Three new clades of putative viral RNA-dependent RNA polymerases with rare or unique catalytic triads discovered in libraries of ORFans from powdery mildews and the yeast of oenological interest Starmerella bacillaris.

High throughput sequencing allowed the discovery of many new viruses and viral organizations increasing our comprehension of virus origin and evolution. Most RNA viruses are currently characterized through similarity searches of annotated virus databases. This approach limits the possibility to detect completely new virus-encoded proteins with no detectable similarities to existing ones, i.e. ORFan proteins. A strong indication of the ORFan viral origin in a metatranscriptome is the lack of DNA corresponding to an assembled RNA sequence in the biological sample. Furthermore, sequence homology among ORFans and evidence of co-occurrence of these ORFans in specific host individuals provides further indication of a viral origin. Here, we use this theoretical framework to report the finding of three conserved clades of protein-coding RNA segments without a corresponding DNA in fungi. Protein sequence and structural alignment suggest these proteins are distantly related to viral RNA-dependent RNA polymerases (RdRP). In these new putative viral RdRP clades, no GDD catalytic triad is present, but the most common putative catalytic triad is NDD and a clade with GDQ, a triad previously unreported at that site. SDD, HDD, and ADD are also represented. For most members of these three clades, we were able to associate a second genomic segment, coding for a protein of unknown function. We provisionally named this new group of viruses ormycovirus. Interestingly, all the members of one of these sub-clades (gammaormycovirus) accumulate more minus sense RNA than plus sense RNA during infection.

Analysis of the virome associated to grapevine downy mildew lesions reveals new mycovirus lineages.

The obligatory biotrophic oomycetes Plasmopara viticola is the causal agent of downy mildew, a destructive disease of grapevine worldwide. So far, chemical fungicides are widely employed to limit this pathogen, but their adverse effects are stimulating the quest for environmentally friendly alternative approaches. Here, we report on the search for mycoviruses that might be later developed as biocontrol agents for this pathogen. Symptomatic leaves were collected from various regions in Spain and Italy and mycelia associated to leaf lesions was harvested. Total RNA extractions were depleted of rRNA and metatranscriptomes were generated using a high-throughput sequencing approach. The virome associated to leaf lesions was then characterized through a bioinformatic pipeline relying on blast searches against current viral databases. Here, we present an inventory of 283 new RNA viruses: 222 positive strand RNA viruses, 29 negative strand RNA viruses, 27 double-stranded RNA viruses and 5 ORFan virus RdRP segments, which could not be reliably assigned to any existing group in the Riboviria. In addition to ORFan viruses, we found other surprising new evolutionary trajectories in this wide inventory of viruses. The most represented viruses in our collection are those in phylum Lenarviricota, and, among them, a group of mycovirus segments distantly related to narnaviruses, but characterized by a polymerase palm domain lacking subdomain C, with the putative GDD catalytic triad. We also provided evidence of a strict association between two RNA segments that form a new mycovirus clade of positive strand RNA in the phylum Kitrinoviricota, order Martellivirales. In the phylum Negarnaviricota, we report for the first time in the order Mononegavirales a clade of viruses that is ambisense, a feature that so far was present only in the order Bunyavirales. Furthermore, in the same phylum we detected the widespread occurrence and abundant accumulation in our libraries of a distinct mycovirus clade distantly related to the Muvirales and Goujanvirales orders, which so far include only viruses infecting invertebrates. Possible new oomycetes-specific virus clades are also described in the phylum Duplornaviricota. These data greatly expand the evolutionary history of mycoviruses adding new layers of diversity to the realm Riboviria.

Physical fitness and white matter microstructure in children with overweight or obesity: the ActiveBrains project.

Recent studies investigated the association of cardiorespiratory fitness with white matter microstructure in children, yet little work has explored to what extent other components of physical fitness (i.e., muscular or motor fitness) are associated with white matter microstructure. Indeed, this association has not been previously explored in children with overweight/obesity who present a different white matter development. Therefore, we aimed to examine associations between physical fitness components and white matter microstructure in children with overweight/obesity. In total, 104 (10.04 ± 1.15 years old; 43 girls) children were included in this cross-sectional study. Physical fitness was assessed using the ALPHA-fitness test battery. Fractional anisotropy (FA) and mean diffusivity were derived from diffusion tensor imaging (DTI). No association was found between physical fitness and global DTI metrics (all P > 0.082). Within individual tracts, all associations became non-significant when analyses were adjusted for multiple comparisons. Using the voxel-wise approach, we identified a small cluster in the left lateral frontal lobe where children with greater upper-body muscular fitness showed higher FA (PFWE-corrected = 0.042). Although our results cannot conclude physical fitness is related to white matter microstructure in children with overweight/obesity; those findings indicate that the association of muscular fitness with white matter microstructure might be more focal on frontal areas of the brain, as opposed to global differences.

