Unfolded protein response IRE1/XBP1 signaling is required for healthy mammalian brain aging.

Aging is a major risk factor to develop neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors involved in synaptic function and pathways linked to neurodegenerative diseases. The genes modified by XBP1 in the aged hippocampus where also altered. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.

Comparative Evaluation of Assays for Broad Detection of Molecular Resistance Mechanisms in Enterobacterales Isolates.

Rapid detection of antimicrobial resistance in both surveillance and diagnostic settings is still a major challenge for the clinical lab, compounded by the rapid evolution of antibiotic resistance mechanisms. This study compares four methods for the broad detection of antibiotic resistance genes in Enterobacterales isolates: two multiplex PCR assays (the Streck ARM-D beta-lactamase kit and the OpGen Acuitas AMR Gene Panel u5.47 (research use only [RUO]) and one microarray assay (the Check-MDR CT103XL assay), with whole-genome sequencing as a reference standard. A total of 65 Gram-negative bacterial isolates, from 56 patients, classified by phenotypic antimicrobial susceptibility testing (AST) as showing resistance to beta-lactam antimicrobials (extended-spectrum beta-lactamase [ESBL] positive or resistance to third-generation cephalosporins or carbapenems) were included in the study. Overall concordance between the molecular assays and sequencing was high. While all three assays had similar performance, the OpGen Acuitas AMR assay had the highest overall percent concordance with sequencing results. The primary differences between the assays tested were the number and diversity of targets, ranging from 9 for Streck to 34 for OpGen. This study shows that commercially available PCR-based assays can provide accurate identification of antimicrobial resistance loci in clinically significant Gram-negative bacteria. Further studies are needed to determine the clinical diagnostic role and potential benefit of such methods.

Survival of the nematophagous fungus Duddingtonia flagrans to in vitro segments of sheep gastrointestinal tract.

The nematophagous fungus Duddingtonia flagrans is used in integrated management of gastrointestinal nematodes in ruminants. The chlamydospores of the fungus, orally administered, pass through the segments of the ruminant digestive tract and, in the feces, capture the nematodes preventing their migration to grasslands. The drastic conditions of the gastrointestinal segments can negatively affect the fungus' biocontrol activity. The aim of this study was to assess the effect of in vitro conditions of the sheep's main gastrointestinal segments on the concentration, viability and nematode predatory ability of D. flagrans chlamydospores. The segments evaluated separately in vitro were the oral cavity, rumen, abomasum, and small intestine. The results showed that chlamydospores concentration was not affected by exposure to the different segments. The viability of the chlamydospores after exposure to the oral cavity (2.53 × 106 CFU/mL) and small intestine (1.24 × 105 CFU/mL) was significantly lower than its control treatment, with values of 6.67 × 106 CFU/mL and 2.31 × 105 CFU/mL respectively. Nematode predatory ability after rumen exposure was reduced by 7% compared to the control treatment, by 25% after abomasum exposure and by 17% after small intestine. This study revealed the individual in vitro effect of each segment of ovine gastrointestinal tract on the integrity of this strain of the fungus D. flagrans affecting its viability and nematode predatory ability under the evaluated conditions. Delivery systems could be designed to protect chlamydospores considering the impact of each gastrointestinal segment.

Clinical Characteristics in Patients with Rheumatoid Arthritis: Differences between Genders.

OBJECTIVE: To compare the clinical characteristics of a group of men and women with rheumatoid arthritis (RA) and determine the differences between genders. MATERIALS AND METHODS: A descriptive and comparative cross-sectional study was developed with a group of 50 men and a control group of 50 women with RA, from a rheumatology center in the city of Guayaquil, Ecuador. Data collected included clinical manifestations, comorbidities, treatment, and disease activity. Clinical and activity differences between sexes were analyzed. RESULTS: Women were more devoted to housework (66%), while men consumed more tobacco (34%) and alcohol (38%). Fatigue (60%), loss of appetite (54%), and weight loss (44%) were more common in women. No differences were found in comorbidities or treatment. Women had higher values of DAS-28 (3.4 vs 2.5), HAQ-DI (1.1 vs 0.4), ESR (33.0 vs 23.2), painful joints (8 vs 3), swollen joints (6 vs 2), and overall physician assessment (3 vs 2). CONCLUSION: The results are similar to other publications that establish that women have a more aggressive disease with greater activity of the disease and disability.

Multiplate and TEG platelet mapping in a population of severely injured trauma patients.

