response requires an efficient connection between glycogen and central carbon metabolism by phosphoglucomutases in cyanobacteria.

Glycogen and starch are the main storage polysaccharides, acting as a source of carbon and energy when necessary. Interconversion of glucose-1-phosphate and glucose-6-phosphate by phosphoglucomutases connects the metabolism of these polysaccharides with central carbon metabolism. However, knowledge about how this connection affects the ability of cells to cope with environmental stresses is still scarce. The cyanobacterium Synechocystis sp. PCC 6803 has two enzymes with phosphoglucomutase activity, PGM (phosphoglucomutase) and PMM/PGM (phosphomannomutase/phosphoglucomutase). In this work, we generated a null mutant of PGM (∆PGM) that exhibits very reduced phosphoglucomutase activity (1% of wild type activity). Although this mutant accumulates moderate amounts of glycogen, its phenotype resembles that of glycogen-less mutants, including high light sensitivity and altered response to nitrogen deprivation. Using an on/off arsenite promoter, we demonstrate that PMM/PGM is essential for growth and responsible for the remaining phosphoglucomutase activity in the ∆PGM strain. Furthermore, overexpression of PMM/PGM in the ∆PGM strain is enough to revoke the phenotype of this mutant. These results emphasize the importance of an adequate flux between glycogen and central carbon metabolism to maintain cellular fitness and indicate that although PGM is the main phosphoglucomutase activity, the phosphoglucomutase activity of PMM/PGM can substitute it when expressed in sufficient amounts.

Excessive Fluid in the Lumbar Facet Joint as a Predictor of Radiological Outcomes After Lateral Lumbar Interbody Fusion.

Background Preoperative segmental instability maybe a predictor of postoperative outcomes when treated with lateral lumbar interbody fusion (LLIF). An abnormal collection of fluid within the facet joint has been described as a sign of segmental instability. The potential relationship between this radiological sign and its prognostic relevance for indirect decompression (ID) has not been investigated. Methods Clinical and radiologic results from patients undergoing LLIF in a single institution between 2007 and 2014 were evaluated retrospectively. Patients were divided into two groups: those presenting with excessive fluid (EF) in the facet joints on T2-MRI and those with a normal amount of facet fluid with less than 1mm, which were controls. Radiological parameters were foraminal height, disc height, Cobb angle, and lumbar lordosis. Results A total of 21 patients (43 operated levels) were evaluated pre- and postoperatively. Mean disc height, mean foraminal height, and coronal Cobb angles were statistically significantly improved after LLIF. Only the EF group showed significant improvement in radiological markers after ID; the mean disc height improved from 5.5±2 to 8.8±1mm (p=0.001), mean foraminal height improved from 16.88±3 to 20.53±3mm (p=0.002), and the mean Cobb angle improved from 27.7±16 to 14±13 (p=0.018). Conclusions Patients undergoing LLIF with the radiological findings of EF in the facet joints demonstrated significant improvement in radiological outcomes of ID. Further studies should validate these findings in larger data sets.

Gustatory function of sensilla chaetica on the labial palps and antennae of three tortricid moths (Lepidoptera: Tortricidae).

In adult Lepidoptera the labial palps are best known for their role in CO2 detection, but they can also bear sensilla chaetica which function is unknown. The number and distribution of sensilla chaetica in labial palps was studied using a bright field microscope. To determine if these sensilla have a gustatory function, we performed single sensillum electrophysiology recordings from palp and antennal sensilla of adult moths of Cydia pomonella (L.), Grapholita molesta (Busck) and Lobesia botrana (Denis and Shieffermüller). Each sensillum was stimulated with 3 doses of one of four test stimulus (sucrose, fructose, KCl and NaCl). Overall, responses (spikes/s-1) increased with dose, and were higher in the palps than in the antennae, and higher to sugars than to salts. With sugars the response increased with concentration in the palp but not in the antenna. With salts there was a drop in response at the intermediate concentration. The number and position of sensilla chaetica on labial palps was variable among individuals. Sensilla were located in the most exposed areas of the palp. Differences in sensilla distribution were detected between species. Such differences among species and between palps and antenna suggest that taste sensilla on the palps have an unforeseen role in adaptation.

A Mouse Model of Ulcerative Cutaneous Leishmaniasis by Leishmania (Viannia) panamensis to Investigate Infection, Pathogenesis, Immunity, and Therapeutics.

