A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity.

The red abalone (Haliotis rufescens) represents North America's most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H. rufescens. The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p-nitrophenyl-ß-D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% (w/v) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a ß-sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.

Activity of imipenem/relebactam against Enterobacterales and Pseudomonas aeruginosa in Spain. SMART 2016-2020.

OBJECTIVE: To determine susceptibility to the novel ß-lactam/ß-lactamase inhibitor combination imipenem/relebactam in clinical isolates recovered from intra-abdominal (IAI), urinary (UTI), respiratory (RTI) and bloodstream (BSI) infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) study in SPAIN during 2016 - 2020. METHODS: Broth microdilution MICs for imipenem/relebactam and comparators were determined by a central laboratory against isolates of Enterobacterales and Pseudomonas aeruginosa. MICs were interpreted using EUCAST-2021 breakpoints. RESULTS: In total, 5,210 Enterobacterales and 1,418 P. aeruginosa clinical isolates were analyzed. Imipenem/relebactam inhibited 98.8% of Enterobacterales. Distinguishing by source of infection susceptibility was 99.1% in BSI, 99.2% in IAI, 97.9% in RTI, and 99.2% in UTI. Of intensive care unit isolates (ICU) 97.4% were susceptible and of non-ICU isolates 99.2% were susceptible. In Enterobacterales, activity against Class A, Class B and Class D carbapenemases was 96.2%, 15.4% and 73.2%, respectively. In P. aeruginosa, imipenem/relebactam was active in 92.2% of isolates. By source of infection it was 94.8% in BSI, 92.9% in IAI, 91.7% in RTI, and 93.1% in UTI. An 88.7% of ICU isolates and 93.6% of non-ICU isolates were susceptible to imipenem/relebactam. Imipenem/relebactam remained active against P. aeruginosa ceftazidime-resistant (76.3%), cefepime-resistant (73.6%), imipenem-resistant (71.5%) and piperacillin-resistant (78.7%) isolates. Of all multidrug-resistant or difficult-to-treat resistance P. aeruginosa isolates, 75.1% and 46.2%, respectively, were susceptible to imipenem/relebactam. CONCLUSIONS: Imipenem/relebactam showed high rates of susceptibility in Enterobacterales and P. aeruginosa isolates from different sources of infection as well as depending on patients' location (ICU or non-ICU scenarios).

Medición automática de la zona avascular foveal de ojos sanos en angiografía por tomografía de coherencia óptica de dominio espectral Heidelberg / Automated measurement of the foveal avascular zone in healthy eyes on Heidelberg spectralis optical coherence tomography angiography

Objetivos Desarrollar y evaluar un método automático de medición del área de la zona avascular foveal (ZAF) en ojos sanos en angiografía por tomografía de coherencia óptica de dominio espectral Heidelberg (HS-OCTA). Este método se denomina Macro Kanno-Saitama modificado (KSMm) y es una evolución de la aproximación Macro Kanno-Saitama (KSM). Métodos Este estudio transversal incluyó 29 ojos de 25 voluntarios sanos a los que se les realizaron dos HS-OCTA de la zona macular en el mismo momento. Se incluyeron todas las imágenes a pesar de su calidad. Los datos de la zona macular del plexo vascular superficial, del plexo capilar intermedio (PCI) y del plexo capilar profundo fueron procesados usando KSMm. El área ZAF se midió dos veces automáticamente usando KSMm y KSM y dos veces manualmente por dos examinadores independientes. Resultados De 174 imágenes, KSM no pudo medir correctamente el 31%, mientras que KSMm pudo medir con éxito todas las imágenes. El coeficiente intraclase intraescaneo varió entre 0,948 y 0,993 para medidas manuales y fue 1 con el método KSMm. Aunque según los gráficos de Bland-Altman la diferencia entre examinadores humanos es menor que entre examinadores humanos y KSMm, los gráficos de dispersión muestran una fuerte correlación entre las medidas realizadas por humanos y las automáticas. Los mejores resultados se obtienen en PCI. Conclusiones Usando KSMm, la medida automática del área ZAF en HS-OCTA es factible y menos humano-dependiente. Resuelve la incapacidad de KSM de medir el área ZAF en imágenes de calidad subóptima que son frecuentes en la práctica clínica diaria. Por lo tanto, el procesamiento KSMm podría contribuir a nuestra comprensión de los tres plexos vasculares (AU)

