Metformin plus chemotherapy versus chemotherapy alone in the first-line treatment of HER2-negative metastatic breast cancer. The MYME randomized, phase 2 clinical trial.

PURPOSE: To investigate the efficacy of metformin (M) plus chemotherapy versus chemotherapy alone in metastatic breast cancer (MBC). METHODS: Non-diabetic women with HER2-negative MBC were randomized to receive non-pegylated liposomal doxorubicin (NPLD) 60 mg/m2 + cyclophosphamide (C) 600 mg/m2 × 8 cycles Q21 days plus M 2000 mg/day (arm A) versus NPLD/C (arm B). The primary endpoint was progression-free survival (PFS). RESULTS: One-hundred-twenty-two patients were evaluable for PFS. At a median follow-up of 39.6 months (interquartile range [IQR] 24.6-50.7 months), 112 PFS events and 71 deaths have been registered. Median PFS was 9.4 months (95% CI 7.8-10.4) in arm A and 9.9 (95% CI 7.4-11.5) in arm B (P = 0.651). In patients with HOMA index < 2.5, median PFS was 10.4 months (95% CI 9.6-11.7) versus 8.5 (95% CI 5.8-9.7) in those with HOMA index ≥ 2.5 (P = 0.034). Grade 3/4 neutropenia was the most common toxicity, occurring in 54.4% of arm A patients and 72.3% of the arm B group (P = 0.019). M induced diarrhea (G2) was observed in 8.8% of patients in Arm A. The effect of M was similar in patients with HOMA index < 2.5 and ≥ 2.5, for PFS and OS. CONCLUSIONS: The MYME trial failed to provide evidence in support of an anticancer activity of M in combination with first line CT in MBC. A significantly shorter PFS was observed in insulin-resistant patients (HOMA ≥ 2.5). Noteworthy, M had a significant effect on CT induced severe neutropenia. Further development of M in combination with CT in the setting of MBC is not warranted.

Identification of oestrogen, progesterone receptor and human epidermal growth factor receptor 2 expression in mediastinal metastases of breast cancer obtained by endobronchial ultrasound-guided transbronchial needle aspiration.

BACKGROUND: In breast cancer patients, the expression statuses of oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are crucial in the choice of treatment. Receptor expression in metastatic lesions can differ from the primary tumour. The aim of our study was to analyse the utility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to obtain samples allowing the identification of ER, PR and HER2 expression in patients with mediastinal metastases of breast cancer. PATIENTS AND METHODS: The clinical files of all patients with a final diagnosis of breast cancer mediastinal metastases diagnosed by EBUS-TBNA in our institution were retrospectively analysed. The ability of EBUS-TBNA to obtain samples that allowed hormone receptor and HER2 expression analysis was calculated. RESULTS: Twenty-four patients were included. ER, PR and HER2 assessments could be performed in 22, 20 and 22 patients, respectively. In 20 of the 24 patients it was possible to investigate all three types of receptor expression. In the remaining four cases, where ER, PR or HER2 expression tests could not be performed, it was due to a lack of tissue. In cases with adequate results for EBUS-TBNA and the primary tumour agreement was greater for ER (16/19) and HER2 (12/14) than PR (8/17). Based on receptor status, there was a change in the choice of treatment for five patients. CONCLUSION: In patients with breast cancer mediastinal metastases, ER, PR and HER2 expression can be assessed in samples obtained by EBUS-TBNA whenever a sufficient tissue sample is collected.

Orientational relaxations in solid (1,1,2,2)tetrachloroethane.

We employ dielectric spectroscopy and molecular dynamic simulations to investigate the dipolar dynamics in the orientationally disordered solid phase of (1,1,2,2)tetrachloroethane. Three distinct orientational dynamics are observed as separate dielectric loss features, all characterized by a simply activated temperature dependence. The slower process, associated to a glassy transition at 156 ± 1 K, corresponds to a cooperative motion by which each molecule rotates by 180° around the molecular symmetry axis through an intermediate state in which the symmetry axis is oriented roughly orthogonally to the initial and final states. Of the other two dipolar relaxations, the intermediate one is the Johari-Goldstein precursor relaxation of the cooperative dynamics, while the fastest process corresponds to an orientational fluctuation of single molecules into a higher-energy orientation. The Kirkwood correlation factor of the cooperative relaxation is of the order of one tenth, indicating that the molecular dipoles maintain on average a strong antiparallel alignment during their collective motion. These findings show that the combination of dielectric spectroscopy and molecular simulations allows studying in great detail the orientational dynamics in molecular solids.

