The drop plate method as an alternative for Azospirillum spp viable cell enumeration within the consensus protocol of the REDCAI network

Abstract Quality evaluation of commercial inoculants is essential to warrant an adequate cropresponse to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cellsas an alternative to the spread plate technique, which is currently proposed in the consensusprotocol of the REDCAI network. Between 14 and 25 private and public laboratories partici-pated in three independent trials. We obtained consistent and robust results that allowed toconfirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensusprotocol.

Resumen La evaluación de la calidad de los inoculantes comerciales es fundamental para garantizar una adecuada respuesta de los cultivos a la inoculación dentro de un marco de bioseguridad. En este sentido, el objetivo de este trabajo fue la estandarización y validación de la técnica de la microgota para la cuantificación de Azospirillum como metodología alternativa a la técnica de siembra en superficie, propuesta actualmente en el protocolo consenso de la Red de Calidad de Inoculantes, REDCAI. Entre 14 y 25 laboratorios, tanto privados como públicos, participaron de tres ensayos independientes. A partir de ellos se obtuvieron resultados reproducibles y robustos que permiten confirmar que ambas técnicas son equivalentes y concluir que la técnica de recuento por la microgota es una alternativa adecuada para ser incluida dentro del mencionado protocolo consenso.

The drop plate method as an alternative for Azospirillum spp. viable cell enumeration within the consensus protocol of the REDCAI network.

Quality evaluation of commercial inoculants is essential to warrant an adequate crop response to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cells as an alternative to the spread plate technique, which is currently proposed in the consensus protocol of the REDCAI network. Between 14 and 25 private and public laboratories participated in three independent trials. We obtained consistent and robust results that allowed to confirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensus protocol.

Experiencia en sedoanalgesia vinculada a procedimientos en un departamento de emergencia pediátrica / Experience in procedural sedation and analgesia in a pediatrics emergency unit / Experiência em sedação e analgesia ligada a procedimentos em um departamento de emergência pediátrica

Resumen: Introducción: la realización de procedimientos dolorosos es cada vez más frecuente en el área de urgencias pediátricas. El control del dolor, mitigar el temor y la ansiedad es una pieza clave en la atención del paciente pediátrico y una de las prioridades del médico emergencista. Objetivo: describir la experiencia en sedoanalgesia con ketamina y fentanilo como único fármaco o asociados a otros para procedimientos dolorosos en el Departamento de Emergencia Pediátrica (DEP) del Centro Hospitalario Pereira Rossell (CHPR). Material y método: estudio descriptivo, retrospectivo. Período: enero de 2011 a julio de 2016. Población: todos los pacientes que recibieron sedoanalgesia con ketamina o fentanilo (solos o asociado a otros fármacos) para realización de procedimientos dolorosos en el DEP-CHPR. Base de datos: historias clínicas. Se midió la eficacia mediante el éxito del procedimiento y la seguridad por la presencia de efectos adversos vinculados a la misma. Resultados: n=352, menores de 15 años; 96 recibieron ketamina y 256 recibieron fentanilo. Media de edad: 7 años, sexo masculino: 245. Dosis media de ketamina 1 mg/kg. Dosis media de fentanilo 1 ɤ/kg. Principales indicaciones de sedoanalgesia: procedimientos ortopédicos (264), toracocentesis (62). Procedimiento con éxito: 352 pacientes. Monitorización cardiovascular y saturometría durante el procedimiento: 100%. Efectos adversos: seis (no requiriendo maniobras de soporte vital avanzado). Ningún paciente cambió su destino final. Conclusiones: la utilización de sedoanalgesia resultó eficaz y segura realizada por el pediatra emergencista capacitado en el manejo farmacológico y de soporte vital avanzado. La monitorización del procedimiento durante y luego de éste es necesaria para pesquisar y resolver precozmente las complicaciones.

Summary: Introduction: painful procedures are gradually increasing frequency in pediatrics emergency units. Controlling pain and mitigating fear and anxiety are of the essence when assisting pediatric patients and one of the priorities of emergency doctors. Objective: to describe using sedoanalgesia with ketamine and fentanyl as the only drug or associated with other drugs during painful procedures, at the Pediatrics Emergency Unit, of the Pereira Rossell Hospital Center. Method: descriptive, retrospective study from January 2011 to July 2016. Population: all patients who received sedoanalgesia with ketamine and fentanyl (as the only drug or associated with other drugs) during painful procedures at the Pediatrics Emergency Unit, of the Pereira Rossell Hospital Center. Database: medical records. Effectiveness was measured by success of the procedure and safety in terms of adverse effects in connection with it. Results: N=352 children younger than 15 years old. 96 received ketamine and 256 received fentanyl. Average age was 7 years old and 245 of them were boys. Average dose of ketamine was 1 mg/kg, average dose of fentanyl was 1 ɤ/kg. Main indications for sedoanalgesia included: orthopaedic procedures (264), thoracentesis (62). Procedures were successful in 352 patients. 100% of cases involved cardiovascular monitoring and blood oxygen saturation meter. Adverse effects: 6 (no advanced life support manoeuvres required). No patient changed final destination. Conclusions: the use of sedoanalgesia was effective and safe, when applied by emergengy pediatricians qualified in the handling of drugs and advanced life support. Monitoring during and after procedure is required to determine and solve complications at an early stage.