Purpose To develop and evaluate an automated method to measure the foveal avascular zone (FAZ) area in healthy eyes on Heidelberg Spectralis Optical Coherence Tomography Angiography (HS-OCTA). This method is referred to as the modified Kanno-Saitama macro (mKSM) which is an evolution of the Kanno-Saitama macro (KSM) approach. Methods This cross-sectional study included 29 eyes of 25 healthy volunteers who underwent HS-OCTA at the macular area twice at the same time. Regardless of the quality of the images, all of them were included. Macular data on the superficial vascular plexus, intermediate capillary plexus (ICP) and deep capillary plexus were processed by mKSM. The FAZ area was measured twice automatically using the mKSM and KSM and twice manually by two independent examiners. Results From 174 images, KSM could not measure correctly 31% while mKSM could successfully measure all of them. Intrascan intraclass coefficient ranged from 0.948 to 0.993 for manual measurements and was 1 for mKSM method. Despite that the difference between human examiners is smaller than between human examiners and mKSM according to Bland-Altman plots, the scatterplots show a strong correlation between human and automatic measurements. The best results are obtained in ICP. Conclusions With mKSM, the automated determination of the FAZ area in HS-OCTA is feasible and less human-dependent. It solves the inability of KSM to measure the FAZ area in suboptimal quality images which are frequent in daily clinical practice. Therefore, the mKSM processing could contribute to our understanding of the three vascular plexuses (AU)

Automated measurement of the foveal avascular zone in healthy eyes on Heidelberg spectralis optical coherence tomography angiography.

PURPOSE: To develop and evaluate an automated method to measure the foveal avascular zone (FAZ) area in healthy eyes on Heidelberg Spectralis Optical Coherence Tomography Angiography (HS-OCTA). This method is referred to as the modified Kanno-Saitama macro (mKSM) which is an evolution of the Kanno-Saitama macro (KSM) approach. METHODS: This cross-sectional study included 29 eyes of 25 healthy volunteers who underwent HS-OCTA at the macular area twice at the same time. Regardless of the quality of the images, all of them were included. Macular data on the superficial vascular plexus, intermediate capillary plexus (ICP) and deep capillary plexus were processed by mKSM. The FAZ area was measured twice automatically using the mKSM and KSM and twice manually by two independent examiners. RESULTS: From 174 images, KSM could not measure correctly 31% while mKSM could successfully measure all of them. Intrascan intraclass coefficient ranged from 0,948 to 0,993 for manual measurements and was 1 for mKSM method. Despite that the difference between human examiners is smaller than between human examiners and mKSM according to Bland-Altman plots, the scatterplots show a strong correlation between human and automatic measurements. The best results are obtained in ICP. CONCLUSIONS: With mKSM, the automated determination of the FAZ area in HS-OCTA is feasible and less human-dependent. It solves the inability of KSM to measure the FAZ area in suboptimal quality images which are frequent in daily clinical practice. Therefore, the mKSM processing could contribute to our understanding of the three vascular plexuses.

Antimicrobial activity of ceftolozane-tazobactam against Enterobacterales and Pseudomonas aeruginosa recovered during the Study for Monitoring Antimicrobial Resistance Trends (SMART) program in Spain (2016-2018).

OBJECTIVE: To analyse the susceptibility to ceftolozane-tazobactam and comparators in Enterobacterales and Pseudomonas aeruginosa isolates recovered from intraabdominal (IAI), urinary (UTI), respiratory (RTI) and bloodstream infection (BSI) in the SMART (Study for Monitoring Antimicrobial Resistance Trends) study. METHODS: The susceptibility of 5,351 isolates collected in 11 Spanish hospitals (2016-2018) were analysed (EUCAST-2020 criteria) by broth microdilution and were phenotypically studied for the presence of extended-spectrum beta-lactamases (ESBL). Ceftolozane-tazobactam and/or carbapenem resistant isolates were genetically characterized for ESBL and carbapenemases. RESULTS: Escherichia coli was the most frequent pathogen (49.3% IAI, 54.9% UTI, 16.7% RTI and 50% BSI), followed by Klebsiella pneumoniae (11.9%, 19.1%, 13.1% and 15.4%, respectively). P. aeruginosa was isolated in 9.3%, 5.6%, 32% and 9%, respectively. The frequency of isolates with ESBLs (2016-2017) was: 30.5% K. pneumoniae, 8.6% E. coli, 2.3% Klebsiella oxytoca and 0.7% Proteus mirabilis. Ceftolozane-tazobactam was very active against non-ESBL-(99.3% susceptible) and ESBL-(95.2%) producing E. coli being less active against K. pneumoniae (98% and 43.1%, respectively) isolates. CTX-M-15 was the most prevalent ESBL in E. coli (27.5%) and K. pneumoniae (51.9%) frequently associated with OXA-48-like carbapenemase. Overall, 93% of P. aeruginosa isolates were susceptible to ceftolozane-tazobactam, preserving this activity (>75%) in isolates resistant to other beta-lactams except in those resistant to meropenen or ceftazidime-avibactam. GES-5, PER-1, VIM-1/2 were the most prevalent enzymes in isolates resistant to ceftolozane-tazobactam. CONCLUSIONS: Ceftolozane-tazobactam showed high activity rates against isolates recovered in the SMART study although it was affected in K. pneumoniae and P. aeruginosa isolates with ESBL and/or carbapenemases.

