A Phase I/IIa Prospective, Randomized, Open-Label Study on the Safety and Efficacy of Nebulized Liposomal Amphotericin for Invasive Pulmonary Aspergillosis.

Although nebulized liposomal amphotericin B (NLAB) is being used in invasive pulmonary aspergillosis (IPA) prophylaxis, no clinical trial has shown its efficacy as a therapeutic strategy. NAIFI is the inaugural randomized, controlled clinical trial designed to examine the safety and effectiveness of NLAB (dosage: 25 mg in 6 mL, three times per week for 6 weeks) against a placebo, in the auxiliary treatment of IPA. Throughout the three-year clinical trial, thirteen patients (six NLAB, seven placebo) were included, with 61% being onco-hematological with less than 100 neutrophils/µL. There were no significant differences noted in their pre- and post-nebulization results of forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and oxygen saturation between the groups. Neither bronchospasm nor serum amphotericin B levels were reported in any patients given NLAB. 18F-Fluorodeoxyglucose positron emission tomography (FDG-PET-TC) was carried out at the baseline and after 6 weeks. A notable decrease in median SUV (standardized uptake value) was observed in NLAB patients after 6 weeks (-3.6 vs. -0.95, p: 0.039, one tail). Furthermore, a reduction in serum substance galactomannan and beta-D-Glucan was identified within NLAB recipients. NLAB is well tolerated and safe for patients with IPA. Encouraging indirect efficacy data have been derived from image monitoring or biomarkers. However, further studies involving more patients are necessary.

An impressive case of isolated thoracic impalement.

Case: A 61-year-old male construction worker was admitted to our Emergency Department due to being impaled in the chest after fall onto the long pole of his cement mixer. He was promptly scanned through the CT then transferred to theatre where unique technique for intubation was utilised prior to performing a Video Assisted Thoracoscopic Surgery exploration and extraction of the foreign object. Discussion: Impalement injuries are classified into Types I or II depending on the direction of movement of the human body in relation to the foreign object. There currently is no consensus on the best management of chest wall injuries involving impalements. Our case utilised Video Assisted Thoracoscopic Surgery as the dominant method of intervention together with highly skilled anaesthetic preparation. Conclusion: The combined expert anaesthetic and surgical approach utilised collectively had a role in ensuring the best possible outcome for the patient.

The Arabidopsis DNA glycosylase MBD4L repairs the nuclear genome in vivo.

DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl-CpG-binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4-like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5-fluorouracil (5-FU) and 5-bromouracil (5-BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5-FU and 5-BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER-associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5-BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5-FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG-GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L-deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions.

Correlation between the Altered Gut Microbiome and Lifestyle Interventions in Chronic Widespread Pain Patients: A Systematic Review.

Background: Lifestyle interventions have a direct impact on the gut microbiome, changing its composition and functioning. This opens an innovative way for new therapeutic opportunities for chronic widespread patients. Purpose: The goal of the present study was to evaluate a correlation between lifestyle interventions and the gut microbiome in patients with chronic widespread pain (CWP). Methods: The systematic review was conducted until January 2023. Pain and microbiome were the two keywords selected for this revision. The search was conducted in PubMed, Chochrane, PEDro and ScienceDirect, where 3917 papers were obtained. Clinical trials with lifestyle intervention in CWP patients were selected. Furthermore, these papers had to be related with the gut microbiome, excluding articles related to other types of microbiomes. Results: Only six articles were selected under the eligibility criteria. Lifestyle interventions were exercise, electroacupuncture and ingesting a probiotic. Conclusions: Lifestyle intervention could be a suitable choice to improve the gut microbiome. This fact could be extrapolated into a better quality of life and lesser levels of pain.

Ambulatory chest drainage with advanced nurse practitioner-led follow-up facilitates early discharge after thoracic surgery.