Citrus Viroids: Symptom Expression and Performance of Washington Navel Sweet Orange Trees Grafted on Carrizo Citrange.

Citrus are natural hosts of several viroid species. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are the causal agents of two well-known diseases of citrus, exocortis and cachexia. Other viroids have been found to induce specific symptoms and different degrees of stunting in trees grafted on trifoliate orange and trifoliate orange hybrids. A field assay was initiated in 1989 to establish the effect of CEVd, HSVd, Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) on Washington navel sweet orange trees grafted on Carrizo citrange rootstock. Here we report the effect of viroid infection on symptom expression, tree size, fruit production and quality evaluated from 2004 to 2007. Vegetative growth was affected by viroid infection with height and canopy volume being reduced. No bark scaling symptoms were observed in CEVd-infected trees albeit they presented lesions and blisters in the roots. Bark cracking symptoms were consistently observed in CBCVd-infected trees that were smaller with enhanced productivity and fruit size. No major effects were found as a result of infection with CBLVd, HSVd, or CDVd. The quality of the fruits was not affected by viroid infection, except for the low diameter of the fruits harvested from HSVd-infected trees. An interesting effect was identified in terms of tree productivity increase (yield/canopy volume) as a result of infection with CEVd, CDVd, and especially CBCVd.

Differences in biological features of breast cancer between Caucasian (Italian) and African (Tanzanian) populations.

Information on hormone receptor and human epidermal growth factor receptor-2 (HER2) expression in breast cancer is acknowledged as mandatory for prognostic stratification and treatment planning. Data on the biological features of African breast cancers are poor. We decided to compare histopathological and biomolecular characteristics (estrogen and progesterone receptor­ER, PgR, and HER2) of Tanzanian and Italian breast cancers. Differences in proliferating index and androgen receptor (AR) expression in triple-negative patients from the two case series were also assessed. Of the 103 consecutive patients seen at the Bugando Medical Center (Mwanza, Tanzania) from 2003 to 2010, who underwent biopsy or surgical resection of primary breast cancer, 69 patients had tissue samples that were evaluable for estrogen receptor (ER), progesterone receptor (PgR), and HER2. Histopathological assessment and biomolecular determinations were performed at the Cancer Institute of Romagna (IRST IRCCS, Meldola, Italy). Caucasian breast cancers were randomly extracted from an electronic database and matched (1:2 ratio) for year of diagnosis and age at diagnosis. Median age of both populations was 51 years (range 27­84). With respect to Caucasian tumors, Tanzanian breast cancers at diagnosis more frequently showed high histological grade (mainly grade 3) (P = 0.03), advanced clinical stage (III or IV) (P\0.001), ER negativity (52.2 %, P\0.001) and high proliferation (P = 0.0002). Triple-negative tumors were over-represented in Tanzanian women. AR was positive in 38.5 and 38 %of triple-negative Tanzanian and Italian breast cancers, respectively. Our results show that histopathological and biomolecular characteristics in Tanzanian and Italian breast cancers differ substantially. The high frequency of poorly differentiated, ER-negative, highly proliferating tumors, together with advanced stage at presentation, could be considered as the main prognostic factors linked to the high mortality rates for breast cancer in the African population.

Benefits of smart pumps for automated changeovers of vasoactive drug infusion pumps: a quasi-experimental study.