Added prognostic value of longitudinal changes of angiogenic factors in early-onset severe pre-eclampsia: a prospective cohort study.

OBJECTIVE: To assess in women with early-onset severe pre-eclampsia whether longitudinal changes in angiogenic factors improve the prediction of adverse outcome. DESIGN: Prospective cohort study. SETTING: Maternity units in two Spanish hospitals. POPULATION: Women with diagnosis of early-onset severe pre-eclampsia. METHODS: Levels of placental growth factor (PlGF), soluble fms-like tyrosine kinase (sFlt-) and sFlt-1/PlGF ratio were measured at admission and before delivery, and average daily change calculated. The association of longitudinal changes of angiogenic factors with the time interval to delivery and with complications was evaluated by logistic and Cox regression. MAIN OUTCOME MEASURES: Interval to delivery and composite of adverse outcomes. RESULTS: We included 63 women, of which 26 (41.3%) had a complication. Longitudinal changes of sFlt-1 were more pronounced in complicated pregnancies (median: 1047 versus 342 pg/ml/day; P = 0.04). On the multivariate analysis, the clinical risk score and sFlt-1 at admission explained 6.2% of the uncertainty for complication; the addition of sFlt-1 longitudinal changes improved this to 25.3% (P = 0.002). The median time from admission to delivery was 4 days (95% CI 1.6-6.04) in those in the highest quartile of sFlt-1 longitudinal changes versus 16 days (95% CI 12.4-19.6) in the remaining women (Log-rank test P < 0.001). CONCLUSIONS: Longitudinal changes in sFlt-1 maternal levels from admission for confirmed early-onset severe pre-eclampsia add to baseline characteristics in the prediction of adverse outcome and interval to delivery. TWEETABLE ABSTRACT: In early-onset severe pre-eclampsia, longitudinal changes in sFlt-1 levels improve the prediction of complications and interval to delivery.

Retinopatía secundaria a mieloma múltiple tratada con bevacizumab / Retinopathy secondary to multiple myeloma treated with bevacizumab

Mujer de 80 años con mieloma múltiple (MM) de tipo IgA, que desarrolló cambios retinianos semejantes a los de retinopatía diabética no proliferativa leve, objetivándose microaneurismas y fluido intrarretiniano. La paciente fue tratada con quimioterapia para MM e inyecciones intravítreas de bevacizumab, siendo controlada durante 22 meses. La terapia antiangiogénica puede ser útil en el control de retinopatía secundaria a MM siempre y cuando se realice el control de la enfermedad sistémica

The case is presented on an 80-year-old woman with IgA multiple myeloma (MM), who developed retinal changes similar to mild non-proliferative diabetic retinopathy, with micro-aneurysms and intraretinal fluid. The patient was treated with systemic chemotherapy for MM, and with bevacizumab intravitreal injections, with control of her ocular disorder for 22 months. Anti-angiogenic therapy can be useful in the control of retinopathy secondary to MM, as long as the systemic disease has been controlled

Retinopathy secondary to multiple myeloma treated with bevacizumab. / Retinopatía secundaria a mieloma múltiple tratada con bevacizumab.

The case is presented on an 80-year-old woman with IgA multiple myeloma (MM), who developed retinal changes similar to mild non-proliferative diabetic retinopathy, with micro-aneurysms and intraretinal fluid. The patient was treated with systemic chemotherapy for MM, and with bevacizumab intravitreal injections, with control of her ocular disorder for 22 months. Anti-angiogenic therapy can be useful in the control of retinopathy secondary to MM, as long as the systemic disease has been controlled.