OBJECTIVES: To demonstrate the safety and feasibility of advanced nurse practitioner-led (ANP-led) outpatient follow-up after discharge with ambulatory chest drains for prolonged air leak and excessive fluid drainage. METHODS: Patients discharged with ambulatory chest drains between January 2017 and December 2019 were retrospectively reviewed. Discharge criteria included air leak < 200 ml/min or fluid drainage > 100 ml/24 h on a digital drain. Patients were reviewed weekly in the clinic by ANPs, a highly skilled cohort of nurses with physician support available. Outcomes included length of stay, duration of air or fluid leak and complications. RESULTS: Two-hundred patients were included, amounting to 368 clinic episodes. The median age was 68 ± 13 years and 119 (60%) were male. 112 (56%) patients underwent anatomical lung resection (total anatomical lung resections during the study period = 917) equating to a discharge with ambulatory chest drain rate of 12.2% in this group. The median length of stay was 6 ± 3 days and 176 (88%) patients were discharged with air leak versus 24 (12%) with excessive fluid drainage. The median time to drain removal was 12 ± 11 days. Complications occurred in 16 patients (8%) and 12 (6%) required readmission. An estimated 2075 inpatient days were saved over the study period equating to an annual cost saving of £123,167 (US$149,032) per annum. CONCLUSIONS: Patients with air leak or excessive fluid drainage can safely be discharged with ambulatory chest drains, allowing them to return to their familiar home environment safely and quickly. ANP-led clinics are a robust and cost-effective follow-up strategy and are associated with a low complication rate.

Association between Sleep Disorders and Sleep Quality in Patients with Temporomandibular Joint Osteoarthritis: A Systematic Review.

BACKGROUND: Osteoarthritis (OA) is a leading cause of disability, the most common form of chronic disease in the temporomandibular joint (TMJ), and the most severe disease type of temporomandibular disorders (TMD). The etiology of TMD is multifactorial, considering parafunctional habits, sleep bruxism, or sleep disturbance as common factors. Insomnia and apnea are the two most frequent forms of sleep disorders in TMD patients. Due to this, the objective of this systematic review was to highlight whether there is currently scientific evidence in the literature describing that patients with temporomandibular joint osteoarthritis (TMJ-OA) are associated with increased sleep disorders or impaired sleep quality. METHODS: This systematic review was completed in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) statement and was registered with PROSPERO prior to completion of the main search. Original observational studies that analyze the association of sleep disorders and sleep quality in patients with TMJ-OA were included in the present review. RESULTS: 770 studies were screened by abstract and title according to inclusion and exclusion criteria, and finally, 7 articles were included in the qualitative synthesis and a total of 772 patients diagnosed with TMJ-OA. CONCLUSIONS: There is insufficient evidence to indicate that patients with TMJ OA are associated with increased sleep disorders or poorer sleep quality.

Convergent Epigenetic Mechanisms Avoid Constitutive Expression of Immune Receptor Gene Subsets.

The gene pool encoding PRR and NLR immune receptors determines the ability of a plant to resist microbial infections. Basal expression of these genes is prevented by diverse mechanisms since their hyperactivity can be harmful. To approach the study of epigenetic control of PRR/NLR genes we here analyzed their expression in mutants carrying abnormal repressive 5-methyl cytosine (5-mC) and histone 3 lysine 9 dimethylation (H3K9me2) marks, due to lack of MET1, CMT3, MOM1, SUVH4/5/6, or DDM1. At optimal growth conditions, none of the mutants showed basal expression of the defense gene marker PR1, but all of them had greater resistance to Pseudomonas syringae pv. tomato than wild type plants, suggesting they are primed to stimulate immune cascades. Consistently, analysis of available transcriptomes indicated that all mutants showed activation of particular PRR/NLR genes under some growth conditions. Under low defense activation, 37 PRR/NLR genes were expressed in these plants, but 29 of them were exclusively activated in specific mutants, indicating that MET1, CMT3, MOM1, SUVH4/5/6, and DDM1 mediate basal repression of different subsets of genes. Some epigenetic marks present at promoters, but not gene bodies, could explain the activation of these genes in the mutants. As expected, suvh4/5/6 and ddm1 activated genes carrying 5-mC and H3K9me2 marks in wild type plants. Surprisingly, all mutants expressed genes harboring promoter H2A.Z/H3K27me3 marks likely affected by the chromatin remodeler PIE1 and the histone demethylase REF6, respectively. Therefore, MET1, CMT3, MOM1, SUVH4/5/6, and DDM1, together with REF6, seemingly contribute to the establishment of chromatin states that prevent constitutive PRR/NLR gene activation, but facilitate their priming by modulating epigenetic marks at their promoters.

Transposon clusters as substrates for aberrant splice-site activation.