BACKGROUND: Manual changeover of vasoactive drug infusion pumps (CVIP) frequently lead to haemodynamic instability. Some of the newest smart pumps allow automated CVIP. The aim of this study was to compare automated CVIP with manual 'Quick Change' relays. METHODS: We performed a prospective, quasi-experimental study, in a university-affiliated intensive care unit (ICU). All adult patients receiving continuous i.v. infusion of vasoactive drugs were included. CVIP were successively performed manually (Phase 1) and automatically (Phase 2) during two 6-month periods. The primary endpoint was the frequency of haemodynamic incidents related to the relays, which were defined as variations of mean arterial pressure >15 mm Hg or heart rate >15 bpm. The secondary endpoints were the nursing time dedicated to relays and the number of interruptions in care because of CVIP. A multivariate mixed effects logistic regression was fitted for analytic analysis. RESULTS: We studied 1329 relays (Phase 1: 681, Phase 2: 648) from 133 patients (Phase 1: 63, Phase 2: 70). Incidents related to CVIP decreased from 137 (20%) in Phase 1 to 73 (11%) in Phase 2 (P<0.001). Automated relays were independently associated with a 49% risk reduction of CVIP-induced incidents (adjusted OR=0.51, 95% confidence interval 0.34-0.77, P=0.001). Time dedicated to the relays and the number of interruptions in care to manage CVIP were also significantly reduced with automated relays vs manual relays (P=0.001). CONCLUSIONS: These results demonstrate the benefits of automated CVIP using smart pumps in limiting the frequency of haemodynamic incidents related to relays and in reducing the nursing workload.

Volumetric evaluations of the maxillary sinus before and post regenerative surgery.

OBJECTIVE: The purpose of this study was to investigate possible differences in the filling of the various recesses of the maxillary sinus after the procedure of sinus floor elevation in relation with the initial volume of the same and if the sinus volume can influence long-term graft dimensions, by using cone beam computed tomography (CBCT) as diagnostic analysis device. PATIENTS AND METHODS: This study included 32 sinuses corresponding to 16 patients referred for bilateral maxillary sinus augmentation procedures needing to increase the volume of the surgical site with the porcine cortical bone for dental implant placement in the posterior maxilla. The sinuses were analyzed by preoperative and postoperative (15 days and 6 months after sinus surgery) cone beam computed tomography. RESULTS: No statistically significant relations were observed between initial sinus dimensions and the entity of the contraction of the graft between T1 (15 days after surgery) and T2 (180 days after surgery). The behavior was the same either for the large sinus (> 15.65 cm3) and for the small one (< 15.65 cm3). Instead, about the filling of the various sinus recesses, a linear regression model was used to explain the difference between the mean preoperative and postoperative surgical spaces; in most of the samples, the filling of the anterior recess was ~15% of the total volume of the graft, i.e., the minor one among the main recesses. CONCLUSIONS: Our findings suggest that: (1) in the procedure of sinus floor augmentation by a lateral approach with deproteinized porcine bone there are no relations between the initial dimensions of the sinus and the long-term dimensional changes of the graft, and (2) that, among the main recesses of the sinus, the anterior one is generally the less filled.

Increased incidence of nonfatal cardiovascular events in stroke patients with sleep apnoea: effect of CPAP treatment.

Obstructive sleep apnoea (OSA) is a risk factor for stroke, but little is known about the effect of OSA and continuous positive airway pressure (CPAP) on the incidence of long-term, nonfatal cardiovascular events (CVE) in stroke patients. A prospective observational study was made in 223 patients consecutively admitted for stroke. A sleep study was performed on 166 of them. 31 had an apnoea/hypopnoea index (AHI) <10 events · h(-1); 39 had an AHI between 10 and 19 events · h(-1) and 96 had an AHI ≥ 20 events · h(-1). CPAP treatment was offered when AHI was ≥ 20 events · h(-1). Patients were followed up for 7 yrs and incident CVE data were recorded. The mean ± SD age of the subjects was 73.3 ± 11 yrs; mean AHI was 26 ± 16.7 events · h(-1). Patients with moderate-to-severe OSA who could not tolerate CPAP (AHI ≥ 20 events · h(-1); n = 68) showed an increased adjusted incidence of nonfatal CVE, especially new ischaemic strokes (hazard ratio 2.87, 95% CI 1.11-7.71; p = 0.03), compared with patients with moderate-to-severe OSA who tolerated CPAP (n = 28), patients with mild disease (AHI 10-19 events · h(-1); n = 36) and patients without OSA (AHI <10 events · h(-1); n = 31). Our results suggest that the presence of moderate-to-severe OSA is associated with an increased long-term incidence of nonfatal CVE in stroke patients and that CPAP reduces the excess of incidence seen in these patients.

Criteria for progressive fibrotic hypersensitivity pneumonitis in a Portuguese patient cohort.