Latencia al parto tras cerclaje de emergencia en gestación múltiple / Latency to delivery after emergency physical-examination indicated cerclage in twin pregnancy

Introducción: Las guías clínicas actuales recomiendan el uso del cerclaje de emergencia (CE) como tratamiento de la insuficiencia cervical con exposición de membranas en gestaciones únicas. Sin embargo, el CE en gestación múltiple es tema de controversia dado que no existen ensayos clínicos randomizados que demuestren su eficacia. Algunos estudios retrospectivos sugieren que el CE también podría prolongar la gestación en embarazos múltiples. El objetivo de nuestro estudio es evaluar los resultados de las gestaciones múltiples que se sometieron a un CE en nuestro centro. Materiales y métodos: Se diseñó un estudio retrospectivo que incluyó los CE realizados en gestaciones gemelares en nuestro centro entre 2007-2016. No fueron tributarias de CE gestaciones con malformaciones fetales, monocoriales-monoamnióticas, triple o superior y finalizaciones activas de la gestación. Variables primarias: latencia al parto espontáneo y edad gestacional al parto. Variables secundarias: mortalidad neonatal, ingreso en UCI neonatal, rotura prematura de membranas pretérmino, corioamnionitis y fallo del cerclaje. Resultados. El estudio incluyó 17 pacientes. La edad gestacional mediana (rango intercuartil) al parto fue de 27,1 (24,5-32,3) semanas y la latencia mediana (rango intercuartil) al parto fue de 43 (21-64) días. Hubo 4/17 (23,5%) casos de parto antes de las 24 semanas de gestación y 2/26 (7,7%) de muerte neonatal. Discusión: Estos resultados muestran que la latencia al parto después del CE en gestación múltiple es remarcable, por lo que podría ser considerado como una opción terapéutica. Sin embargo, se requiere evidencia basada en estudios randomizados para hacer una recomendación firme

Introduction: Current guidelines support the use of physical-examination indicated cerclage (PEIC) as a treatment for cervical insufficiency and membrane exposure in single pregnancies. However, PEIC in twin pregnancies is a controversial issue as no data from random clinical trial are available to demonstrate its efficacy. Few studies suggest that PEIC may prolong pregnancy also in twin pregnancies. The aim of this study was to evaluate the results of twin pregnancies that underwent a PEIC in our health centre. Material and methods: A retrospective review was performed on women that underwent a PEIC from 2007-2016 in our centre. Women were not eligible if they were carrying foetuses with major foetal anomalies, more than two foetuses or monochorionic-monoamniotic pregnancies, or three or more foetuses or requesting an elective termination of pregnancy. Primary outcomes: latency to spontaneous delivery and gestational age (GA) at delivery. Secondary outcomes: neonatal mortality and Neonatal Intensive Care Unit admission, preterm premature rupture of membranes (PPROM), chorioamnionitis and cerclage displacement. Results: The study included a total of 17 women. The median (inter-quartile range) gestational age at delivery was 27.1 (24.5-32.3) weeks, and median (inter-quartile range) latency, from cervical cerclage to delivery, was 43 (21-64) days. There were 4/17 (23.5%) cases of delivery before 24 weeks of pregnancy, and 2/26 (7.7%) cases of neonatal death. Discussion: These results suggest that latency to delivery after PEIC in twins is remarkable. Therefore, it could be considered as an optional management. Nevertheless, evidence based on random clinical trial is required to make firm recommendations on its formal use

Tunable room-temperature spin galvanic and spin Hall effects in van der Waals heterostructures.

Spin-orbit coupling stands as a powerful tool to interconvert charge and spin currents and to manipulate the magnetization of magnetic materials through spin-torque phenomena. However, despite the diversity of existing bulk materials and the recent advent of interfacial and low-dimensional effects, control of this interconversion at room temperature remains elusive. Here, we demonstrate strongly enhanced room-temperature spin-to-charge interconversion in graphene driven by the proximity of WS2. By performing spin precession experiments in appropriately designed Hall bars, we separate the contributions of the spin Hall and the spin galvanic effects. Remarkably, their corresponding conversion efficiencies can be tailored by electrostatic gating in magnitude and sign, peaking near the charge neutrality point with an equivalent magnitude that is comparable to the largest efficiencies reported to date. Such electric-field tunability provides a building block for spin generation free from magnetic materials and for ultra-compact magnetic memory technologies.

First-trimester prediction of small-for-gestational age in pregnancies at false-positive high or intermediate risk for fetal aneuploidy.