OBJECTIVES: The objectives of this study were to compare thromboelastography platelet mapping (TEG PM) with impedance aggregometry (Multiplate, MP) in a single trauma population and relate their results clinically. BACKGROUND: Platelet function as measured by thromboelastography and impedance aggregometry demonstrates significant reductions that persist for days following traumatic injury. However, no study compares these devices and the correlation between them is not known. METHODS: In level 1 trauma patients, TEG PM and MP were conducted at their initial presentation to the emergency department. Within-device repeatability and between-device association were determined using correlation analyses. Demographic variables, Injury Severity Score, blood product transfusion, laboratory test results and mortality rate were recorded. RESULTS: Ninety-two patients were enrolled. Within-device repeatability was high for TEG PM and MP for arachidonic acid (AA) and adenosine diphosphate (ADP) activation pathways. When comparing TEG PM with MP, results correlated poorly in the ADP pathway (Spearman's rho = 0·11, P = 0·44) and moderately in the AA pathway (Spearman's rho = 0·56, P < 0·0001). TEG PM was predictive of blood product transfusion and correlated with increased base deficit, whereas MP was only predictive of mortality. CONCLUSIONS: Intra-device variability was low for TEG PM and MP, but the two point-of-care devices measuring platelet function correlate poorly with each other in injured trauma patients. Each device also had different clinical associations.

Practical approach to the evaluation of industrial wastewater treatment by the application of advanced microbiological techniques.

In cork industry, the operation of boiling raw cork generates large volumes of wastewater named Cork Boiling Wastewater (CBW). The main characteristics are the low biodegradability and medium to low acute toxicity, resulting in the necessity of designing advanced biological treatments by possible conventional activated sludge adaptation. In order to evaluate the variation of bacterial population along that process, a study based on optical microscopy, plate count, DNA extraction, qPCR and massive sequencing techniques was performed. Results showed a diminution of the total and volatile solids (TSS and VSS), jointly with a decrease in DNA concentration, general bacteria (16 S) and ammonia-oxidizing bacteria (AOB). After a few hours of testing, diverse microbiological species died while others showed a possible adaptation of the biological system, accompained by a dissolved organic carbon (DOC) reduction. In addition, toxicity tests based on activated sludge showed the development of chronic toxicity through the contact time. Combination of classical and advanced microbiological techniques, such as quantitative real time Polymerase Chain Reaction (qPCR) and metagenomics, was essential to predict the variation of species during the experiment and to conclude if effective biological adaptation could be finally attained for the target complex wastewater.

Chemical acetylation of nixtamalized maize flour and structural, rheological and physicochemical properties of flour, dough and tortillas.

Nixtamalized maize tortilla is a basic food for the Mexican population. It has high energy due to high starch, which may be modified to decrease its bioavailability and to produce changes in the characteristics of flours. For this research, nixtamalized maize flour was prepared and subjected to an acetylation chemical process, with and without prior hydrolysis. Raw maize flour, traditionally nixtamalized maize flour, acetylated-nixtamalized maize flour (AF) and acetylated-hydrolyzed nixtamalized maize flour (AHF) were prepared and evaluated. These flours were used for dough and tortilla preparation and analyzed for degree of substitution (DS), physicochemical properties, structure, thermal, rheological, morphological and texture properties. FTIR spectra and DS showed the presence of acetyl groups. AHF showed the highest value for water absorption index. The resistant starch increased 0.27 and 0.42% for AF and AHF samples. The gelatinization enthalpy (∆Hg) for AF was greater than other flours. AF tortillas showed better characteristics than the traditional ones and their consumption was recommended since showed better RS.

Study of the Huntington's disease IT-15 gene in different ethnic groups in Ecuador.

This study aims to establish the current state of the IT-15 (HTT) gene in different Ecuadorian ethnic groups and patients by determining CAG triplet repeats, compared with the ethnicity of individuals. A total of 412 individuals were studied using nested polymerase chain reaction and Sanger sequencing: 75 individuals were indigenous (Kichwas), 211 mestizos, and 65 Afro-Ecuadorians. We included 31 patients who were clinically diagnosed with Huntington's disease (HD) and relatives of the affected patients (n = 30). Moreover, we correlated the presence of HD in Ecuadorian patients with 46 genetic ancestry-informative insertion-deletion polymorphic markers. We found that 77.20% had <28 CAG repetitions, 18.80% had mutable alleles, 2.27% had incomplete penetrance, and 1.70% reflected >39 repetitions. The average of CAG repetitions was 24 ± 3 for indigenous people; 28 ± 2 for mestizos; and 24 ± 3.2 repetitions for the Afro-Ecuadorians. The ancestral component showed that the main ancestry corresponded to Native Americans (0.873) and European ascendants (0.145), Africans were less represented in the evaluated population (0.018). There was a significant difference between the number of CAG repeats in mestizos and indigenous people (P < .01), suggesting that the Ecuadorian mestizo population has a risk factor for the gene mutation.