Laparoscopic Living Donor Hepatectomy for Pediatric Liver Transplantation: the First 7 Cases in Spain.

Herein we report on laparoscopic donor hepatectomy (left lateral sectionectomy) for pediatric living donor liver transplantation by using a pure laparoscopic approach. Seven laparoscopic living donor procedures were performed during the period March 2016 to February 2017 at our institution. The average age of donors was 33.3 years. Preoperative liver function was normal in all donors. Four donors required 1 or more Pringle maneuver(s). The etiology was biliary atresia (n = 3), metabolic disorders (n = 2) (OTC deficiency), Alagille syndrome (n = 1), and neonatal ductopenia (n = 1). The graft was implanted orthotopically in 6 patients; we performed an auxilliary transplantation in a patient with an OTC deficiency. The time of donor surgery was 363 minutes. Dindo-Clavien complications among donors were type I (n = 1), type IIa (n = 1), and type IIb (n = 2). The mean hospital stay for the recipients was 14 days. The mean donor stay was 3.7 days. Perioperative donor and recipient mortality was 0%. Graft survival was 87.5% with 1 graft loss secondary to inadequate venous outflow. In conclusion, we can propose the laparoscopic approach in experienced centers as a "standard of practice" due to its minimal complication rate and short hospital stay.

Use of Peritoneovenous Shunt for the Management of Refractory Ascites.

BACKGROUND: Guidelines for the management of refractory ascites (RA) recommend transjugular intrahepatic portosystemic shunting (TIPS), diuretics, and paracentesis as the main strategies, discouraging use of surgical peritoneovenous shunts (PVSs). However, PVSs, including both Denver (DS) or saphenoperitoneal (SPS) modalities, may still have indications. Herein we report our experience with PVSs in the context of modern surgical and anesthetic management. METHODS: In our unit, PVSs are offered to patients with ascites refractory to diuretics in which TIPS are contraindicated. Heart function and spontaneous bacterial peritonitis must be assessed before surgical indication. RESULTS: Seven procedures were performed on 5 patients (6-DS, 1-SPS) in 2013. Their mean age was 61 (range, 54-68) years. In 3 patients, the indication was RA without options for liver transplant; 2 patients were on the waiting list for liver transplantation, which were performed to improve renal function and quality of life (QOL). The median hospital stay was 6.5 (range, 3-12) days. All patients were alive after 12 months. One patient died 2 years after the first DS and another later died due to liver insufficiency with patency of the DS. The ascites was well-controlled in 4 of 5 patients at up to 48 months of follow-up. Decreases in diuretics doses, proper weight maintenance, and a dramatic improvement in QOL (measured by a modified Ascites Symptom Inventory-7 [ASI-7] test) were observed after the procedures. CONCLUSION: PVSs are useful for the treatment of patients with RA who develop resistance to common therapies, leading to a major improvement in QOL. These surgical procedures should be included in the armamentarium of experienced liver surgeons.

Physical fitness and psychological health in overweight/obese children: A cross-sectional study from the ActiveBrains project.