Resumo Introdução: a realização de procedimentos dolorosos é cada vez mais frequente na área de emergência pediátrica. Controlar a dor, mitigar o medo e a ansiedade é um elemento-chave no atendimento ao paciente pediátrico e uma das prioridades do médico de emergência. Objetivo: descrever a experiência em sedação e analgesia com ketamina e fentanil como fármaco único ou associado a outros para procedimentos dolorosos, no Serviço de Emergência Pediátrica (DEP) do Centro Hospitalar Pereira Rossell (CHPR) Material e métodos: estudo descritivo retrospectivo. Período: janeiro de 2011 a julho de 2016. População: todos os pacientes que receberam sedação e analgesia com ketamina ou fentanil (isoladamente ou em combinação com outras drogas) para realização de procedimentos dolorosos no DEP-CHPR. Banco de dados: prontuários médicos. A eficácia foi medida pelo sucesso do procedimento e a segurança pela presença de efeitos adversos associados. Resultados: n = 352 crianças menores de 15 anos. 96 receberam ketamina e 256 receberam fentanil. Idade média: 7 anos, sexo masculino: 245. Dose média de ketamina 1 mg / kg. Dose média de fentanil 1 ɤ/kg. Principais indicações para sedação e analgesia: procedimentos ortopédicos (264), toracocentese (62). Procedimento com sucesso: 352 pacientes. Monitorização cardiovascular e oximetria durante o procedimento: 100%. Efeitos adversos: 6 (não requer manobras de suporte avançado de vida). Nenhum paciente mudou seu destino final. Conclusões: o uso da sedação e analgesia foi eficaz e seguro realizado por pediatra de emergência capacitado em manejo farmacológico e suporte avançado de vida. O monitoramento do procedimento durante e após é necessário para investigar e resolver precocemente as complicações.

In vivo imaging of injured cortical axons reveals a rapid onset form of Wallerian degeneration.

BACKGROUND: Despite the widespread occurrence of axon and synaptic loss in the injured and diseased nervous system, the cellular and molecular mechanisms of these key degenerative processes remain incompletely understood. Wallerian degeneration (WD) is a tightly regulated form of axon loss after injury, which has been intensively studied in large myelinated fibre tracts of the spinal cord, optic nerve and peripheral nervous system (PNS). Fewer studies, however, have focused on WD in the complex neuronal circuits of the mammalian brain, and these were mainly based on conventional endpoint histological methods. Post-mortem analysis, however, cannot capture the exact sequence of events nor can it evaluate the influence of elaborated arborisation and synaptic architecture on the degeneration process, due to the non-synchronous and variable nature of WD across individual axons. RESULTS: To gain a comprehensive picture of the spatiotemporal dynamics and synaptic mechanisms of WD in the nervous system, we identify the factors that regulate WD within the mouse cerebral cortex. We combined single-axon-resolution multiphoton imaging with laser microsurgery through a cranial window and a fluorescent membrane reporter. Longitudinal imaging of > 150 individually injured excitatory cortical axons revealed a threshold length below which injured axons consistently underwent a rapid-onset form of WD (roWD). roWD started on average 20 times earlier and was executed 3 times slower than WD described in other regions of the nervous system. Cortical axon WD and roWD were dependent on synaptic density, but independent of axon complexity. Finally, pharmacological and genetic manipulations showed that a nicotinamide adenine dinucleotide (NAD+)-dependent pathway could delay cortical roWD independent of transcription in the damaged neurons, demonstrating further conservation of the molecular mechanisms controlling WD in different areas of the mammalian nervous system. CONCLUSIONS: Our data illustrate how in vivo time-lapse imaging can provide new insights into the spatiotemporal dynamics and synaptic mechanisms of axon loss and assess therapeutic interventions in the injured mammalian brain.

The Endophytic Strain Klebsiella michiganensis Kd70 Lacks Pathogenic Island-Like Regions in Its Genome and Is Incapable of Infecting the Urinary Tract in Mice.

