Analysis of the expression of the Serpina1 gene in SARS-CoV-2 infection: study of a new biomarker.

INTRODUCTION: The SERPINA1 gene encodes the protein Alpha-1 Antitrypsin (AAT1). Possible imbalances between the concentrations of proteases and antiproteases (AAT1) can lead to the development of serious pulmonary and extrapulmonary pathologies. In this work we study the importance of this possible imbalance in patients with COVID-19. OBJECTIVES: To correlate the severity of the symptoms of SARS-COV-2 infection with the AAT1 concentrations at diagnosis of the disease. METHODS: An observational, prospective, cross-sectional, non-interventional, analytical study was carried out where 181 cases with COVID-19 admitted to the "Lozano Blesa" University Clinical Hospital of Zaragoza were selected. The concentration of AAT1 was studied in all of them and this was correlated with the clinical aspects and biochemical parameters at hospital admission. RESULTS: 141 cases corresponded to patients with severe COVID and 40 patients with mild COVID. AAT1 levels were positively correlated with the days of hospitalization, severity, C-Reactive Protein, ferritin, admission to Intensive Care, and death, and presented a negative correlation with the number of lymphocytes/mm3. AAT1 concentrations higher than 237.5 mg/dL allowed the patient to be classified as "severe" (S72%; E78%) and 311.5 mg/dL were associated with the risk of admission to Intensive Care or Exitus (S67%; E79%). CONCLUSIONS: Levels of the SERPINA1 gene expression product, AAT1, correlate with the severity of COVID-19 patients at diagnosis of the disease, being useful as a prognostic biomarker.

[Prognosis of patients with COVID-19 presenting acute ischaemic stroke and receiving interventional treatment]. / Pronóstico de pacientes COVID-19 con ictus isquémico y tratamiento neurointervencionista.

Objetivo: Presentar nuestra experiencia y analizar el pronóstico de pacientes COVID-19 con ictus isquémico agudo por oclusión de grandes vasos tratados con neurointervencionismo (NIV) en la unidad de ictus. Material y métodos: Se incluyeron todos los pacientes consecutivos con ictus isquémico agudo debido a oclusión de grandes vasos tratados por NIV en nuestra institución entre marzo y abril de 2020, durante el brote de COVID-19. Se realizó una comparación entre pacientes con COVID-19 y pacientes sin infección por coronavirus. Se comunican los resultados clínicos iniciales y a corto plazo. Resultados: Del 1 de marzo al 30 de abril se realizaron 25 procedimientos de NIV por ictus isquémico agudo en nuestra institución. Ocho pacientes eran COVID-19 y 17 eran pacientes no COVID-19. La edad media de los pacientes con COVID-19 fue de 70,1 ± 12,23 años, y 7 fueron hombres (87,5%, p = 0,006). Mientras que todos los pacientes sin COVID procedían de urgencias, solo 5 pacientes con COVID-19 (62,5%) fueron atendidos desde urgencias por ictus (p = 0,01). Tres pacientes procedían de hospitalización. La tasa de mortalidad en pacientes sin COVID-19 fue del 5,8%, pero en pacientes con COVID-19 fue considerablemente alta (50%). Ningún parámetro analítico difirió entre ambos grupos. No se registraron hemorragias en esta serie.En comparación con el mismo período del año pasado, se observó una disminución de la actividad neurointervencionista del 39%. Conclusiones: La mejor terapia médica y de NIV desembocó en malos resultados y una mortalidad dramática. La pandemia de COVID-19 dificultó significativamente el funcionamiento normal de los servicios de urgencias y la atención de estos pacientes con ictus.

Lung isolation in patients with previous lung resections: Selective sequential lobar blockade using a Fuji Uniblocker® endobronchial blocker. / Aislamiento pulmonar en pacientes con resecciones pulmonares previas: bloqueo lobular selectivo secuencial con bloqueador bronquial Fuji Uniblocker®.