OBJECTIVES: To examine the associations of physical fitness (i.e. cardiorespiratory fitness, muscular strength, and speed/agility) with psychological distress and psychological well-being in overweight/obese pre-adolescent children. DESIGN: 110 overweight/obese children (10.0±1.1years old, 61 boys) from the ActiveBrains project (http://profith.ugr.es/activebrains) participated in this cross-sectional study. METHODS: Physical fitness was evaluated by the ALPHA battery test. Cardiorespiratory fitness was additionally evaluated by a maximal incremental treadmill. Stress was assessed by the Children's Daily Stress Inventory, anxiety by the State-Trait Anxiety Inventory, depression by the Children Depression Inventory, positive affect and negative affect by the Positive and Negative Affect Scale for Children, happiness by the Subjective Happiness Scale, optimism by the Life Orientation Test, and self-esteem by the Rosenberg Self-Esteem questionnaire. Linear regression adjusted for sex and peak height velocity was used to examine associations. RESULTS: Absolute upper-body muscular strength was negatively associated with stress and negative affect (ß=-0.246, p=0.047; ß=-0.329, p=0.010, respectively). Furthermore, absolute lower-body muscular strength was negatively associated with negative affect (ß=-0.301, p=0.029). Cardiorespiratory fitness, expressed by the last completed lap, and relative upper-body muscular strength were positively associated with optimism (ß=0.220, p=0.042; ß=0.240, p=0.017, respectively). Finally, absolute upper-body muscular strength was positively associated with self-esteem (ß=0.362, p=0.003) independently of sex and weight status (p for interactions >0.3), and absolute lower-body muscular strength was also positively associated with self-esteem (ß=0.352, p=0.008). CONCLUSIONS: Muscular strength was associated with psychological distress (i.e. stress and negative affect) and psychological well-being (i.e. optimism and self-esteem) as well as cardiorespiratory fitness was associated with optimism. Therefore, increased levels of physical fitness, specifically muscular strength, could have significant benefits for overweight/obese children psychological health.

Apparent Diffusion Coefficient Histograms of Human Papillomavirus-Positive and Human Papillomavirus-Negative Head and Neck Squamous Cell Carcinoma: Assessment of Tumor Heterogeneity and Comparison with Histopathology.

BACKGROUND AND PURPOSE: Head and neck squamous cell carcinoma associated with human papillomavirus infection represents a distinct tumor entity. We hypothesized that diffusion phenotypes based on the histogram analysis of ADC values reflect distinct degrees of tumor heterogeneity in human papillomavirus-positive and human papillomavirus-negative head and neck squamous cell carcinomas. MATERIALS AND METHODS: One hundred five consecutive patients (mean age, 64 years; range, 45-87 years) with primary oropharyngeal (n = 52) and oral cavity (n = 53) head and neck squamous cell carcinoma underwent MR imaging with anatomic and diffusion-weighted sequences (b = 0, b = 1000 s/mm2, monoexponential ADC calculation). The collected tumor voxels from the contoured ROIs provided histograms from which position, dispersion, and form parameters were computed. Histogram data were correlated with histopathology, p16-immunohistochemistry, and polymerase chain reaction for human papillomavirus DNA. RESULTS: There were 21 human papillomavirus-positive and 84 human papillomavirus-negative head and neck squamous cell carcinomas. At histopathology, human papillomavirus-positive cancers were more often nonkeratinizing (13/21, 62%) than human papillomavirus-negative cancers (19/84, 23%; P = .001), and their mitotic index was higher (71% versus 49%; P = .005). ROI-based mean and median ADCs were significantly lower in human papillomavirus-positive (1014 ± 178 × 10-6 mm2/s and 970 ± 187 × 10-6 mm2/s, respectively) than in human papillomavirus-negative tumors (1184 ± 168 × 10-6 mm2/s and 1161 ± 175 × 10-6 mm2/s, respectively; P < .001), whereas excess kurtosis and skewness were significantly higher in human papillomavirus-positive (1.934 ± 1.386 and 0.923 ± 0.510, respectively) than in human papillomavirus-negative tumors (0.643 ± 0.982 and 0.399 ± 0.516, respectively; P < .001). Human papillomavirus-negative head and neck squamous cell carcinoma had symmetric normally distributed ADC histograms, which corresponded histologically to heterogeneous tumors with variable cellularity, high stromal component, keratin pearls, and necrosis. Human papillomavirus-positive head and neck squamous cell carcinomas had leptokurtic skewed right histograms, which corresponded to homogeneous tumors with back-to-back densely packed cells, scant stromal component, and scattered comedonecrosis. CONCLUSIONS: Diffusion phenotypes of human papillomavirus-positive and human papillomavirus-negative head and neck squamous cell carcinomas show significant differences, which reflect their distinct degree of tumor heterogeneity.

Spontaneous liver rupture as first sign of polyarteritis nodosa.