Klebsiella spp. have been isolated from many different environmental habitats but have mainly been associated with nosocomial acquired diseases in humans. Although there are many recently published sequenced genomes of members of this genus, there are very few studies on whole genome comparisons between clinical and non-clinical isolates, and it is therefore still an open question if a strain found in nature is capable of infecting humans/animals. Klebsiella michiganensis Kd70 was isolated from the intestine of larvae of Diatraea saccharalis but genome analysis revealed multiple genes associated with colonization and growth promotion in plants suggesting an endophytic lifestyle. Kd70 cells labeled with gfp confirmed capability of root colonization and soil application of Kd70 promoted growth in greenhouse grown sugarcane. Further genomic analysis showed that the Kd70 genome harbored fewer mammalian virulence factors and no pathogen island-like regions when compared to clinical isolates of this species, suggesting attenuated animal/human pathogenicity. This postulation was corroborated by in vivo experiments in which it was demonstrated that Kd70 was unable to infect the mouse urinary tract. This is to the best of our knowledge the first experimental example of a member of a pathogenic Klebsiella spp. unable to infect a mammalian organism. A proteomic comparison deduced from the genomic sequence between Kd70 and several other K. michiganensis strains showed a high similarity with isolates from many different environments including clinical strains, and demonstrated the existence of conserved genetic lineages within this species harboring members from different ecological niches and geographical locations. Furthermore, most genetic differences were found to be associated with genomic islands of clinical isolates, suggesting that evolutionary adaptation of animal pathogenicity to a large extent has depended on horizontal gene transfer. In conclusion our results demonstrate the importance of conducting thorough in vivo pathogenicity studies before presupposing animal/human virulence of non-clinical bacterial isolates.

Nicotine protects rat hypoglossal motoneurons from excitotoxic death via downregulation of connexin 36.

Motoneuron disease including amyotrophic lateral sclerosis may be due, at an early stage, to deficit in the extracellular clearance of the excitatory transmitter glutamate. A model of glutamate-mediated excitotoxic cell death based on pharmacological inhibition of its uptake was used to investigate how activation of neuronal nicotinic receptors by nicotine may protect motoneurons. Hypoglossal motoneurons (HMs) in neonatal rat brainstem slices were exposed to the glutamate uptake blocker DL-threo-ß-benzyloxyaspartate (TBOA) that evoked large Ca2+ transients time locked among nearby HMs, whose number fell by about 30% 4 h later. As nicotine or the gap junction blocker carbenoxolone suppressed bursting, we studied connexin 36 (Cx36), which constitutes gap junctions in neurons and found it largely expressed by HMs. Cx36 was downregulated when nicotine or carbenoxolone was co-applied with TBOA. Expression of Cx36 was preferentially observed in cytosolic rather than membrane fractions after nicotine and TBOA, suggesting protein redistribution with no change in synthesis. Nicotine raised the expression of heat shock protein 70 (Hsp70), a protective factor that binds the apoptotic-inducing factor (AIF) whose nuclear translocation is a cause of cell death. TBOA increased intracellular AIF, an effect blocked by nicotine. These results indicate that activation of neuronal nicotinic receptors is an early tool for protecting motoneurons from excitotoxicity and that this process is carried out via the combined decrease in Cx36 activity, overexpression of Hsp70 and fall in AIF translocation. Thus, retarding or inhibiting HM death may be experimentally achieved by targeting one of these processes leading to motoneuron death.

Nicotinic receptors modulate the onset of reactive oxygen species production and mitochondrial dysfunction evoked by glutamate uptake block in the rat hypoglossal nucleus.

In several neurodegenerative diseases, glutamate-mediated excitotoxicity is considered to be a major process to initiate cell degeneration. Indeed, subsequent to excessive glutamate receptor stimulation, reactive oxygen species (ROS) generation and mitochondrial dysfunction are regarded as two major gateways leading to neuron death. These processes are mimicked in an in vitro model of rat brainstem slice when excitotoxicity is induced by DL-threo-ß-benzyloxyaspartate (TBOA), a specific glutamate-uptake blocker that increases extracellular glutamate. Our recent study has demonstrated that brainstem hypoglossal motoneurons, which are very vulnerable to this damage, were neuroprotected from excitotoxicity with nicotine application through the activation of nicotinic acetylcholine receptors (nAChRs) and subsequent inhibition of ROS and mitochondrial dysfunction. The present study examined if endogenous cholinergic activity exerted any protective effect in this pathophysiological model and how ROS production (estimated with rhodamine fluorescence) and mitochondrial dysfunction (measured as methyltetrazolium reduction) were time-related during the early phase of excitotoxicity (0-4h). nAChR antagonists did not modify TBOA-evoked ROS production (that was nearly doubled over control) or mitochondrial impairment (25% decline), suggesting that intrinsic nAChR activity was insufficient to contrast excitotoxicity and needed further stimulation with nicotine to become effective. ROS production always preceded mitochondrial dysfunction by about 2h. Nicotine prevented both ROS production and mitochondrial metabolic depression with a delayed action that alluded to a complex chain of events targeting these two lesional processes. The present data indicate a relatively wide time frame during which strong nAChR activation can arrest a runaway neurotoxic process leading to cell death.