Transposed elements (TEs) have dramatically shaped evolution of the exon-intron structure and significantly contributed to morbidity, but how recent TE invasions into older TEs cooperate in generating new coding sequences is poorly understood. Employing an updated repository of new exon-intron boundaries induced by pathogenic mutations, termed DBASS, here we identify novel TE clusters that facilitated exon selection. To explore the extent to which such TE exons maintain RNA secondary structure of their progenitors, we carried out structural studies with a composite exon that was derived from a long terminal repeat (LTR78) and AluJ and was activated by a C > T mutation optimizing the 5' splice site. Using a combination of SHAPE, DMS and enzymatic probing, we show that the disease-causing mutation disrupted a conserved AluJ stem that evolved from helix 3.3 (or 5b) of 7SL RNA, liberating a primordial GC 5' splice site from the paired conformation for interactions with the spliceosome. The mutation also reduced flexibility of conserved residues in adjacent exon-derived loops of the central Alu hairpin, revealing a cross-talk between traditional and auxilliary splicing motifs that evolved from opposite termini of 7SL RNA and were approximated by Watson-Crick base-pairing already in organisms without spliceosomal introns. We also identify existing Alu exons activated by the same RNA rearrangement. Collectively, these results provide valuable TE exon models for studying formation and kinetics of pre-mRNA building blocks required for splice-site selection and will be useful for fine-tuning auxilliary splicing motifs and exon and intron size constraints that govern aberrant splice-site activation.

What are the Top 10 Unanswered Questions in Molecular Plant-Microbe Interactions?

This article is part of the Top 10 Unanswered Questions in MPMI invited review series.The past few decades have seen major discoveries in the field of molecular plant-microbe interactions. As the result of technological and intellectual advances, we are now able to answer questions at a level of mechanistic detail that we could not have imagined possible 20 years ago. The MPMI Editorial Board felt it was time to take stock and reassess. What big questions remain unanswered? We knew that to identify the fundamental, overarching questions that drive our research, we needed to do this as a community. To reach a diverse audience of people with different backgrounds and perspectives, working in different areas of plant-microbe interactions, we queried the more than 1,400 participants at the 2019 International Congress on Molecular Plant-Microbe Interactions meeting in Glasgow. This group effort resulted in a list of ten, broad-reaching, fundamental questions that influence and inform our research. Here, we introduce these Top 10 unanswered questions, giving context and a brief description of the issues. Each of these questions will be the subject of a detailed review in the coming months. We hope that this process of reflecting on what is known and unknown and identifying the themes that underlie our research will provide a framework to use going forward, giving newcomers a sense of the mystery of the big questions and inspiring new avenues and novel insights.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Pharmacokinetics of echinocandins in suspected candida peritonitis: A potential risk for resistance.

INTRODUCTION: A possible increase in Candida resistance, especially in Candida glabrata, has been speculated according to poor diffusion of echinocandins to peritoneal fluid. MATERIALS/METHODS: Peritoneal and serum concentrations of caspofungin, micafungin and anidulafungin were analysed in surgical patients with suspected candida peritonitis. After 4 days of starting therapy, serum and peritoneal samples (through peritoneal drainage) were obtained at baseline, 1, 6, 12 and 24 h of drug administration. Micafungin and anidulafungin concentrations were determined using high-performance liquid chromatography (HPLC/F), whereas caspofungin concentrations were established by bioassay. RESULTS: Twenty-three critically ill patients with suspected abdominal fungal infection who were receiving an echinocandin were prospectively recruited. No specific criteria were applied to prescribe one specific echinocandin. No special clinical differences were observed among the three groups of patients. All were receiving antibiotic therapy, 80% required inotropic drugs, and fungal peritonitis was confirmed in 74% of them. The AUC0_24h (mg × h/L) obtained in serum and peritoneal fluid were: 126.84 and 34.38, 98.52 and 18.83, and 66.9 and 8.78 for anidulafungin, micafungin and caspofungin, respectively. The median concentration in peritoneal fluid ranged from 0.66 to 1.82 µg/mL for anidulafungin, 0.68-0.88 µg/mL for micafungin and 0.21-0.46 µg/mL for caspofungin. CONCLUSION: The results showed moderate penetration of echinocandins into the peritoneal fluid of these patients. These levels are below the threshold of resistance mutant selection published by other authors. This could justify a potential risk of resistance in patients with prolonged treatment with echinocandins and suboptimal control of abdominal infection.

OXR2 Increases Plant Defense against a Hemibiotrophic Pathogen via the Salicylic Acid Pathway.