Background: Hypersensitivity pneumonitis (HP) is a syndrome caused by sensitisation to inhaled antigens that leads to an abnormal immune response in the airways and lung parenchyma. Some patients previously diagnosed with certain types of fibrotic interstitial lung diseases (f-ILDs), including fibrotic HP (f-HP), are susceptible to develop a progressive fibrosing phenotype (PF-ILD), despite initial state-of-the-art management. Objectives: To characterise a cohort of patients with a multidisciplinary diagnosis (MTD) of chronic f-HP, who were followed up in an ILD outpatient clinic of a hospital in Portugal, and to assess the prevalence of PF-ILD criteria in these patients. Methods: Data were collected from all patients with a definite or provisional diagnosis of f-HP after a multidisciplinary team discussion. Patients were followed up between December 2014 and July 2019. Data included clinical characteristics, high-resolution chest tomography (HRCT) disease patterns, lung function tests, bronchoalveolar lavage and further immunological work-up, biopsy reports (conventional transbronchial lung biopsy, transbronchial lung cryobiopsy or surgical video-assisted thoracoscopic lung biopsy), all ILD multidisciplinary team records and diagnostic confidence levels. Patients were assessed according to PF-ILD criteria as defined in the INBUILD trial. Results: We identified 83 patients with an MTD of HP, who had been followed up for at least 12 months. Of these, 63 (75.9%) were diagnosed with f-HP. Of the 63 f-HP patients, 33.3% (n=21) fulfilled the predefined criteria for PF-HP: 66.7% had a relative decline of ≥10% forced vital capacity (FVC); 5% a relative decline of 5 - 9% FVC, with worsening symptoms or increased fibrosis on HRCT; and 23.8% had worsening respiratory symptoms with radiological progression. Conclusion: This single-centre cohort study demonstrated that a third of f-HP patients presented with PF-ILD, as determined by progression during initial standard-of-care treatment. A usual interstitial pneumonia (UIP)/UIP-like pattern was present in >70% of patients with f-HP, and two-thirds of these patients had an FVC decline of ≥10%. PF-HP patients were also more exacerbation prone. According to recent trial data, this segment of patients can be considered possible candidates for antifibrotic treatment, with a reasonable prospect of effectiveness. Further efforts should focus on refining knowledge of longitudinal behaviour of large multicentric cohorts of f-HP patients, establishing a consensual and uniform definition of progression for use in clinical practice, as well as developing prognostic prediction tools to better (and early) inform the disease course.

Reactive astrocytes and Wnt/ß-catenin signaling link nigrostriatal injury to repair in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease.

Emerging evidence points to reactive glia as a pivotal factor in Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of basal ganglia injury, but whether astrocytes and microglia activation may exacerbate dopaminergic (DAergic) neuron demise and/or contribute to DAergic repair is presently the subject of much debate. Here, we have correlated the loss and recovery of the nigrostriatal DAergic functionality upon acute MPTP exposure with extensive gene expression analysis at the level of the ventral midbrain (VM) and striata (Str) and found a major upregulation of pro-inflammatory chemokines and wingless-type MMTV integration site1 (Wnt1), a key transcript involved in midbrain DAergic neurodevelopment. Wnt signaling components (including Frizzled-1 [Fzd-1] and ß-catenin) were dynamically regulated during MPTP-induced DAergic degeneration and reactive glial activation. Activated astrocytes of the ventral midbrain were identified as candidate source of Wnt1 by in situ hybridization and real-time PCR in vitro. Blocking Wnt/Fzd signaling with Dickkopf-1 (Dkk1) counteracted astrocyte-induced neuroprotection against MPP(+) toxicity in primary mesencephalic astrocyte-neuron cultures, in vitro. Moreover, astroglial-derived factors, including Wnt1, promoted neurogenesis and DAergic neurogenesis from adult midbrain stem/neuroprogenitor cells, in vitro. Conversely, lack of Wnt1 transcription in response to MPTP in middle-aged mice and failure of DAergic neurons to recover were reversed by pharmacological activation of Wnt/ß-catenin signaling, in vivo, thus suggesting MPTP-reactive astrocytes in situ and Wnt1 as candidate components of neuroprotective/neurorescue pathways in MPTP-induced nigrostriatal DAergic plasticity.

[Impact of exacerbations on the clinical course of chronic obstructive pulmonary disease]. / Impacto de las exacerbaciones en la evolución de la EPOC.