[Morgagni-Larrey anterior diaphragmatic hernias. Review of 13 cases]. / Hernias diafragmáticas anteriores. Revisión de 13 casos.

OBJECTIVE: A review of anterior congenital diaphragmatic hernias (CDH) treated at our center, analysis of our experience and evolution in the surgical technique in pediatric patients over the last 15 years. MATERIAL AND METHODS: A retrospective descriptive study of patients of our center with anterior CDH (Morgagni, Larrey and Morgagni-Larrey) between 2000 and 2015, through a systematic review of clinical records. The studied variables were: age at diagnosis, age at treatment, diagnostic methods, symptoms, surgical technique, evolution, complications and relapses. Posterior CDH (Bochdalek) and patients older than 14 years old were excluded. RESULTS: 13 patients (8 females) with anterior diaphragmatic hernia. Mean age at diagnosis was 29.4 months (range fetal age 4 months-14 years old) and mean age at surgery 18.5 m (range 1 day-14 years). Three left hernias (Larrey hernia) and n= 2central hernias (Morgagni-Larrey). Two patients were diagnosed prenatally, the rest of them were casual diagnoses. Symptoms: n= 6 asymptomatic, n= 4respiratory symptoms, n= 1abdominal pain, n= 1both symptoms and n= 1 hydrops fetalis. In all cases, a chest X-ray was performed, CT in n= 5 cases and US in other n= 5 patients. The surgical approach was laparoscopic in n= 7 (1 conversion due to bleeding), thoracoscopic in n= 1 (requiring conversion to laparotomy due to technical difficulty), thoracotomy in n= 1patients and laparotomy in n= 4. Prosthetic patches were necessary in n= 2 patients. During the follow-up (3.9 years, range: 1-8), there were n= 2 recurrences (laparoscopy and thoracotomy) and n= 1 exitus 2 years after surgery, due to pathology not associated with diaphragmatic hernia. CONCLUSIONS: The minimally invasive approach is considered of choice in the literature. Our group considers that most patients with anterior CDH should be considered for a laparoscopic repair. The laparoscopic approach is easily reproducible and the conversion rate is low when it is performed by expert surgeons. However, the low prevalence of this pathology would require comparative and long-term multicenter studies to obtain statistically significant conclusions.

OBJETIVO: Revisión de las hernias diafragmáticas congénitas (HDC) anteriores intervenidas en nuestro centro, análisis de nuestra experiencia y evolución de la técnica quirúrgica a lo largo de 15 años en el paciente pediátrico. MATERIAL Y METODOS: Estudio descriptivo retrospectivo de pacientes pediátricos intervenidos de HDC anterior (Morgagni, Larrey y Morgagni-Larrey) en nuestro centro entre 2000 y 2015, mediante revisión sistemática de historias clínicas. Variables estudiadas: edad al diagnóstico, edad al tratamiento, pruebas diagnósticas, síntomas, técnica quirúrgica, evolución, complicaciones y recidivas. Fueron excluidas las hernias posteriores (Bochdaleck) y los pacientes mayores de 14 años. RESULTADOS: 13 pacientes (8 mujeres) con hernia diafragmática anterior. Media de edad al diagnóstico: 29,4 meses (rango: 4 meses de edad gestacional-14 años). Media de edad en el momento de la intervención: 18,5 meses (rango: 1 día-14 años). Tres hernias izquierdas (hernia de Larrey) y 2 centrales (Morgagni-Larrey). Dos diagnósticos prenatales, siendo el resto hallazgos casuales. Síntomas: n= 6 asintomáticos, n= 4 síntomas respiratorios, n= 1 dolor abdominal, n= 1 ambos síntomas, n= 1 hidrops fetalis. En todos los casos se realizó radiografía de tórax, en n= 5 casos TAC y en otros n= 5 ecografía. El abordaje quirúrgico fue laparoscópico en n= 7 (1 conversión por hemorragia), toracoscópico en n= 1 casos (precisando conversión a laparotomía por dificultad técnica), toracotomía en n= 1 y laparotomía en n= 4. Fue necesario el uso de parches protésicos en 2 pacientes. Seguimiento: media 3,9 años (rango: 1-8). Dos recidivas (laparoscopia y toracotomía) y n= 1 exitus a los 2 años de la intervención por patología no asociada a la hernia diafragmática. CONCLUSIONES: El abordaje mínimamente invasivo es considerado de elección en la literatura. Nuestro grupo defiende que la mayoría de los pacientes con HDC anterior deben considerarse para una reparación laparoscópica por ser fácilmente reproducible, con una tasa de conversión baja en manos de cirujanos expertos. Sin embargo, la baja prevalencia de esta patología requeriría estudios multicéntricos comparativos y prolongados en el tiempo para obtener conclusiones estadísticamente significativas.