Arabidopsis (Arabidopsis thaliana) OXIDATION RESISTANCE2 (AtOXR2) is a mitochondrial protein belonging to the Oxidation Resistance (OXR) protein family, recently described in plants. We analyzed the impact of AtOXR2 in Arabidopsis defense mechanisms against the hemibiotrophic bacterial pathogen Pseudomonas syringae oxr2 mutant plants are more susceptible to infection by the pathogen and, conversely, plants overexpressing AtOXR2 (oeOXR2 plants) show enhanced disease resistance. Resistance in these plants is accompanied by higher expression of WRKY transcription factors, induction of genes involved in salicylic acid (SA) synthesis, accumulation of free SA, and overall activation of the SA signaling pathway. Accordingly, defense phenotypes are dependent on SA synthesis and SA perception pathways, since they are lost in isochorismate synthase1/salicylic acid induction deficient2 and nonexpressor of pathogenesis-related genes1 (npr1) mutant backgrounds. Overexpression of AtOXR2 leads to faster and stronger oxidative burst in response to the bacterial flagellin peptide flg22 Moreover, AtOXR2 affects the nuclear localization of the transcriptional coactivator NPR1, a master regulator of SA signaling. oeOXR2 plants have increased levels of total glutathione and a more oxidized cytosolic redox cellular environment under normal growth conditions. Therefore, AtOXR2 contributes to establishing plant protection against infection by P. syringae acting on the activity of the SA pathway.

The N-terminal domain of Arabidopsis proline dehydrogenase affects enzymatic activity and protein oligomerization.

Proline dehydrogenase (ProDH) is a flavoenzyme that catalyzes the oxidation of proline (Pro) into Δ1-pyrroline-5-carboxylate (P5C). In eukaryotes, ProDH coordinates with different Pro metabolism enzymes to control energy supply or stress responses signaling. Heterologous expression and crystallization of prokaryotic enzymes provided key data on their active center, folding capacity and oligomerization status. In contrast, eukaryotic ProDHs have not been crystallized so far, and their study as recombinant proteins remains limited. Plants contain two isoforms of ProDH with non-redundant functions. To contribute to the study of these enzymes, we describe the modeling, expression in E. coli, purification, and characterization of the Arabidopsis isoenzymes, AtProDH1 and AtProDH2. The 3D model suggested that both proteins adopt a distorted barrel structure (ßα) with a cap formed by N-terminal α helices. The expression of two types of N-terminal deletion proteins indicated that this domain affected enzyme activity. Full-length enzymes had Km values similar to those of native proteins, whereas truncated proteins were inactive. Moreover, the first α helix proved to be necessary for AtProDH1 and AtProDH2 activities. Interestingly, both isoenzymes were able to oligomerize and this also required the first N-terminal α helix. Thus, we report the first insights into structure-function relationship of plant ProDHs demonstrating that the N-terminus, although not directly involved in catalysis, controls enzyme arrangement and activity. The resources generated here could be useful to analyze other plant ProDH features, such as its coordination with other enzymes, and differences between ProDH1 and ProDH2, providing new information on its effects on stress tolerance.

Robust increase of leaf size by Arabidopsis thaliana GRF3-like transcription factors under different growth conditions.

An increase in crop yield is essential to reassure food security to meet the accelerating global demand. Several genetic modifications can increase organ size, which in turn might boost crop yield. Still, only in a few cases their performance has been evaluated under stress conditions. MicroRNA miR396 repress the expression of GROWTH-REGULATING FACTOR (GRF) genes that codes for transcription factors that promote organ growth. Here, we show that both Arabidopsis thaliana At-GRF2 and At-GRF3 genes resistant to miR396 activity (rGRF2 and rGRF3) increased organ size, but only rGRF3 can produce this effect without causing morphological defects. Furthermore, introduction of At-rGRF3 in Brassica oleracea can increase organ size, and when At-rGRF3 homologs from soybean and rice are introduced in Arabidopsis, leaf size is also increased. This suggests that regulation of GRF3 activity by miR396 is important for organ growth in a broad range of species. Plants harboring rGRF3 have larger leaves also under drought stress, a condition that stimulates miR396 accumulation. These plants also showed an increase in the resistance to virulent bacteria, suggesting that the size increment promoted by rGRF3 occurs without an obvious cost on plant defenses. Our findings indicate that rGRF3 can increase plant organ size under both normal and stress conditions and is a valuable tool for biotechnological applications.

Differential control and function of Arabidopsis ProDH1 and ProDH2 genes on infection with biotrophic and necrotrophic pathogens.