The clinical course of chronic obstructive pulmonary disease (COPD) is marked by frequent exacerbations. Not all patients suffer exacerbations and not all decompensations have the same consequences; however, in the last few years, these episodes of clinical instability - especially if recurrent - have come to be viewed as a major element in the natural history of the disease insofar as they generate heavy workload and high costs, síntonegatively affect patients' quality of life, contribute to the multidimensional progression of the disease and, finally, affect its prognosis. The present article reviews the current scientific evidence on the impact of exacerbations on the clinical course of COPD and analyzes this impact from a multidimensional perspective. The need to base the management of COPD on clinical phenotypes is discussed, emphasizing the importance of the exacerbation phenotype, a clinical phenotype characterized by frequent exacerbations. This phenotype is often associated with the presence of cough and chronic expectoration, latent bronchial infection and bronchiectasis and leads to higher morbidity and mortality.

Early combination treatment with existing HIV antivirals: an effective treatment for COVID-19?

OBJECTIVE: Since no effective therapy exists, we aimed to test existing HIV antivirals for combination treatment of Coronavirus disease 19 (COVID-19). MATERIALS AND METHODS: The crystal structures of SARS-CoV-2 main protein (Mpro) (PDB ID: 6Y2F) and SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) (PDB ID: 7BV2) both available from Protein Data Bank were used in the study. Automated Docking by using blind and standard method both on Mpro and RdRp bound to the modified template-primer RNA was performed with AutoDock 4.2.6 program suite. Lamarckian genetic algorithm (LGA) was used for structures docking. All inhibitors were docked with all bonds completely free to rotate. RESULTS: Our molecular docking findings suggest that lopinavir, ritonavir, darunavir, and atazanavir activated interactions with the key binding sites of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) protease with a better inhibition constant (Ki) for lopinavir, ritonavir, and darunavir. Furthermore, we evidenced the ability of remdesivir, tenofovir, emtricitabine, and lamivudine to be incorporated in SARS-CoV-2 RdRp in the same protein pocket where poses the corresponding natural nucleoside substrates with comparable Ki and activating similar interactions. In principle, the four antiviral nucleotides might be used effectively against SARS-CoV-2. CONCLUSIONS: The combination of a protease inhibitor and two nucleoside analogues, drugs widely used to treat HIV infection, could be evaluated in clinical trials for the treatment of COVID-19.

Film-free laser forward printing of transparent and weakly absorbing liquids.

A laser-based technique for printing transparent and weakly absorbing liquids is developed. Its principle of operation relies in the tight focusing of short laser pulses inside the liquid and close to its free surface, in such a way that the laser radiation is absorbed in a tiny volume around the beam waist, with practically no absorption in any other location along the beam path. If the absorbed energy overcomes the optical breakdown threshold, a cavitation bubble is generated, and its expansion results in the propulsion of a small fraction of liquid which can be collected on a substrate, leading to the printing of a microdroplet for each laser pulse. The technique does not require the preparation of the liquid in thin film form, and its forward mode of operation imposes no restriction concerning the optical properties of the substrate. These characteristics make it well suited for printing a wide variety of materials of interest in diverse applications. We demonstrate that the film-free laser forward printing technique is capable of printing microdroplets with good resolution, reproducibility and control, and analyze the influence of the main process parameter, laser pulse energy. The mechanisms of liquid printing are also investigated: time-resolved imaging provides a clear picture of the dynamics of liquid transfer which allows understanding the main features observed in the printed droplets.

First Report of Citrus viroid V in Moro Blood Sweet Orange in Iran.