A mouse model of cutaneous leishmaniasis (CL) by Leishmania (Viannia) panamensis (L(V)p) that reproduces the characteristics of the human disease remains elusive. Here we report the development of a CL model that uses a mouse-adapted L(V)p isolate to reproducibly induce a dermal disease with a remarkable similarity to human CL. BALB/c mice infected intradermally in the ear with 105 stationary UA-946 L(V)p promastigotes develop a progressive cutaneous disease that exhibits the typical ulcerated lesions with indurated borders observed in CL patients. Although most of parasites in the inoculum die within the first week of infection, the survivors vigorously multiply at the infection site during the following weeks, paralleling disease appearance and aggravation. Regional lymphadenopathy as well as lymphatic dissemination of parasites to draining lymph nodes (dLN) was evidenced early after infection. Viable parasites were also isolated from spleen at later timepoints indicating systemic parasitic dissemination, but, strikingly, no signs of systemic disease were observed. Increasing numbers of myeloid cells and T lymphocytes producing IFNγ and IL-4 were observed in the dLN as disease progressed. A mixed adaptive L(V)p-specific T cell-mediated response was induced, since ex vivo recall experiments using dLN cells and splenocytes revealed the production of type 1 (IFNγ, IL-2), type 2 (IL-4, IL-13), regulatory (IL-10), and inflammatory (GM-CSF, IL-3) cytokines. Humoral adaptive response was characterized by early production of IgG1- followed by IgG2a-type of L(V)p-specific antibodies. IFNγ/IL-4 and IgG2a/IgG1 ratios indicated that the initial non-protective Th2 response was redirected toward a protective Th1 response. In situ studies revealed a profuse recruitment of myeloid cells and of IFNγ- and IL-4-producing T lymphocytes to the site of infection, and the typical histopathological changes induced by dermotropic Leishmania species. Evidence that this model is suitable to investigate pharmacological and immunomodulatory interventions, as well as for antigen discovery and vaccine development, is also presented. Altogether, these results support the validity and utility of this novel mouse model to study the pathogenesis, immunity, and therapeutics of L(V)p infections.

Reptile vector-borne diseases of zoonotic concern.

Reptile vector-borne diseases (RVBDs) of zoonotic concern are caused by bacteria, protozoa and viruses transmitted by arthropod vectors, which belong to the subclass Acarina (mites and ticks) and the order Diptera (mosquitoes, sand flies and tsetse flies). The phyletic age of reptiles since their origin in the late Carboniferous, has favored vectors and pathogens to co-evolve through millions of years, bridging to the present host-vector-pathogen interactions. The origin of vector-borne diseases is dated to the early cretaceous with Trypanosomatidae species in extinct sand flies, ancestral of modern protozoan hemoparasites of zoonotic concern (e.g., Leishmania and Trypanosoma) associated to reptiles. Bacterial RVBDs are represented by microorganisms also affecting mammals of the genera Aeromonas, Anaplasma, Borrelia, Coxiella, Ehrlichia and Rickettsia, most of them having reptilian clades. Finally, reptiles may play an important role as reservoirs of arborivuses, given the low host specificity of anthropophilic mosquitoes and sand flies. In this review, vector-borne pathogens of zoonotic concern from reptiles are discussed, as well as the interactions between reptiles, arthropod vectors and the zoonotic pathogens they may transmit.

Lethality caused by ADP-glucose accumulation is suppressed by salt-induced carbon flux redirection in cyanobacteria.

Cyanobacteria are widely distributed photosynthetic organisms. During the day they store carbon, mainly as glycogen, to provide the energy and carbon source they require for maintenance during the night. Here, we generate a mutant strain of the freshwater cyanobacterium Synechocystis sp. PCC 6803 lacking both glycogen synthases. This mutant has a lethal phenotype due to massive accumulation of ADP-glucose, the substrate of glycogen synthases. This accumulation leads to alterations in its photosynthetic capacity and a dramatic decrease in the adenylate energy charge of the cell to values as low as 0.1. Lack of ADP-glucose pyrophosphorylase, the enzyme responsible for ADP-glucose synthesis, or reintroduction of any of the glycogen synthases abolishes the lethal phenotype. Viability of the glycogen synthase mutant is also fully recovered in NaCl-supplemented medium, which redirects the surplus of ADP-glucose to synthesize the osmolite glucosylglycerol. This alternative metabolic sink also suppresses phenotypes associated with the defective response to nitrogen deprivation characteristic of glycogen-less mutants, restoring the capacity to degrade phycobiliproteins. Thus, our system is an excellent example of how inadequate management of the adenine nucleotide pools results in a lethal phenotype, and the influence of metabolic carbon flux in cell viability and fitness.