Arabidopsis contains two proline dehydrogenase (ProDH) genes, ProDH1 and ProDH2, encoding for homologous and functional isoenzymes. Although ProDH1 has been studied extensively, especially under abiotic stress, ProDH2 has only started to be analysed in recent years. These genes display distinctive expression patterns and show weak transcriptional co-regulation, but are both activated in pathogen-infected tissues. We have demonstrated previously that Arabidopsis plants with silenced ProDH1/2 expression fail to trigger defences against the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. tomato AvrRpm1 (Pst-AvrRpm1), and that ProDH1 and ProDH2 are differentially regulated by salicylic acid (SA). In the current work, we used prodh1 and prodh2 single-mutant plants to assess the particular contribution of each gene to resistance against Pst-AvrRpm1 and the necrotrophic fungal pathogen Botrytis cinerea. In addition, we studied the sensitivity of ProDH1 and ProDH2 to the jasmonic acid (JA) defence pathway. We found that ProDH1 and ProDH2 are both necessary to achieve maximum resistance against Pst-AvrRpm1 and B. cinerea. However, ProDH2 has a major effect on early restriction of B. cinerea growth. Interestingly, ProDH1 is up-regulated by SA and JA, whereas ProDH2 is only activated by JA, and both genes display transcriptional inter-regulation at basal and infection conditions. These studies provide the first evidence of the contribution of ProDH2 to disease resistance, and describe the differential regulation and non-redundant but complementary function of both enzyme isoforms in infected tissues, providing support for a fundamental role of ProDH in the control of biotrophic and necrotrophic pathogens.

Arabidopsis Proline Dehydrogenase Contributes to Flagellin-Mediated PAMP-Triggered Immunity by Affecting RBOHD.

Plants activate different defense systems to counteract the attack of microbial pathogens. Among them, the recognition of conserved microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) by pattern-recognition receptors stimulates MAMP- or PAMP-triggered immunity (PTI). In recent years, the elicitors, receptors, and signaling pathways leading to PTI have been extensively studied. However, the contribution of organelles to this program deserves further characterization. Here, we studied how processes altering the mitochondrial electron transport chain (mETC) influence PTI establishment. With particular emphasis, we evaluated the effect of proline dehydrogenase (ProDH), an enzyme that can load electrons into the mETC and regulate the cellular redox state. We found that mETC uncouplers (antimycin or rotenone) and manganese superoxide dismutase deficiency impair flg22-induced responses such as accumulation of reactive oxygen species (ROS) and bacterial growth limitation. ProDH mutants also reduce these defenses, decreasing callose deposition as well. Using ProDH inhibitors and ProDH inducers (exogenous Pro treatment), we showed that this enzyme modulates the generation of ROS by the plasma membrane respiratory burst NADPH oxidase homolog D. In this way, we contribute to the understanding of mitochondrial activities influencing early and late PTI responses and the coordination of the redox-associated mitochondrial enzyme ProDH with defense events initiated at the plasma membrane.

The synthetic cationic lipid diC14 activates a sector of the Arabidopsis defence network requiring endogenous signalling components.

Natural and synthetic elicitors have contributed significantly to the study of plant immunity. Pathogen-derived proteins and carbohydrates that bind to immune receptors, allow the fine dissection of certain defence pathways. Lipids of a different nature that act as defence elicitors, have also been studied, but their specific effects have been less well characterized, and their receptors have not been identified. In animal cells, nanoliposomes of the synthetic cationic lipid 3-tetradecylamino-tert-butyl-N-tetradecylpropionamidine (diC14) activate the TLR4-dependent immune cascade. Here, we have investigated whether this lipid induces Arabidopsis defence responses. At the local level, diC14 activated early and late defence gene markers (FRK1, WRKY29, ICS1 and PR1), acting in a dose-dependent manner. This lipid induced the salicylic acid (SA)-dependent, but not jasmonic acid (JA)-dependent, pathway and protected plants against Pseudomonas syringae pv. tomato (Pst), but not Botrytis cinerea. diC14 was not toxic to plant or pathogen, and potentiated pathogen-induced callose deposition. At the systemic level, diC14 induced PR1 expression and conferred resistance against Pst. diC14-induced defence responses required the signalling protein EDS1, but not NDR1. Curiously, the lipid-induced defence gene expression was lower in the fls2/efr/cerk1 triple mutant, but still unchanged in the single mutants. The amidine headgroup and chain length were important for its activity. Given the robustness of the responses triggered by diC14, its specific action on a defence pathway and the requirement for well-known defence components, this synthetic lipid is emerging as a useful tool to investigate the initial events involved in plant innate immunity.