OBJECTIVES: To explore the risk of small-for-gestational age (SGA) and fetal growth restriction (FGR) and to test the performance of first-trimester screening for SGA and FGR in women with a false-positive high or intermediate risk for aneuploidy. METHODS: This was a prospective cohort study of women with a singleton pregnancy attending for a routine first-trimester scan. The risks of aneuploidy and preterm SGA (defined as birth weight < 10th percentile with delivery before 37 weeks) were determined according to Fetal Medicine Foundation algorithms. In non-malformed euploid pregnancies, the predictive performance of both the aneuploidy and preterm SGA risks was evaluated for SGA, FGR (defined as birth weight < 3rd centile), preterm SGA and early SGA (delivery before 34 weeks), using receiver-operating-characteristics (ROC) curve analysis, in those with a high or intermediate risk of aneuploidy and in the overall population. RESULTS: A total of 2053 pregnancies were included in the analysis, of which 191 (9.3%) were at high or intermediate risk for aneuploidy (≥ 1/1000) and 304 (14.8%) were at high risk for preterm SGA (≥ 1/100). In total, there were 140 (6.8%) cases of SGA, 61 (3.0%) of FGR, 44 (2.1%) of preterm SGA and 33 (1.6%) of early SGA. Among women with a false-positive high or intermediate risk for aneuploidy, the rates of SGA, FGR, preterm SGA and early SGA were 13.6% (26/191), 7.9 % (15/191), 6.8% (13/191) and 5.8% (11/191), respectively. Compared with women with a first-trimester low risk for preterm SGA, regardless of aneuploidy risk, those with a high risk for preterm SGA and a high or intermediate risk for aneuploidy had relative risks for SGA, FGR, preterm SGA and early SGA of 6 (95% CI, 3.9-9), 9.2 (95% CI, 5.1-16.5), 13.4 (95% CI, 6.9-26.1) and 17.6 (95% CI, 8.1-38.2), respectively. The predictive performance for SGA of the preterm SGA algorithm was higher in women at high or intermediate risk for aneuploidy than in the overall population (area under the ROC curve (AUC), 0.8 vs 0.7; P < 0.001). Among women at high or intermediate risk for aneuploidy, the predictive performance of the preterm SGA algorithm was better than that of the aneuploidy algorithm for SGA (AUC, 0.80 vs 0.58; P = 0.003), preterm SGA (AUC, 0.85 vs 0.65; P = 0.013) and early SGA (AUC, 0.86 vs 0.60; P = 0.002). CONCLUSION: In women with a first-trimester false-positive high or intermediate risk of aneuploidy, further screening for SGA allows stratification of the risk for fetal growth disorders. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

Persistently altered liver test results in hepatitis C patients after sustained virological response with direct-acting antivirals.

Guidelines recommend evaluating persistent alteration of liver tests in HCV-infected patients after sustained virological response (SVR) and its influence on liver disease progression. We studied the prevalence, etiology, associated factors and evolutionary implications of persistent alteration of liver tests in HCV patients after direct-acting antivirals (DAA)-induced SVR. This was a prospective study of HCV-infected patients and SVR after DAA. Those with another previously diagnosed liver disease were excluded. Persistent alteration of liver tests was defined as any increase in ALT, AST or GGT at SVR12 and SVR24. Causes were determined according to standard clinical practice, including liver biopsy and follow-up transient elastography. A total of 1112 patients were included (70.8% males, median age 53 years, 38.8% cirrhosis, 34.9% interferon-experienced, 56.8% HIV-coinfected). Persistent alteration of liver tests was detected in 130/1112 patients (11.7% [95%CI: 9.7-13.6]). Its frequency differed between HCV-monoinfected (45/480: 9.4% [95%CI: 6.7-12.1]) and HIV-coinfected (85/632: 13.5% [95%CI: 10.7-16.2]) (P = .046). In multivariable analysis, cirrhosis (OR 2.12; 95%CI: 1.28-3.53; P = .004) and baseline transient elastography values (OR 1.03; 95%CI: 1.01-1.04; P = .000) were associated with persistent alteration of liver tests. The main etiologies were clinical diagnosis suggestive of nonalcoholic fatty liver disease in 47 (36.2%), alcohol in 30 (23.1%) and drug consumption in 19 (14.6%). Baseline and follow-up transient elastography was performed in 594 patients and showed a significantly different decrease in patients who did or did not have a persistent alteration of liver tests (-21.1% vs -30%, respectively; P = .003), independently of sex, HIV status or baseline TE value. In conclusion, persistent alteration of liver tests is not infrequent after SVR. It is associated with cirrhosis and baseline transient elastography, and the main cause is fatty liver. According to transient elastography changes, persistent alteration of liver tests seems to affect the course of liver disease.