Extremely low neonicotinoid doses alter navigation of pest insects along pheromone plumes.

The prevailing use of neonicotinoids in pest control has adverse effects on non-target organisms, like honeybees. However, relatively few studies have explored the effect of sublethal neonicotinoid levels on olfactory responses of pest insects, and thus their potential impact on semiochemical surveillance and control methods, such as monitoring or mating disruption. We recently reported that sublethal doses of the neonicotinoid thiacloprid (TIA) had dramatic effects on sex pheromone release in three tortricid moth species. We present now effects of TIA on pheromone detection and, for the first time, navigational responses of pest insects to pheromone sources. TIA delayed and reduced the percentage of males responding in the wind tunnel without analogous alteration of electrophysiological antennal responses. During navigation along an odor plume, treated males exhibited markedly slower flights and, in general, described narrower flight tracks, with an increased susceptibility to wind-induced drift. All these effects increased in a dose-dependent manner starting at LC0.001 - which would kill just 10 out of 106 individuals - and revealed an especially pronounced sensitivity in one of the species, Grapholita molesta. Our results suggest that minimal neonicotinoid quantities alter chemical communication, and thus could affect the efficacy of semiochemical pest management methods.

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection.

Francisella noatunensis subsp. orientalis is a pathogen of tilapia and other warm-water fish for which no vaccines are commercially available. In this study, a whole cell formalin-inactivated vaccine was developed for the first time using the highly virulent isolate STIR-GUS-F2f7 and the oil-based adjuvant Montanide™ ISA 763A VG. The efficacy of the vaccine was assessed in red Nile tilapia via intraperitoneal (i.p.) injection using homologous experimental infection and correlates of protection such as seral antibody production and bacterial loads in the spleen. For immunization, fish were i.p. injected with 0.1 ml of the vaccine, the adjuvant alone or PBS. At 840 degree days post-vaccination, all fish were i.p. injected with 4.0 × 103 CFU/fish of pathogenic bacteria. The RPS at the end of the trial was 100% in the vaccinated group with significantly higher survival than in the adjuvant and control groups. The RPS in the adjuvant group was 42%, and no significant difference was seen in survival between this and the PBS group. Moreover, significantly higher antibody titres in the serum and significantly lower bacterial loads in the spleen were detected in the vaccinated fish by ELISA and qPCR, respectively. These findings highlight the potential of autogenous vaccines for controlling francisellosis in tilapia.

Sublethal Effects of Neonicotinoid Insecticide on Calling Behavior and Pheromone Production of Tortricid Moths.

In moths, sexual behavior combines female sex pheromone production and calling behavior. The normal functioning of these periodic events requires an intact nervous system. Neurotoxic insecticide residues in the agroecosystem could impact the normal functioning of pheromone communication through alteration of the nervous system. In this study we assess whether sublethal concentrations of the neonicotinoid insecticide thiacloprid, that competitively modulates nicotinic acetylcholine receptors at the dendrite, affect pheromone production and calling behavior in adults of three economically important tortricid moth pests; Cydia pomonella (L.), Grapholita molesta (Busck), and Lobesia botrana (Denis & Schiffermüller). Thiacloprid significantly reduced the amount of calling in C. pomonella females at LC0.001 (a lethal concentration that kills only 1 in 105 individuals), and altered its calling period at LC1, and in both cases the effect was dose-dependent. In the other two species the effect was similar but started at higher LCs, and the effect was relatively small in L. botrana. Pheromone production was altered only in C. pomonella, with a reduction of the major compound, codlemone, and one minor component, starting at LC10. Since sex pheromones and neonicotinoids are used together in the management of these three species, our results could have implications regarding the interaction between these two pest control methods.

Comparative Effect of Three Neurotoxic Insecticides With Different Modes of Action on Adult Males and Females of Three Tortricid Moth Pests.