First Report of Fusarium equiseti Causing Damping-Off Disease on Aleppo Pine in Algeria.

The Aleppo pine (Pinus halepensis Mill.) is a conifer native to the Mediterranean region. In 2008 and 2009, a survey of Aleppo pine seedling diseases was performed in three forest nurseries from Relizane, Sidi Bel Abbes, and Tlemcen provinces in northwestern Algeria. Aleppo pine seedlings showed symptoms of pre- and post-emergence damping-off disease, with an incidence of 64 to 77%. Four composite samples were taken from each location. Disinfested root and root collar segments, approximately 5 mm in length, were cultured on potato dextrose agar (PDA) and incubated at 25°C, and hyphal tips were transferred to PDA. Fusarium equiseti (Corda) Sacc. (teleomorph: Gibberella intricans Wollenw.) was identified from roots of two seedlings from the Sidi Bel Abbes nursery. Morphological identification was done according to Fusarium keys (2). PDA colonies with abundant, loosely floccose, whitish aerial mycelium and beige pigmentation were observed. Macroconidia with usually 5 to 6 septa, 31 to 45 µm long. A pronounced dorsiventral curvature, tapered and elongated apical cell, and prominent foot shape were observed. Microconidia were absent. Chlamydospores were produced in hyphae, most often intercalary, solitary, in pairs, frequently forming chains or clusters, globose (7 to 13 µm). To confirm the identity of this fungus, the internal transcribed spacer of F3RS1 and F19RS1 isolates of F. equiseti were amplified and sequenced using ITS1 and ITS4 primers (4), GenBank accession nos. JX114784 and JX114791, respectively. Those sequences bore 100% (HQ671182) similarity with sequences of F. equiseti in GenBank. Pathogenicity tests were performed to fulfill Koch's postulates. Inoculum was produced by adding a 5-mm-diameter plug from a 7-day-old CMA petri dish culture to a previously sterilized 500 ml flask (237.5 g sand, 12.5 g cornmeal, 80 ml sterile distilled water), shaken over 9 days at 25°C, and mixed with sterile sandy clay soil at 1:3 (v:v). Infested soil was then transferred to 500 ml pots, and 10 Aleppo pine seeds were planted per pot. A completely randomized design was used with three replicates per isolate and three control pots with a similar non-infested soil. After 1 month at 25°C the two tested isolates caused typical damping-off symptoms (collar rot) on seedlings and were re-isolated from recently infected tissues. The percentages of the inoculated plants that became infected were 59 to 65% among isolates (0% in control pots). To our knowledge, infection by F. equiseti is a first report on Aleppo pine in northwestern Algeria, Northern Africa, and globally, and on conifers in the Mediterranean region (1,3). In Algeria, F. equiseti is associated with black pepper (Piper nigrum L.) (3). These findings highlight the moderate impact of F. equiseti on the production of Aleppo seedling stock for reforestation activities in northwestern Algeria. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory. ARS, USDA, Beltsville, MD. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , February 20, 2013. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) D. W. Minter. Cybertruffle's Robigalia, Observations of Fungi and their Associated Organisms. Retrieved from http://www.cybertruffle.org.uk/robigalia/eng/ , February 20, 2013. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Novel Inositol 1,4,5-Trisphosphate Receptor Inhibitor Antagonizes Hepatic Stellate Cell Activation: A Potential Drug to Treat Liver Fibrosis.

Liver fibrosis, characterized by excessive extracellular matrix (ECM) deposition, can progress to cirrhosis and increases the risk of liver cancer. Hepatic stellate cells (HSCs) play a pivotal role in fibrosis progression, transitioning from a quiescent to activated state upon liver injury, wherein they proliferate, migrate, and produce ECM. Calcium signaling, involving the inositol 1,4,5-trisphosphate receptor (IP3R), regulates HSC activation. This study investigated the efficacy of a novel IP3R inhibitor, desmethylxestospongin B (dmXeB), in preventing HSC activation. Freshly isolated rat HSCs were activated in vitro in the presence of varying dmXeB concentrations. The dmXeB effectively inhibited HSC proliferation, migration, and expression of fibrosis markers without toxicity to the primary rat hepatocytes or human liver organoids. Furthermore, dmXeB preserved the quiescent phenotype of HSCs marked by retained vitamin A storage. Mechanistically, dmXeB suppressed mitochondrial respiration in activated HSCs while enhancing glycolytic activity. Notably, methyl pyruvate, dimethyl α-ketoglutarate, and nucleoside supplementation all individually restored HSC proliferation despite dmXeB treatment. Overall, dmXeB demonstrates promising anti-fibrotic effects by inhibiting HSC activation via IP3R antagonism without adverse effects on other liver cells. These findings highlight dmXeB as a potential therapeutic agent for liver fibrosis treatment, offering a targeted approach to mitigate liver fibrosis progression and its associated complications.