Selection of candidate reference genes and validation for real-time PCR studies in rice plants exposed to low temperatures.

Rice is a cereal that presents a great ability to adapt to different soil and climate conditions. However, as it is a tropical crop with C3 metabolism, it performs better in warm temperatures with high solar radiation. Tolerance to stress caused by low temperatures is a highly complex process that involves various metabolic pathways and cellular compartments, resulting in general or specific effects on plant growth and development. In order to observe the true effect of a particular stress on genetic expression, reference genes need to be chosen for real-time PCRs, the expression levels of which should remain stable independent of the situation imposed. In this paper, the expression stability was evaluated of the actin 11 (ACT11), ubiquitin-conjugating enzyme 2 (UBC-E2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta tubilin (ß-Tubulin), eukaryotic initiation factor 4α (eIF-4-α), eukaryotic initiation factor 1α (eIF-1-α), ubiquitin 10 (UBQ10), ubiquitin 5 (UBQ5), aquaporin (TIP41), and cyclophilin genes, in two rice genotypes cultivated in low temperature (13°C) conditions in vegetative stage (V4). The analysis material (leaves) was collected after 0, 6, 24, 48, and 72 h of exposure to the stress. In this study, the geNorm, BestKeeper, ΔCt, NormFinder, and RefFinder methods were used to evaluate the expression stability of the candidate reference genes. The results revealed that the most indicated genes for all the analysis methods were UBQ10 and UBQ5 for BRS Bojuru and BRS Pampa, respectively. On the other hand, the eIF-1-α gene presents the least expression stability and is not indicated for studies of rice plants subjected to low temperatures. The validation with the antioxidant system genes SODCc1-Cu/Zn, CATC, APX2, and GR2 confirmed the importance of using previously tested normalizing genes for adequate real-time PCR results.

Prevalence and distribution of hepatitis C virus genotypes in Spain during the 2000-2015 period (the GEHEP 005 study).

We report the largest study on the prevalence and distribution of HCV genotypes in Spain (2000-2015), and we relate them with clinical, epidemiological and virological factors. Patients from 29 hospitals in 10 autonomous communities (Andalusia, Aragon, Castilla-Leon, Catalonia, Galicia, Canary Islands, Madrid Community, Valencian Community, Murcia Region and Basque Country) have been studied. Annual distribution of HCV genotypes and subtypes, as well as gender, age, transmission route, HIV and/or HBV coinfection, and treatment details were recorded. We included 48595 chronically HCV-infected patients with the following characteristics: median age 51 years (IQR, 44-58), 67.9% male, 19.1% HIV-coinfected, 23.5% HBV-coinfected. Parenteral transmission route was the most frequent (58.7%). Genotype distribution was 66.9% GT1 (24.9% subtype 1a and 37.9% subtype 1b), 2.8% GT2, 17.3% GT3, 11.4% GT4 and 0.1% GT5 and 0.02% GT6. LiPA was the most widely HCV genotyping test used (52.4%). HCV subtype 1a and genotypes 3 and 4 were closely associated with male gender, parenteral route of infection and HIV and HBV coinfection; in contrast, subtype 1b and genotype 2 were associated with female gender, nonparenteral route and mono-infection. Age was related to genotype distribution, and different patterns of distribution and biodiversity index were observed between different geographical areas. Finally, we describe how treatment and changes in transmission routes may have affected HCV genotype prevalence and distribution patterns. We present the most recent data on molecular epidemiology of hepatitis C virus in Spain. This study confirms that genotype distributions vary with age, sex, HIV and HBV coinfection and within geographical areas and epidemiological groups.

Expressão gênica de IGF-I e GHR no fígado e no músculo do peito de codornas de corte suplementadas com diferentes níveis de metionina em duas gerações sucessivas / IGF-I and GHR gene expression in liver and breast muscle of meat quails supplemented with different levels of methionine in two successive generations