Insecticides are the dominant pest management method in fruit and vegetable crops worldwide owing to their quick effect, low cost, and relatively easy application, but they bear negative effects on human health and the environment. Insecticide mode of action (MoA), target species, and sex are variables that could affect insecticide-induced mortality. We recorded the mortality caused by three neurotoxic insecticides with different modes of action (chlorpyrifos [organophosphate, acetylcholinesterase inhibitor], λ-cyhalothrin [pyrethroid, sodium channel modulator], and thiacloprid [neonicotinoid, nicotinic acetylcholinesterase receptor agonist]) applied topically to adult males and females of three economically important tortricid species [Cydia pomonella (L.), Grapholita molesta (Busck), and Lobesia botrana (Denis & Schiffermüller)] that strongly depend on insecticide use for their control. Concentration and dose-mortality curves were recorded at 24 and 48 h postapplication. Large mortality differences between insecticides (maximum 7,800-fold for LD50) were followed by much lower, yet important, differences between species (maximum 115-fold), and sexes (maximum 41.5-fold). Significant interactions between the three factors indicate that they are not independent from each other. Interestingly, with the organophosphate chlorpyrifos, males of the three species were less susceptible than females, which was unexpected, as females are larger than males. Higher female sensitivity to organophosphates has been reported previously but only in G. molesta, not in other moth species. Our results highlight the importance of taking into account sex in dose-mortality studies with adult moths.

Redox regulation of glycogen biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803: analysis of the AGP and glycogen synthases.

Glycogen constitutes the major carbon storage source in cyanobacteria, as starch in algae and higher plants. Glycogen and starch synthesis is linked to active photosynthesis and both of them are degraded to glucose in the dark to maintain cell metabolism. Control of glycogen biosynthesis in cyanobacteria could be mediated by the regulation of the enzymes involved in this process, ADP-glucose pyrophosphorylase (AGP) and glycogen synthase, which were identified as putative thioredoxin targets. We have analyzed whether both enzymes were subjected to redox modification using purified recombinant enzymes or cell extracts in the model cyanobacterium Synechocystis sp. PCC 6803. Our results indicate that both AGP and glycogen synthases are sensitive to copper oxidation. However, only AGP exhibits a decrease in its enzymatic activity, which is recovered after reduction by DTT or reduced thioredoxin (TrxA), suggesting a redox control of AGP. In order to elucidate the role in redox control of the cysteine residues present on the AGP sequence (C45, C185, C320, and C337), they were replaced with serine. All AGP mutant proteins remained active when expressed in Synechocystis, although they showed different electrophoretic mobility profiles after copper oxidation, reflecting a complex pattern of cysteines interaction.

Proatherogenic macrophage activities are targeted by the flavonoid quercetin.

Many studies have demonstrated that the flavonoid quercetin protects against cardiovascular disease (CVD) and related risk factors. Atherosclerosis, the underlying cause of CVD, is also attenuated by oral quercetin administration in animal models. Although macrophages are key players during fatty streak formation and plaque progression and aggravation, little is known about the effects of quercetin on atherogenic macrophages. Here, we report that primary bone marrow-derived macrophages internalized less oxidized low-density lipoprotein (oxLDL) and accumulated less intracellular cholesterol in the presence of quercetin. This reduction of foam cell formation correlated with reduced surface expression of the oxLDL receptor CD36. Quercetin also targeted the lipopolysaccharide-dependent, oxLDL-independent pathway of lipid droplet formation in macrophages. In oxLDL-stimulated macrophages, quercetin inhibited reactive oxygen species production and interleukin (IL)-6 secretion. In a system that evaluated cholesterol crystal-induced IL-1ß secretion via nucleotide-binding domain and leucine-rich repeat containing protein 3 inflammasome activation, quercetin also exhibited an inhibitory effect. Dyslipidemic apolipoprotein E-deficient mice chronically treated with intraperitoneal quercetin injections had smaller atheromatous lesions, reduced lipid deposition, and less macrophage and T cell inflammatory infiltrate in the aortic roots than vehicle-treated animals. Serum levels of total cholesterol and the lipid peroxidation product malondialdehyde were also reduced in these mice. Our results demonstrate that quercetin interferes with both key proatherogenic activities of macrophages, namely foam cell formation and pro-oxidant/proinflammatory responses, and these effects may explain the atheroprotective properties of this common flavonoid.

Loss of heterozygosity in the short arm of human chromosome 3 in sporadic lung cancer / Pérdida de heterocigocidad en el brazo corto del cromosoma 3 humano en cáncer esporádico de pulmón