17-ß-estradiol and phytoestrogens elicit NO production and vasodilatation through PI3K, PKA and EGF receptors pathways, evidencing functional selectivity.

Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-ß-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2',7'-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.

Assessing geospatial models to explain the occurrence of clandestine graves in Mexico.

We present an assessment of several geospatial layers proposed as models for detecting clandestine graves in Mexico. The analyses were based on adapting the classical ROC curves to geospatial data (gROC) using the fraction of the predicted area instead of the false positive rate. Grave locations were obtained for ten Mexican states that represent the most conflicting regions in Mexico, and 30 layers were computed to represent geospatial models for grave detection. The gROC analysis confirmed that the travel time from urban streets to grave locations was the most critical variable for detecting graves, followed by nighttime light brightness and population density, whereas, contrary to the rationale, a previously proposed visibility index is less correlated with grave locations. We were also able to deduce which variables are most relevant in each state and to determine optimal thresholds for the selected variables.

Mitochondrial division inhibitor (mdivi-1) induces extracellular matrix (ECM)-detachment of viable breast cancer cells by a DRP1-independent mechanism.

Increasing evidence supports the hypothesis that cancer progression is under mitochondrial control. Mitochondrial fission plays a pivotal role in the maintenance of cancer cell homeostasis. The inhibition of DRP1, the main regulator of mitochondrial fission, with the mitochondrial division inhibitor (mdivi-1) had been associated with cancer cell sensitivity to chemotherapeutics and decrease proliferation. Here, using breast cancer cells we find that mdivi-1 induces the detachment of the cells, leading to a bulk of floating cells that conserved their viability. Despite a decrease in their proliferative and clonogenic capabilities, these floating cells maintain the capacity to re-adhere upon re-seeding and retain their migratory and invasive potential. Interestingly, the cell detachment induced by mdivi-1 is independent of DRP1 but relies on inhibition of mitochondrial complex I. Furthermore, mdivi-1 induces cell detachment rely on glucose and the pentose phosphate pathway. Our data evidence a novel DRP1-independent effect of mdivi-1 in the attachment of cancer cells. The generation of floating viable cells restricts the use of mdivi-1 as a therapeutic agent and demonstrates that mdivi-1 effect on cancer cells are more complex than anticipated.

First Report of Globisporangium ultimum Causing Pythium Damping-Off on Aleppo Pine in Algeria, Africa, and the Mediterranean Region.

Globisporangium ultimum (Trow) Uzuhashi, Tojo & Kakish. (syn. Pythium ultimum Trow, syn. P. ultimum Trow var. ultimum) is a known oomycetal species from Pythium s.l. causing damping-off and/or root rot on a great variety of plants throughout the world, including some pine species (Pinus L.) and conifers (2,3). Aleppo pine (Pinus halepensis Mill.) is a common native forest tree in the Mediterranean region. Pre- and post-emergence damping-off disease symptoms were observed during 2008 and 2009 in four forest nurseries from northwestern Algeria (Relizane, Sidi Belabes, and Tlemcen departments). This disease occurred under cool conditions, and Aleppo pines were significantly affected, reducing seedling emergence. Disinfected segments, about 5 mm in length, from decayed root and collar, were cultured on CMA at 25°C. This oomycetal species was identified based on the species description in Pythium keys (3,4). For the molecular identification, PCR was used to amplify the ITS region of Pythium isolates. It was amplified with the flanking primers ITS1 and ITS4, and these products were directly sequenced. Sequence data were compared to known sequences deposited in the NCBI non redundant database to confirm morphological identification. A BLAST search identified U3CR, U7CR, U1RT, U2CR, U4CR, U14CR, U7RT, and U17RT isolates (GenBank Accession Nos. JX191921, 22, 27, 29, 31, and 33 to 35, respectively) as G. ultimum based on 100% similarity with corresponding sequence of the reference isolate no. UZ056 MAFF240024 (AB468781) (3). Phytopathogenicity testing was conducted in a petri dish and pot experiment. In the petri dish experiment, a 3 mm diameter plug was transferred from a 7-day-old CMA colony to the center of a CMA petri dish, with three replicates per isolate, and three control plates were inoculated with sterile agar plugs. After 72 h, 10 Aleppo pine seeds were placed equally spaced to 1 cm from the edge of each plug. After 7 days at 22°C in the dark, germination inhibition (46.1 to 87.6%) and root growth inhibition (62.3 to 92.2%) were calculated. In the control plates, germination failure (13.4%) and root length (27.7 cm) were observed. For the pot experiment, inocula were produced by adding a 5 mm diameter plug from a 7-day-old CMA culture to a previously sterilized 500 ml flask containing 237.5 g sand, 12.5 g cornmeal, and 80 ml SDW. Nine-day-old inoculum was mixed with sterile soil at a rate of 1:3 (v:v). Inoculum was transferred to 500 ml pot, and 10 Aleppo pine seeds were planted, with three replicates per isolate, and three control pots were used. After 2 weeks, all of the isolates tested caused typical symptoms of Aleppo pine Pythium damping-off, the percentage of inoculated plants that became infected was 36.6 to 83.3%. In the control pots, no infected plants were observed. To our knowledge, this is the first report of G. ultimum causing damping-off on Aleppo pine in Algeria, Africa, and the Mediterranean Region. Before, Aleppo pine damping-off caused by G. ultimum was reported in Australia (1). References: (1) R. P. Cook and A. J. Dubé. Host-pathogen index of plant diseases in South Australia. SADA, Melbourne, Australia, 1989. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory. ARS, USDA, Bestville, MD. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , June 24, 2012. (3) S. Uzuhashi et al. Mycoscience 51:337, 2010. (4) A. J. van der Plaats-Niterink. Stud. Mycol. 21:1, 1981.