Este estudo foi desenvolvido com o objetivo de avaliar a expressão gênica do fator de crescimento semelhante à insulina I (IGF-I) e do receptor do hormônio do crescimento (GHR) no fígado e no músculo do peito de codornas de corte, alimentadas com dietas contendo diferentes níveis de suplementação de metionina, em duas gerações sucessivas. Foram utilizadas codornas dos 22 aos 42 dias de idade, distribuídas em três e cinco tratamentos na primeira e na segunda geração, respectivamente. Ao final, as aves foram abatidas por deslocamento cervical, sendo coletados fígado e músculo do peito para extração de RNA total. O cDNA foi amplificado usando primers específicos para os genes analisados. Os resultados mostraram que a expressão dos genes GHR e IGF-I sofreu influência da suplementação. No quinto tratamento, em que apenas a primeira geração recebeu uma suplementação acima do padrão das exigências para o período, houve uma expressão significativamente maior do GHR tanto no músculo do peito como no fígado e igualmente do IGF-I no músculo, levando a concluir que o excesso de metionina na dieta torna-se tóxica para as aves. Apesar de a expressão dos genes ter sofrido influência da adição de metionina nos níveis estudados, não foi observada diferença no consumo alimentar, na conversão alimentar e no peso das aves.(AU)

This study was conducted to evaluate the gene expression of the insulin-like I growth factor (IGF-I) and growth hormone receptor (GHR), in the liver and chest muscle of slaughter quails fed with diets containing different levels of methionine supplementation, in two successive generations. Twenty-two to 42 day-old quails were used, distributed in three and five treatments in the first and second generation, respectively. At the end, the birds were killed by cervical dislocation, and their liver and chest muscle were collected for total RNA extraction. The cDNA was amplified using specific primers for the genes analyzed. The results showed that the expression of GHR gene and IGF-I were influenced by the supplementation. In the fifth treatment, where only the first generation received supplementation above the standard requirements for the period, there was a significantly higher expression of GHR both in muscle chest and in the liver, and also IGF-I on muscle, leading to the conclusion that the excess dietary methionine becomes toxic to birds. Despite the gene´s expression seeming to be influenced by the addition of methionine levels in the study, there was no difference in feed intake, feed conversion and weight of the birds.(AU)

Changes in gene expression and catalase activity in Oryza sativa L. under abiotic stress.

Different rice (Oryza sativa L.) genotypes were subjected to high salinity and low temperature (150 mM NaCl and 13°C, respectively) for 0, 6, 24, 48, or 72 h. We evaluated the simultaneous expression of the genes OsCATA, OsCATB, and OsCATC, correlated gene expression with enzyme activity, and verified the regulation of these genes through identification of cis-elements in the promoter region. The hydrogen peroxide content increased in a tolerant genotype and decreased in a sensitive genotype under both stress conditions. Lipid peroxidation increased in the tolerant genotype when exposed to cold, and in the sensitive genotype when exposed to high salinity. Catalase activity significantly increased in both genotypes when subjected to 13°C. In the tolerant genotype, OsCATA and OsCATB were the most responsive to high salinity and cold, while in the sensitive genotype, OsCATA and OsCATC responded positively to saline stress, as did OsCATA and OsCATB to low temperature. Cis-element analysis identified different regulatory sequences in the catalase promoter region of each genotype. The sensitive genotype maintained a better balance between hydrogen oxyacid levels, catalase activity, and lipid peroxidation under low temperature than the resistant genotype. OsCATA and OsCATB were the most responsive in the salt-tolerant genotype to cold, OsCATA and OsCATC were the most responsive to saline stress, and OsCATA and OsCATB were the most responsive to chilling stress in the sensitive genotype. There were positive correlations between catalase activity and OsCATB expression in the tolerant genotype under saline stress and in the sensitive genotype under cold stress.

Implantes intravítreos de dexametasona en pacientes con edema macular diabético refractario a ranibizumab en monoterapia o en combinación de tratamientos / Dexamethasone intravitreal implants for diabetic macular edema refractory to ranibizumab monotherapy or combination therapy

OBJETIVO: Estudiar la eficacia y seguridad a nivel local del implante de dexametasona intravítreo como tratamiento del edema macular diabético (EMD) refractario a ranibizumab intravítreo en monoterapia o en combinación de tratamientos. MÉTODOS: Estudio retrospectivo de pacientes con EMD refractario a inyecciones intravítreas de ranibizumab, en monoterapia o en combinación con otros tratamientos, a quienes se ha administrado un implante de dexametasona intravítreo. Los parámetros analizados fueron la agudeza visual (AV) medida en optotipos según el Early Treatment Diabetic Retinopathy Study (ETDRS) y el grosor foveal determinado mediante tomografía de coherencia óptica de dominio espectral (SD-OCT) previos al tratamiento, 2 meses después y al final del período de seguimiento. RESULTADOS: Se incluyeron 14 ojos de 14 pacientes con una edad media de 64 años (DE: 9,5; rango 41-78) y un seguimiento medio de 7,6 meses. La AV media aumentó de 53 a 59 letras a los 2 meses (p = 0,03), con 57 letras al final del período de seguimiento (p = 0,3). El grosor foveal medio disminuyó de 502 a 304 μ a los 2 meses (p = 0,001), con 376 μ al final del período de seguimiento (p = 0,009). El 43% de los pacientes fueron tratados con un nuevo implante intravítreo de dexametasona. El 21,4% de los pacientes presentaron hipertensión ocular que se pudo controlar con medicación tópica. CONCLUSIONES: El implante de dexametasona intravítreo es eficaz y seguro localmente para el tratamiento del EMD en pacientes refractarios al ranibizumab en monoterapia o en combinación con otros tratamientos