Viroids are nonencapsidated, small, circular, single-stranded RNAs that replicate autonomously when inoculated in their host plants in which they may elicit diseases (sensitive hosts) or replicate as latent infections (tolerant hosts). Citrus viroid V (CVd-V) was initially identified in Spain (1) and later found to be present in the United States, Nepal, and the Sultanate of Oman (2). CVd-V is a member of the Apscaviroid genus within the Pospiviroidae family. Like other members of this genus, CVd-V has a restricted host range but it is able to infect a wide range of citrus and citrus related species (1,2). Within the framework of a comprehensive survey of the sanitary status of the citrus industry in Iran, a sample from a private orchard of symptomless Moro blood sweet orange (Citrus sinensis) trees grafted on Mexican lime (C. aurantifolia) located at Javanan in the southern inland region was found to be infected with CVd-V. Briefly, RNAs of nucleic acid preparations from bark tissues were separated by 5% polyacrylamide gel electrophoresis (PAGE), electrotransferred to positively charged nylon membranes, immobilized by UV cross-linking, and hybridized with a full length CVd-V specific digoxigenin (DIG)-labeled DNA probe (2). A positive identification of CVd-V was made in these extracts. This positive detection of CVd-V was confirmed by reverse transcription-PCR using CVd-V specific primers of opposite polarity (5'-GACGAAGGCCGGTGAGCAGTAAGCC-3') and (5'-GACGACGACAGGTGAGTACTTTC-3') corresponding to CVd-V positions 90 to 114 and 69 to 89, respectively. Analysis of the sequence of the 293-bp amplicon (Genbank Accession No. GQ466068) revealed 99% identity with the reference sequence (Genbank Accession No. NC010165) of CVd-V. The rod-like predicted minimum free energy secondary structure of this new variant has 68.3% paired nucleotides. The changes with respect to the reference CVd-V variant are: (i) a deletion (48→-U) located in a loop of the V domain; (ii) a substitution (155A→C) located in a loop of the TR domain of the viroid secondary structure; and (iii) two compensatory substitutions located in the upper (46A→G) and lower (244U→C) strands of the viroid secondary structure. As shown earlier, the genome of CVd-V allows little variation with a large loop located in the segment I of the secondary structure (2) being the most amenable for mutations/changes. Among the viroids that have been found naturally infecting citrus, the members of the genus Apscaviroid are not associated with specific diseases but they cause a reduction of tree size and fruit harvest (3), an effect that is enhanced when several viroids coinfect the same plant (4). Therefore, the presence of CVd-V should be considered in further indexing tests aimed at the production and distribution of pathogen-free plants in Iran. References: (1) P. Serra et al. Virology 370:102, 2008. (2) P. Serra et al. Phytopathology 98:1199, 2008. (3) C. Vernière et al. Plant Dis. 88:1189, 2004. (4) C. Vernière et al. Phytopathology 96:356, 2006.

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells.

Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.

A novel hybridization approach for detection of citrus viroids.

Citrus plants are natural hosts of several viroid species all belonging to the family Pospiviroidae. Previous attempts to detect viroids from field-grown species and cultivars yielded erratic results unless analyses were performed using Etrog citron a secondary bio-amplification host. To overcome the use of Etrog citron a number of RT-PCR approaches have been proposed with different degrees of success. Here we report the suitability of an easy to handle northern hybridization protocol for viroid detection of samples collected from field-grown citrus species and cultivars. The protocol involves: (i) Nucleic acid preparations from bark tissue samples collected from field-grown trees regardless of the growing season and storage conditions; (ii) Separation in 5% PAGE or 1% agarose, blotting to membrane and fixing; (iii) Hybridization with viroid-specific DIG-labelled probes and detection with anti-DIG-alkaline phosphatase conjugate and autoradiography with the CSPD substrate. The method has been tested with viroid-infected trees of sweet orange, lemon, mandarin, grapefruit, sour orange, Swingle citrumello, Tahiti lime and Mexican lime. This novel hybridization approach is extremely sensitive, easy to handle and shortens the time needed for reliable viroid indexing tests. The suitability of PCR generated DIG-labelled probes and the sensitivity achieved when the samples are separated and blotted from non-denaturing gels are discussed.

Molecular and biological characterization of natural variants of Citrus dwarfing viroid.

Citrus dwarfing viroid has been proposed as an agent to control tree size in high-density plantations. Thirty-three field isolates have been characterized, and the most frequent sequence/s have been identified. Five distinct variants were selected for biological characterization. Symptom expression analysis demonstrated a good correlation between leaf/stem symptoms and plant growth. The discriminating nucleotide sequence differences included two deletions and an insertion resulting in a reorganization of the base pairing of the terminal left loop, two (G42 --> A and C52 --> U) changes found in one of the variants, and as many as thirteen changes located in the right and left regions flanking the CCR.

Effect of a Field-Source Mixture of Citrus Viroids on the Performance of 'Nules' Clementine and 'Navelina' Sweet Orange Trees Grafted on Carrizo Citrange.