Lung isolation is essential during thoracic surgery, as it allows the thoracic surgeon to visualise and work in the surgical field. The occurrence of hypoxaemia during lung isolation is common, and is even more so in patients with decreased pulmonary functional reserve. The clinical cases are presented of 2 patients with a history of left pulmonary resections (1st left lower lobectomy, 2nd left lower lobectomy and left upper lobe segmentectomy), in which sequential selective lobar blockade was performed with Fuji Uniblocker® endobronchial blocker for performing right lung atypical resections (right upper lobe, middle lobe, and right lower lobe). In our experience the technique was successful, the surgical field was optimal and no intra- or post-operative complications were found. This technique may be an alternative to traditional lung isolation in patients with compromised respiratory function (low functional reserve or previous contralateral lung resections).

Expression of a conifer glutamine synthetase gene in transgenic poplar

The assimilation of ammonium into organic nitrogen catalyzed by the enzyme glutamine synthetase (GS; EC 6.3.1.2) has been suggested to be the limiting step for plant nitrogen utilization (H-M. Lam et al. 1995, Plant Cell 7: 887-898). We have developed a molecular approach to increase glutamine production in transgenic poplar by the overexpression of a conifer GS gene. A chimeric construct consisting of the cauliflower mosaic virus 35S promoter fused to pine cytosolic GS cDNA and nopaline synthetase polyadenylation region was transferred into pBin19 for transformation of a hybrid poplar clone (INRA 7171-B4, Populus tremula x P. alba) via Agrobacterium tumefaciens. Transformed poplar lines were selected by their ability to grow on selective medium containing kanamycin. The presence of the introduced gene in the poplar genome was verified by Southern blotting and polymerase chain reaction analysis. Transgene expression was detected in all selected poplar lines at the mRNA level. The detection of the corresponding polypeptide (41 kDa) and increased GS activity in the transgenics suggest that pine transcripts are correctly processed by the angiosperm translational machinery and that GS1 subunits are assembled in functional holoenzymes. Expression of the pine GS1 gene in poplar was associated with an increase in the levels of total soluble protein and an increase in chlorophyll content in leaves of transformed trees. Furthermore, the mean net growth in height of GS-overexpressing clones was significantly greater than that of non-transformed controls, ranging from a 76% increase in height at 2 months to a 21.3% increase at 6 months. Our results suggest that the efficiency of nitrogen utilization may be engineered in trees by genetic manipulation of glutamine biosynthesis.

High-level expression of Pinus sylvestris glutamine synthetase in Escherichia coli. Production of polyclonal antibodies against the recombinant protein and expression studies in pine seedlings.

In a previous work we reported the molecular characterization of a glutamine synthetase (GS; EC 6.3.1.2.) complementary DNA from a woody plant (Cantón et al. (1993) Plant Mol. Biol. 22, 819-828). The isolated cDNA (pGSP114) encoding a Scots pine (Pinus sylvestris) cytosolic subunit, has been subcloned into the expression vector pET3c to overproduce the GS polypeptide in Escherichia coli cells. The recombinant GS protein showed the same molecular size as a native Scots pine GS subunit. Antibodies against the pET3c-GSP114 encoded protein were raised in rabbits by injecting purified preparations and specificity was determined by immunoprecipitation of GS activity present in pine crude extracts. In spite of the antibodies were able to recognize both cytosolic and chloroplastic GS in tomato plants, they were unable to immunodetect chloroplastic GS in green cotyledons of pine seedlings and cytosolic GS was the unique recognized polypeptide. Unlike to that found in other plant species, cytosolic GS was strongly expressed in green tissues as determined by protein and Northern analysis. Our results suggest a key role for cytosolic GS in photosynthetic tissues of conifers.

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings.

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163-168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a lambda gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm, Pinus sylvestris.

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a lambda gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78-82%) than to chloroplastic (GS2) (71-75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.

Accumulation of glutamine synthetase during early development of maritime pine (Pinus pinaster) seedlings.