OBJECTIVE: To determine the effectiveness and local safety of dexamethasone intravitreal implants as a treatment in diabetic macular edema (DME) refractory to intravitreal injections of ranibizumab monotherapy or combination therapy. METHODS: A retrospective study conducted on patients with DME refractory to ranibizumab monotherapy or combined with other treatments treated with dexamethasone intravitreal implants. The parameters analyzed were visual acuity (VA) by ETDRS (Early Treatment Diabetic Retinopathy Study) charts and foveal thickness by spectral-domain optical coherence tomography (SD-OCT) before the treatment, 2 months after treatment, and at the end of the follow-up. RESULTS: A total of 14 eyes of 14 patients were included, with a mean age of 64 years (SD: 9.5; range 41-78) and a mean follow-up of 7.6 months. The mean VA improved from 53 letters to 59 letters at 2 months (P=.03), and 57 at the end of the follow-up period (P=.3). The mean foveal thickness decreased from 502 μ to 304 μ at 2 months (P=.001), and 376 μ at the end of the follow-up period (P=.009). Further treatment with intravitreal dexamethasone was required in 43% of the patients, and 21% had increased intraocular pressure, which was controlled with topical medication. CONCLUSIONS: Intravitreal dexamethasone implant is an effective and locally safe treatment for the management of DME refractory to ranibizumab monotherapy or combined with other treatments

Expression of LTP genes in response to saline stress in rice seedlings.

Saline stress is one of the primary factors limiting increased rice productivity in the southern region of Brazil. Farming can be affected by salinity that is due to both the origin of the soils as well as the irrigation water. Lipid transfer proteins (LTPs) have many physiological functions, including in the response to saline stress. Therefore, the objective of this study was to quantify the relative expression of 11 genetic isoforms that encode LTP1-type proteins in rice genotypes tolerant and sensitive to saline stress in the vegetative period. When the plants reached development stage V4, alternating irrigation was started with nutritive solution and water containing 150 mM NaCl. The LTP7 gene showed an increase in expression by 13.81-fold after 96 h of stress exposure in the saline-tolerant group, whereas the LTP10 gene expression level was increased by 71.10-fold after 96 h in the saline-sensitive group. The LTP26, LTP23, and LTP18 genes showed increased expression in both genotypes; however, the expression levels and response times were different. Thus, LTP7 and LTP10 showed the highest response to salinity. The LTP18, LTP23, and LTP26 genes were negatively correlated with the response to salinity.

Dexamethasone intravitreal implants for diabetic macular edema refractory to ranibizumab monotherapy or combination therapy.

OBJECTIVE: To determine the effectiveness and local safety of dexamethasone intravitreal implants as a treatment in diabetic macular edema (DME) refractory to intravitreal injections of ranibizumab monotherapy or combination therapy. METHODS: A retrospective study conducted on patients with DME refractory to ranibizumab monotherapy or combined with other treatments treated with dexamethasone intravitreal implants. The parameters analyzed were visual acuity (VA) by ETDRS (Early Treatment Diabetic Retinopathy Study) charts and foveal thickness by spectral-domain optical coherence tomography (SD-OCT) before the treatment, 2 months after treatment, and at the end of the follow-up. RESULTS: A total of 14 eyes of 14 patients were included, with a mean age of 64 years (SD: 9.5; range 41-78) and a mean follow-up of 7.6 months. The mean VA improved from 53 letters to 59 letters at 2 months (P=.03), and 57 at the end of the follow-up period (P=.3). The mean foveal thickness decreased from 502 µ to 304 µ at 2 months (P=.001), and 376 µ at the end of the follow-up period (P=.009). Further treatment with intravitreal dexamethasone was required in 43% of the patients, and 21% had increased intraocular pressure, which was controlled with topical medication. CONCLUSIONS: Intravitreal dexamethasone implant is an effective and locally safe treatment for the management of DME refractory to ranibizumab monotherapy or combined with other treatments.