COMPARACIÓN DE MÉTODOS CONVENCIONALES Y MOLECULARES PARA LA DETECCIÓN DE Giardia lamblia EN HECES HUMANAS / COMPARISON OF CONVENTIONAL AND MOLECULAR METHODS FOR DETECTION OF Giardia lamblia IN HUMAN FEACES

Los protozoarios del género Giardia representan algunos de los parásitos humanos más comunes en el mundo y están entre los principales causantes de infecciones gastrointestinales y enfermedades diarreicas en humanos. La detección del parásito se fundamenta generalmente en los métodos por concentración y microscopía convencional, pero estas técnicas presentan limitaciones por su baja sensibilidad e inespecificidad en el diagnóstico. En procura de mejorar los métodos de diagnóstico, las técnicas moleculares se perfilan como una alternativa promisoria. En este estudio se analizaron 88 muestras de heces provenientes de pacientes de una empresa prestadora de servicios de salud (ASSBASALUD) de la ciudad de Manizales (Caldas). Para la detección de Giardia lamblia en heces se compararon tres métodos diferentes por medio del porcentaje de positividad: los métodos convencionales de concentración de la muestra y observación microscópica, análisis por inmunoensayo (ELISA indirecto) y finalmente la amplificación de dos secuencias génicas nucleares por PCR. Se obtuvieron tres muestras positivas por concentración y microscopía convencional, dos por inmunoensayo y 26 por técnicas moleculares. El estudio sugiere que las pruebas diagnósticas rutinarias basadas en microscopía convencional e inmunoensayo, tienen más bajo porcentaje de detección de este parásito y que esta deficiencia puede ser compensada por medio de la implementación de métodos de diagnóstico molecular basados en PCR, como una estrategia complementaria de apoyo en el diagnóstico de este protozoo.

The protozoa of the genus Giardia represent one of the most common human parasites in the world and are among the main causes of gastrointestinal infections and diarrheal diseases in humans. Parasite detection is generally based on concentration and conventional microscopy methods, but these techniques have limitations due to their low sensitivity and specificity in diagnosis. In an attempt to improve the diagnostic methods, molecular techniques are emerging as a promising alternative. In this study 88 stool samples from patients of a company that provides health services (ASSBASALUD) in the city of Manizales (Caldas) were analyzed. In order to detect Giardia lamblia in stool, three different methods were compared using the positivity percent: conventional methods for concentration of the sample and microscopic observation, analysis by immunoassay (indirect ELISA) and finally the amplification of two nuclear gene sequences by PCR. Three positive samples were obtained by concentration and conventional microscopy, two by immunoassay and 26 by molecular techniques. The study suggests that routine diagnostic tests based on conventional microscopy and immunoassay have lower detection rate of this parasite and that this deficiency can be compensated by means of the implementation of molecular diagnostic methods based on PCR, as a complementary strategy to support the diagnosis of this protozoan.

Context of action of proline dehydrogenase (ProDH) in the Hypersensitive Response of Arabidopsis.

BACKGROUND: Proline (Pro) dehydrogenase (ProDH) potentiates the oxidative burst and cell death of the plant Hypersensitive Response (HR) by mechanisms not yet elucidated. ProDH converts Pro into ∆1 pyrroline-5-carboxylate (P5C) and can act together with P5C dehydrogenase (P5CDH) to produce Glu, or with P5C reductase (P5CR) to regenerate Pro and thus stimulate the Pro/P5C cycle. To better understand the effects of ProDH in HR, we studied the enzyme at three stages of the defense response differing in their ROS and cell death levels. In addition, we tested if ProDH requires P5CDH to potentiate HR. RESULTS: Control and infected leaves of wild type and p5cdh plants were used to monitor ProDH activity, in vivo Pro catabolism, amino acid content, and gene expression. Wild type plants activated ProDH at all HR stages. They did not consume Pro during maximal ROS accumulation, and maintained almost basal P5C levels at all conditions. p5cdh mutants activated ProDH as wild type plants. They achieved maximum oxidative burst and cell death levels producing normal HR lesions, but evidenced premature defense activation. CONCLUSION: ProDH activation has different effects on HR. Before the oxidative burst it leads to Pro consumption involving the action of P5CDH. During the oxidative burst, ProDH becomes functionally uncoupled to P5CDH and apparently works with P5CR. The absence of P5CDH does not reduce ROS, cell death, or pathogen resistance, indicating this enzyme is not accompanying ProDH in the potentiation of these defense responses. In contrast, p5cdh infected plants displayed increased ROS burst and earlier initiation of HR cell death. In turn, our results suggest that ProDH may sustain HR by participating in the Pro/P5C cycle, whose action on HR must be formally evaluated in a future.