Introduction: Loss of Heterozygocity (LOH) in the short arm of human chromosome 3 (3p) is a frequent event in different types of sporadic tumors, including lung cancer (LC). Aim: To determine 3p LOH in LC samples using 17 microsatellite markers. Methodology: In a pilot study on volunteers, thirteen LC biopsies (tumor tissue) and 4 ml of blood (normal tissue) from the same patient were collected. DNA extraction and Polymerase Chain Reaction (PCR) were performed with 17 microsatellite markers to analyze LOH. Amplified fragments were run on 6% denaturalizing polyacrilamide gels and were visualized by using silver stain. Descriptive analysis was performed for each region on the 3p chromosome. Results: All tumors were informative for one or more of the analyzed markers. LOH was found in one or more loci in eleven samples (84.6%). The markers with major LOH were UBE1L (23.1%), D3S1317, D3S1300, D3S1284, D3S1274, D3S3049, and D3S1577 (15.4%). Three samples showed microsatellite instability (changes in the length of the microsatellite) in different loci. The percentages of LOH for the regions of 3p were: 17.6 % for 3p24-25, 11.62% for 3p21-22, 20% for 3p13-14, and 18.42% for the 3p12 region. Conclusions: Chromosomal regions with allelic loss were identified where probably other GSTs involved in the development of the LC are localized. It should increases sample size and marker number in order to narrow a minimal region and to identify a unknown gene involved in LC.

Introducción: La pérdida de heterocigocidad (LOH) en el brazo corto del cromosoma 3 (3p) humano es un evento frecuente en diferentes tipos de tumores esporádicos, incluyendo cáncer de pulmón (CP). Objetivo: Determinar la LOH de 3p en muestras de CP, con 17 marcadores microsatelitales. Metodología: En un estudio piloto en voluntarios, se recolectaron 13 biopsias de CP (tejido tumoral) y 4 ml de sangre periférica (tejido normal) del mismo paciente, se extrajo el ADN y se realizaron reacciones en cadena de la polimerasa (PCR) con 17 marcadores microsatelitales para analizar LOH. Los fragmentos amplificados se corrieron en geles de poliacrilamida desnaturalizante al 6% y se visualizaron por medio de la coloración de tinción de plata. El análisis descriptivo se realizó para cada región estudiada en el cromosoma 3p. Resultados: Todos los tumores fueron informativos para uno o más de los marcadores analizados. Se encontró LOH en uno o más loci en 11 muestras (84.6%). Los marcadores con mayores LOH fueron UBE1L (23.1%), D3S1317, D3S1300, D3S1284, D3S1274, D3S3049 y D3S1577 con 15.4%. Tres muestras presentaron inestabilidad microsatelital (cambios en la longitud del microsatélite) en diferentes loci. Los porcentajes de LOH para las regiones de 3p fueron: 17.6 % para 3p24-25, 11.6% para 3p21-22, 20% para 3p13-14 y 18.4% para la región 3p12. Conclusiones: Se identificaron regiones cromosómicas con pérdida alélica donde es probable que se localicen otros GST involucrados en el desarrollo de CP, diferentes de los ya identificados como VHL, RASSF1A, FHIT y DUTTI, entre otros. Se debe aumentar el número de muestras y de marcadores para delimitar una región mínima e identificar algún gen no descrito implicado en la carcinogénesis de pulmón.

[Transanesthetic anaphylactic shock induced by propofol and fentanyl: case report]. / Choque anafiláctico transanestésico por propofol y fentanil: comunicación de un caso.

We report an anaphylactic shock case secondary to propofol and fentanyl exposition, demonstrated by skin tests. A male patient, 19 years old, was admitted in the operating room for resection of residual juvenile nasal angyofibroma. The anesthetic induction was done with atropin 800 microg, midazolam 2 mg, fentanyl 200 microg, propofol 150 mg, and neuromuscular block with rocuronium 30 mg. One minute after the application of drugs, hypotension of 60/30 mmHg and tachycardia of 130 was observed. By the second minute the blood pressure dropped to 40/20 mmHg, tachycardia got 135, facial edema and generalized wheals occurred and the plestimography and oxymetry record were absent. Once resolved the event and the patient recovered, skin tests were performed with positive results to propofol and fentanyl, so that they were excluded in the next surgical intervention that concluded without incidents and with success.

Histoplasmosis pulmonar: infección pulmonar primaría: histoplastoma / Pulmonary histoplasmosis: primary pulmonary infection: histoplasmoma

La histoplasmosis es una micosis de origen pulmonar primario, adquirida por vía inhalatoria. En la mayoría de los casos, la infección pasa inadvertida o se manifiesta por síntomas respiratorios leves. El histoplasmoma es una forma relativamente común de histoplasmosis pulmonar aguda, de aspecto nodular, generalmente acompañada de calcificación, la cual puede aumentar en tamaño y simular una neoplasia pulmonar. presenta un caso de un paciente inmunosuprimido con esta forma de micosis pulmonar.