Seedlings of Pinus pinaster Alton accumulated chlorophyll (Chl) when grown in complete darkness. Contents of Chl a and Chl b increased during germination, reaching similar levels in light- and dark-grown plants. Glutamine-synthetase (GS; EC 6.3.1.2) activity was detected in the embryo and its level increased markedly in cotyledons of dark-germinated seedlings. Similar levels of GS activity were observed when the seeds were germinated in the presence of white light. Only one GS form, which eluted at about 0.1 M KCl, was found by ion-exchange chromatography. A predominant GS polypeptide of 43 kDa was detected in cotyledons, and its steady-state level increased with development in a lightindependent fashion. In roots and needles, a related GS polypeptide of 43 kDa was the unique species detectable by western blot analysis. Immunoblots of soluble proteins from isolated chloroplasts showed low abundance of GS protein, indicating that glutamine synthesis in pine cotyledons occurs mainly in the cytosol. Nitrogen-feeding experiments carried out with detached shoots indicated that neither NO 3 (-) nor NH 4 (+) regulate GS levels and the polypeptide pattern. Our results indicate that environmental factors, such as light and nitrogen supply, have a limited role in GS accumulation during pine development.

[The EUROPAN project: conclusions of the first seminar on nursing methods]. / Proyecto EUROPAN: conclusiones del primer seminario sobre metodología de enfermería.

The First Meeting of the Spanish European Project on Nursing was held in León from May 24th to 26th 1990. This seminar was attended by nursing professionals from 11 Spanish hospitals participating in the Project. During the meeting, an important discussion was held on the protocols used with regard to practical aspects, in order to unify criteria and homologate the results obtained by the different centres. By means of this seminar, Spanish professionals made an important contribution to the design of this Spanish project, not only as collaborators but also as active participants in the research conducted.

[Technic for recovery of instruments fractured in the root canal, using cyanoacrylate cement (in vitro)]. / Técnica de recuperación de instrumentos fracturados dentro del conducto radicular, por medio del uso de cemento de cianoacrilato (in vitro).

A technique for retrieving and extracting mental fragments from root canals is presented, in which adhesives of cyanoacrylate esters are applied by dripping through hypodermic needles with their bevel edges previously trimmed in two sharp, angled slopes to facilitate cutting through the dentine around the metal fragment imbedded in the root canal, and prepared with high and low-speed funneled burrs. Besides being simple to handle, this technique has the advantage of employing equipment which is easy to acquire and construct.

Behaviour of persistent organochlorine micropollutants during primary sedimentation of waste water.

The removal of polychlorinated biphenyls and the organochlorine insecticides gamma-HCH, aldrin, dieldrin and endrin during primary sedimentation of raw sewage has been studied using a pilot plant facility. A matrix of sixteen experiments was performed, where the pilot plant was operated at four different hydraulic loadings with four different influent suspended solids loadings. Significant removals of micropollutants into the primary sludge were observed, although no particular relationship with the removal of suspended solids was evident. It is concluded that a proportion of the organic microcontaminants present in raw sewage were associated with non-settleable solids and were also in the dissolved form. The significance of the behaviour of organochlorine micropollutants in waste water treatment processes and the hydrological cycle are discussed in the context of water re-use operations.

Lesiones del torax en los politraumatizados. Estudio particular de las lesiones del esqueleto toracico. / Thoracic lesions in polytraumatized patients. Specific study of lesions of the thoracic skeleton

Se describe la experiencia obtenida en el hospital docente "General Calixto Garcia" acerca de las lesiones toracicas de los politraumatizados y se hace un analisis especial de los traumatismos oseos de la pared toracica basado en dicha experiencia con 115 pacientes hospitalizados en 1978, y 79 en 1981. El 51,6% de los traumatismos del torax en hospitalizados en nuestro servicio ocurrieron en politraumatizados.El 73,2% se preto en pacientes que estaban entre los 21 y 60 anos de edad, y el 79,7% en el sexo masculino. Las causas mas frecuentes de estos traumatismos fueron los accidentes del transito y las precipitaciones o caidas. Hubo lesiones de la pared toracica en el 88,2% de los politraumatizados, y el 16,3% de ellos tenia tambien lesiones viscerales del torax. Las lesiones mas frecuentemente asociadas a los traumatismos toracicos fueron las craneoencefalicas y tambien las que mas contribuyeron a la mortalidad. La mortalidad general en los politraumatizados con lesiones toracicas oscilo entre el 12,2% y el 12,7% y fue nueve veces superior a la ocurrida en los pacientes con traumatismos aislados del torax. Las lesiones del esqueleto del torax que mayor mortalidad produjeron, fueron el torax batiente y la fractura del esternon