First Report of Fusarium redolens as a Causal Agent of Aleppo Pine Damping-Off in Algeria.

Aleppo pine (Pinus halepensis Mill.) is a common native coniferous tree in the natural forests of the Mediterranean region. In 2008 and 2009, a survey of Aleppo pine seedling diseases was performed in three forest nurseries from Relizane, Sidi Bel Abbes, and Tlemcen departments in northwestern Algeria. Aleppo pine seedlings showed symptoms of pre- and post-emergence damping-off, resulting in severe crop losses. The problem was widespread with a disease incidence of 64 to 77%. Fusarium redolens Wollenw. was isolated from Relizane and Sidi Bel Abbes forest nurseries. Disinfested root and root collar segments, approximately 5 mm in length, were cultured on potato dextrose agar (PDA) and incubated at 25°C. Morphological identification was done according to Fusarium keys (2). PDA colonies consisted of flat mycelium with sparse white aerial hyphae. Macroconidia with three to five septa, 24 to 43.8 µm long, widest upper third, hooked apical cell, and foot shaped basal cell were observed. Microconidia with zero to one septa, 6.8 to 10.4 µm long, oval to cylindrical, and produced on monophialides were also observed. Chlamydospores were produced abundantly in terminal and intercalary chains, in 3- to 4-week-old cultures. To confirm the identity of the fungus, the internal transcribed spacer (ITS) of F5RS3, F91SR, F55RS1, F8RS3, and F09SS1 isolates of F. redolens were amplified and sequenced using ITS 1 and ITS 4 primers (3). GenBank Accession Nos. are JX051323 to 26, and JX114783, respectively. Those sequences bore 99% (JF311916) and 100% (U34565) similarity with sequences of F. redolens in GenBank. A Fusarium pathogenicity assay was used to complete Koch's postulates. Inoculum was produced by adding a 5 mm diam. agar disc from a 7-day-old CMA petri dish culture to a previously sterilized 500 ml flask (237.5 g of sand, 12.5 g of cornmeal, and 80 ml of deionized H2O). Isolates were allowed to colonize the medium for 9 days, and flasks were shaken every day. The inoculum was mixed with sterile soil at a rate of 3:1 (v:v). Infested soil was then transferred to 500 ml pots, and 10 Aleppo pine seeds were planted. A completely randomized design was used with three replicates. After 1 month, all tested isolates caused typical damping-off symptoms on seedlings. The percentage of the inoculated plants that became infected was 53 to 91%. To our knowledge, this is the first report of F. redolens being pathogenic on Aleppo pine in northwestern Algeria and throughout the world. In Algeria, F. redolens has been reported on tomato (Solanum lycopersicum L.) (1), suggesting that it is adapted to the conditions of this area and could become a major threat to regional plant production. The annual economic impact of this disease was estimated at approximately US$50,000 per forest nursery. References: (1) N. Hamini-Kadar et al. New Dis. Rep. 22:3, 2010. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, Iowa, 2006. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Fusarium chlamydosporum Causing Damping-Off Disease on Aleppo Pine in Algeria.