Nicotinic receptor activation contrasts pathophysiological bursting and neurodegeneration evoked by glutamate uptake block on rat hypoglossal motoneurons.

KEY POINTS: Impaired uptake of glutamate builds up the extracellular level of this excitatory transmitter to trigger rhythmic neuronal bursting and delayed cell death in the brainstem motor nucleus hypoglossus. This process is the expression of the excitotoxicity that underlies motoneuron degeneration in diseases such as amyotrophic lateral sclerosis affecting bulbar motoneurons. In a model of motoneuron excitotoxicity produced by pharmacological block of glutamate uptake in vitro, rhythmic bursting is suppressed by activation of neuronal nicotinic receptors with their conventional agonist nicotine. Emergence of bursting is facilitated by nicotinic receptor antagonists. Following excitotoxicity, nicotinic receptor activity decreases mitochondrial energy dysfunction, endoplasmic reticulum stress and production of toxic radicals. Globally, these phenomena synergize to provide motoneuron protection. Nicotinic receptors may represent a novel target to contrast pathological overactivity of brainstem motoneurons and therefore to prevent their metabolic distress and death. ABSTRACT: Excitotoxicity is thought to be one of the early processes in the onset of amyotrophic lateral sclerosis (ALS) because high levels of glutamate have been detected in the cerebrospinal fluid of such patients due to dysfunctional uptake of this transmitter that gradually damages brainstem and spinal motoneurons. To explore potential mechanisms to arrest ALS onset, we used an established in vitro model of rat brainstem slice preparation in which excitotoxicity is induced by the glutamate uptake blocker dl-threo-ß-benzyloxyaspartate (TBOA). Because certain brain neurons may be neuroprotected via activation of nicotinic acetylcholine receptors (nAChRs) by nicotine, we investigated if nicotine could arrest excitotoxic damage to highly ALS-vulnerable hypoglossal motoneurons (HMs). On 50% of patch-clamped HMs, TBOA induced intense network bursts that were inhibited by 1-10 µm nicotine, whereas nAChR antagonists facilitated burst emergence in non-burster cells. Furthermore, nicotine inhibited excitatory transmission and enhanced synaptic inhibition. Strong neuroprotection by nicotine prevented the HM loss observed after 4 h of TBOA exposure. This neuroprotective action was due to suppression of downstream effectors of neurotoxicity such as increased intracellular levels of reactive oxygen species, impaired energy metabolism and upregulated genes involved in endoplasmic reticulum (ER) stress. In addition, HMs surviving TBOA toxicity often expressed UDP-glucose glycoprotein glucosyltransferase, a key element in repair of misfolded proteins: this phenomenon was absent after nicotine application, indicative of ER stress prevention. Our results suggest nAChRs to be potential targets for inhibiting excitotoxic damage of motoneurons at an early stage of the neurodegenerative process.

Azospirillum brasilense siderophores with antifungal activity against Colletotrichum acutatum.

Anthracnose, caused by the fungus Colletotrichum acutatum is one of the most important diseases in strawberry crop. Due to environmental pollution and resistance produced by chemical fungicides, nowadays biological control is considered a good alternative for crop protection. Among biocontrol agents, there are plant growth-promoting bacteria, such as members of the genus Azospirillum. In this work, we demonstrate that under iron limiting conditions different strains of A. brasilense produce siderophores, exhibiting different yields and rates of production according to their origin. Chemical assays revealed that strains REC2 and REC3 secrete catechol type siderophores, including salicylic acid, detected by thin layer chromatography coupled with fluorescence spectroscopy and gas chromatography-mass spectrometry analysis. Siderophores produced by them showed in vitro antifungal activity against C. acutatum M11. Furthermore, this latter coincided with results obtained from phytopathological tests performed in planta, where a reduction of anthracnose symptoms on strawberry plants previously inoculated with A. brasilense was observed. These outcomes suggest that some strains of A. brasilense could act as biocontrol agent preventing anthracnose disease in strawberry.