A field-source mixture of citrus viroids was characterized and shown to contain Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). Sequencing results showed that: (i) CEVd contained the PL and PR characteristic of class A variants; (ii) HSVd was a noncachexia variant; (iii) CBLVd was related to CVd-Ia variants; (iv) CDVd was a mixture of two types (CVd-IIIa and CVd-IIIb) of variants. The presence of the same type of variants in inoculated clementine (Citrus clementina 'Nules') and sweet orange (C. sinensis 'Navelina') trees on Carrizo citrange (Poncirus trifoliata × C. sinensis) rootstocks was confirmed. The effect of infection was determined by assessing the performance of infected and noninfected trees growing in the field. Infection resulted in small trees with reduced canopy, yielding a reduced crop. Fruit characteristics were also affected: (i) clementine and sweet orange fruits from infected trees were larger than those from noninfected trees; (ii) clementine fruits from infected trees differed in shape from those of noninfected trees; (iii) sweet orange fruits from infected trees had maturity indexes and juice contents higher than those from noninfected trees; (iv) in both species, the density of the juice, the amount of soluble solids, and the acidity of the fruits from infected trees were lower than those of fruits from noninfected trees. Infected trees had a poorly developed root system with fibrous roots containing fewer amyloplasts than noninfected trees. The results of an in vitro assay on the induction and development of roots in cultured explants are discussed.

Loss of aromatase cytochrome P450 function as a risk factor for Parkinson's disease?

The final step in the physiological synthesis of 17beta estradiol (E(2)) is aromatization of precursor testosterone by a CYP19 gene product, cytochrome P450 estrogen aromatase in the C19 steroid metabolic pathway. Within the central nervous system (CNS) the presence, distribution, and activity of aromatase have been well characterized. Developmental stage and injury are known modulators of brain enzyme activity, where both neurons and glial cells reportedly have the capability to synthesize this key estrogenic enzyme. The gonadal steroid E(2) is a critical survival, neurotrophic and neuroprotective factor for dopaminergic neurons of the substantia nigra pars compacta (SNpc), the cells that degenerate in Parkinson's disease (PD). In previous studies we underlined a crucial role for the estrogenic status at the time of injury in dictating vulnerability to the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our ongoing studies address the contribution of brain aromatase and extragonadal E(2) as vulnerability factors for PD pathology in female brain, by exposing aromatase knockout (ArKO, -/-) female mice which are unable to synthesize estrogens to MPTP. Our initial results indicate that aromatase deficiency from early embryonic life significantly impairs the functional integrity of SNpc tyrosine hydroxylase-positive neurons and dopamine transporter innervation of the caudate-putamen in adulthood. In addition, ArKO females exhibited a far greater vulnerability to MPTP-induced nigrostriatal damage as compared to their Wt type gonadally intact and gonadectomized counterparts. Characterization of this novel implication of P450 aromatase as determining factor for PD vulnerability may unravel new avenues for the understanding and development of novel therapeutic approaches for Parkinson's disease.

Anesthetic management of the diaphragmatic pacemaker placement surgery. Our experience in the Institut Guttmann. / Manejo anestésico de la cirugía de implante de marcapasos diafragmático. Experiencia en el Institut Guttmann.

BACKGROUND: The diaphragm pacemaker (DP) has proven its utility in replacing mechanical ventilation in patients with chronic spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS). The DP improves patient quality of life and reduces morbidity and mortality and their associated health care costs. The anesthetic management of these patients and the particularities of the surgical procedure are challenging. The aim of our study is to analyze anesthetic management and intraoperative complications in patients undergoing DP placement in our hospital. METHODS: We performed a chart review of patients treated between December 2007 and July 2017, recording the patients' preoperative status, anesthetic management and intraoperative complications. RESULTS: The study included 16 patients (5 pediatric) undergoing DP implantation for chronic SCI (63%), ALS (25%) and other neurologic conditions (12%). Abdominal laparoscopy was performed under general anesthesia, with intravenous (87%) or inhalational (13%) induction and maintenance using total intravenous (50%) or balanced (50%) anesthesia. Rocuronium was administered in one case to permit airway management. Complications included: hypotension (50%), difficulties in mechanical ventilation during laparoscopy (31%), pneumothorax (12.5%) and autonomic dysreflexia (6%). CONCLUSIONS: DP placement under general anesthesia is a safe intervention in both adult and pediatric patients. Complications derived from both the underlying disease and the surgical technique may appear, and must be rapidly identified and treated to obtain a satisfactory surgical outcome.