The Aleppo pine (Pinus halepensis Mill.) is a conifer native to the Mediterranean region. In 2008 and 2009, a survey of Aleppo pine seedling diseases was performed in three forest nurseries from the Relizane, Sidi Bel Abbes, and Tlemcen departments in northwestern Algeria. One- to two-month-old Aleppo pine seedlings showed symptoms of damping-off in pre- and post-emergence (typical seedling collar rot). The problem was widespread with a disease incidence of 64 to 77% and an annual impact of US$50,000. Disinfested root and root collar segments (from four composite samples per location), approximately 5 mm in length, were cultured on PDA and incubated at 25°C and day/night light. Two (from 21) isolates were identified morphologically (2) as the anamorph Fusarium chlamydosporum Wollenw. & Reinking and isolated from collar rots of Relizane forest nursery seedlings. Colony development on PDA media was fast; 32 mm diameter colonies developed after 3 days. Colonies were white. Mycelia were floccose, fairly dense, off-white, and turned a lilac color in older portions of the colony. Macroconidia were thick-walled and moderately curved with unequal dorsiventral curvature (the lower wall is almost straight), short, curved and pointed apical cell, usually notched, but occasionally foot shaped basal cell, 3- to 5-septate, and 2 × 8 to 21 µm. Microconidia were abundant, 0-septate, and 2 × 6 to 9 µm. Chlamydospores were abundant, formed rapidly in single chains or clusters, and 8 to 15 µm diameter. To confirm the identity of this fungus, the internal transcribed spacer of F12RR and F4SR isolates of F. chlamydosporum were amplified and sequenced using ITS1 and ITS4 primers (4). Sequences were deposited in GenBank under accessions JX114795 and JX114789, respectively. Those sequences bore 99% similarity with reference sequence AY213655 (2) and 100% with HQ671187, also found 99 to 100% similarity with F. equiseti (Corda) Sacc. but with different conidia. Pathogenicity tests were performed to fulfill Koch's postulates. Inoculum was produced by adding a 5 mm diam. plug from a 7-day-old CMA petri dish culture to a previously sterilized 500 ml flask (237.5 g sand, 12.5 g cornmeal, 80 ml SDW), shaken over 9 days, and mixed with sterile soil at 1:3 (v:v). Infested soil was then transferred to 500 ml pots, and 10 seeds were planted. A completely randomized design was used with three replicates per isolate and three control pots. After 1 month, two tested isolates caused typical damping-off symptoms on seedlings. The percentage of the plants that became infected was 65 to 77%. To our knowledge (1,3), this is the first report of F. chlamydosporum on Aleppo pine in northwestern Algeria. It is also the first report of this fungal species affecting the Aleppo pine throughout the world, and on conifers in Africa and the Mediterranean region (1,3). References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Syst. Mycol. Microbiol. Lab. ARS, USDA, Beltsville, MD. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , February 20, 2013. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) D. W. Minter. Cybertruffle's Robigalia, Observations of Fungi and their Associated Organisms. Retrieved from http://www.cybertruffle.org.uk/robigalia/eng/ , February 20, 2013. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Fusarium acuminatum Causing Damping-Off Disease on Aleppo Pine in Algeria.

The Aleppo pine (Pinus halepensis Mill.) is a conifer native to the Mediterranean region. In autumn and spring of 2008 to 2009, a survey of Aleppo pine seedling diseases was carried out in three forest nurseries from the Relizane, Sidi Bel Abbes, and Tlemcen departments in northwestern Algeria. Aleppo pine seedlings were potted from the soil. In all three nurseries, 1- to 2-month old seedlings showed symptoms of damping-off disease in pre- and post-emergence (collar rot) with a disease incidence of 64, 77, and 72%, respectively. Disinfected collar segments, about 5 mm in length, were plated on PDA and petri dishes incubated at 25°C. A Fusarium sp. was consistently isolated from tissues and all isolates were morphologically identified as Fusarium acuminatum Ellis & Everh. (teleomorph: Gibberella acuminata Wollenw.) according to Fusarium keys (2). Colony growth was 43 mm after 3 days on PDA; the aerial mycelium was white, developing a brownish tinge in the center on PDA; macroconidia were formed in orange sporodochia, broadly falcate, strongly septate, 3 to 5 septa, the apical cell with an incurved elongation, distinct foot shape, 3 to 4 × 20 to 50 µm; microconidia were usually absent for isolates other than F12SS1, reniform, septate, 5 to 6 × 6 to 10 µm, in monophialides; chlamydospores were formed in chains, 6 to 13 µm. For the molecular identification, ITS regions of Fusarium isolates were amplified with the primers ITS1 and ITS4, and products were directly sequenced in both strands using the same primers ITS 1 and ITS4. Sequences were compared to known sequences deposited in the NCBI non redundant database to confirm morphological identification. An NCBI BLAST search identified isolates F12SS1, F14SS3, F30SS3, and F25SR as F. acuminatum based on 100% similarity with corresponding sequences. GenBank Accession Nos. were JX114788, JX114785, JX114782, and JX114790, respectively. Pathogenicity tests were performed to fulfill Koch's postulates. Inocula were produced by adding a 5-mm diameter plug from a 7-day-old CMA petri dish culture to a previously sterilized 500-ml flask (237.5 g sand, 12.5 g cornmeal, 80 ml SDW), shaken over 9 days, and mixed with sterile soil at 1:3 (v:v). The inocula were transferred to a 500-ml pot, and 10 Aleppo pine seeds were planted with three replicates. After 1 month, all tested isolates caused typical symptoms on seedlings and the proportion of infected seedlings per each isolate was 50, 53.33, 56.66, 60, and 63.33%, respectively. There are many reports of F. acuminatum associated to conifer seedlings in nurseries (1,3) and most of them are conflicting because in some reports this species is considered non-pathogenic or only a seed contaminant and others consider it as a pathogen. To our knowledge, F. acuminatum is a first report on the Aleppo pine in northwestern Algeria, northern Africa. It is also the first report of this fungal species affecting the Aleppo pine throughout the world, and on conifers in Africa and the Mediterranean region. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory. ARS, USDA., Bestville, Maryland, USA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , June 18, 2012. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, Iowa, USA, 2006. (3) D. W. Minter. Cybertruffle's Robigalia, Observations of Fungi and their Associated Organisms. Retrieved from http://www.cybertruffle.org.uk/robigalia/eng/ , June 18, 2012.

[Analysis of efficacy in diaphragmatic plication with minimally invasive surgery]. / Análisis de efectividad de la plicatura diafragmática mínimamente invasiva.

OBJECTIVE: Diaphragmatic paralysis may result difficulty for respiratory compromise and extubation. We study the effectiveness of diaphragmatic plication held in its two modalities: videothoracoscopy assisted surgery (VATS) and percutaneous with needle. MATERIAL AND METHODS: Retrospective study of diaphragmatic plication performed in our center in the past three years. Review of clinical histories, analyzing the variables: sex and age, etiology and laterality, surgical indications, technique, ventilation support, complications and outcome. Data Analysis with SPSS 18.0. RESULTS: From 2009-2012, 24 diaphragmatic plication were realized (7 left, 13 right, 2 bilateral) in 22 children (15 males). The mean age was 10.4 months (2 to 23.1). In 68.1% paralysis was secondary to thoracic surgery. In 12 patients was performed VATS and in 5 percutaneous with needle. Surgical indications were: respiratory distress (69.1%) with intercurrent processes (pneumonia, bronchiolitis, and atelectasis) and failure of extubation in 27.3%. After diaphragmatic plication in 90.9% respiratory symptoms remitted. Extubation was accomplished in all patients, requiring ventilator support for 12-96 hours. Mean hospital stay was 7.5 days (3 to 13.5). There were no complications derived from surgery. There were two recurrences (9.2%) and only one was reoperated (4.6%). The percutaneous technique decreased the required postoperative analgesia and improved cosmetic outcome in 90.9%. CONCLUSIONS: The diaphragmatic plication by minimally invasive surgery reduces operative morbidity and facilitates extubation of ventilator-dependent patients with diaphragmatic paralysis. The percutaneous technique improves the cosmetic results and reduces pain after surgery, without finding differences with VATS.

Impact of platelet-derived mitochondria transfer in the metabolic profiling and progression of metastatic MDA-MB-231 human triple-negative breast cancer cells.

Introduction: An active role of platelets in the progression of triple-negative breast cancer (TNBC) cells has been described. Even the role of platelet-derived extracellular vesicles on the migration of MDA-MB-231 cells has been reported. Interestingly, upon activation, platelets release functional mitochondria into the extracellular environment. However, the impact of these platelet-derived mitochondria on the metabolic properties of MDA-MB-231 cells remains unclear. Methods: MDA-MB-231 and MDA-MB-231-Rho-0 cells were co-cultured with platelets, which were isolated from donor blood. Mitochondrial transfer was assessed through confocal microscopy and flow cytometry, while metabolic analyses were conducted using a Seahorse XF HS Mini Analyzer. The mito-chondrial DNA (mtDNA) copy number was determined via quantitative PCR (qPCR) following platelet co-culture. Finally, cell proliferation and colony formation assay were performed using crystal violet staining. Results and Discussion: We have shown that platelet-derived mitochondria are internalized by MDA-MB-231 cells in co-culture with platelets, increasing ATP production, oxygen (O2) consumption rate (OCR), cell proliferation, and metabolic adaptability. Additionally, we observed that MDA-MB-231 cells depleted from mtDNA restore cell proliferation in uridine/pyruvate-free cell culture medium and mitochondrial O2 consumption after co-culture with platelets, indicating a reconstitution of mtDNA facilitated by platelet-derived mitochondria. In conclusion, our study provides new insights into the role of platelet-derived mitochondria in the metabolic adaptability and progression of metastatic MDA-MB-231 TNBC cells.