Polyarteritis nodosa (PAN) is one of the systemic vasculitis that affects the media wall of arteries of small and medium diameter. Diagnosis proves difficult due to the unspecific symptoms that dominate the clinical profile. Liver involvement is very diverse, ranging from the development of cirrhotic liver disease to acute abdomen presentation that requires surgery because of liver rupture. The management of these patients requires an expert multidisciplinary team. There are several cases in the literature that describe a sudden liver rupture as the first manifestation of a PAN. In this paper we present the case of a 75 years old patient without any previous disease, who is subjected to major hepatic resection for spontaneous liver rupture.

Optimizing the management of patients with BCLC stage-B hepatocellular carcinoma: Modern surgical resection as a feasible alternative to transarterial chemoemolization.

OBJECTIVE: To analyse the impact of liver resection (LR) in patients with Hepatocellular Carcinoma (HCC) within the Barcelona-Clinic-Liver-Cancer (BCLC)-B stage. METHODS: Analysis of patients with BCLC-B HCC treated with LR or transarterial chemoembolization (TACE) between 2007 and 2012 in our hospital. Survival/recurrence analyses were performed by log-rank tests and Cox multivariate models. Further analyses were specifically obtained for the HCC subclassification (B1-2-3-4) proposed recently. RESULTS: Eighty patients were treated (44-TACE/36-LR). Number of nodules was [1.8(1.1)], being multinodular in 50% of cases. Although resected patients had a higher hospital stay than those who underwent TACE (14 ± 13 vs 7 ± 6; P = 0.004), the rate and severity of complications was lower measured by Dindo-Clavien scale (P < 0.05). Overall survival was 40% with a median follow-up of 29.5 months (0.07-96.9). Five-years survival rates were 62.9%, 28.1% and 15.4%, respectively (P = 0.004) for B1, B2 and B3-4 stages. Cox model showed that only total bilirubin [OR = 2.055(1.23-3.44)] and BCLC subclassification B3-4 [OR = 2.439(1.04-5.7)] and B2 [OR = 2.79(1.35-5.77)] vs B1 were independent predictors of 5-years-survival. In B1 patients, surgical approach led a significant decrease in 5-years recurrence-rate (25% vs 60%; P = 0.018). In the surgical subgroup analysis, better results were observed if well/moderate differentiation combined with no microvascular-invasion (VI) in 5-years-survival (84.6%; P = 0.001) and -recurrence (23.1%; P = 0.041), respectively. These survival and recurrence trends were remarkable in B1 stages. CONCLUSIONS: Management of Intermediate BCLC-B HCC stage should be more complex and include updated criteria regarding B-stage subclassifications, VI and tumour differentiation. Modern surgical resection would offer improved survival benefit with acceptable safety in selected BCLC-B stage patients.

Características en tomografía computarizada de la isquemia cecal aislada / Computed tomography characteristics of isolated caecal ischaemia

Objetivo: Valorar la sobrecarga férrica mediante el cálculo del valor T2* en el hígado y el miocardio en los pacientes con hemocromatosis secundaria. Evaluar la correlación de los valores obtenidos con los niveles de ferritina en sangre y la concentración de hierro hepático (CHH) calculada mediante resonancia magnética (RM), y la correlación de los valores T2* entre sí. Material y métodos: Se incluyeron 16 pacientes (13 varones y 3 mujeres), evaluados entre los a˜nos 2008 y 2009, con una edad media de 61 a˜nos. Quince eran pacientes politransfundidos y uno estaba diagnosticado de anemia sideroblástica hereditaria. Se estudió la ferritina en sangre, la CHH por RM, la función cardíaca mediante RM y el valor T2* mediante secuencias multieco en el hígado (TR/TE1/ TE/n◦ecos/ : 21/1,18/1.0/20/35◦) y el miocardio (26/1,04/0.8/30/60◦). Se realizó el análisis de correlación-regresión de los valores T2* cardíaco y hepático con los valores de ferritina y CHH, y entre sí. Resultados: Trece pacientes mostraron valores de ferritina superiores a 1.000 ng/ml (mediana/mínimo/máximo: 1.762/294/3.785 ng/ml). Trece pacientes presentaron CHH elevada, mayor de 80 mol/g (mediana/mínimo/máximo: 125,4/41,2/241,5 mol/g). En todos los casos la función cardíaca estaba preservada. En 15 pacientes el valor T2* hepático fue menor de 6,3 ms. Solo en un caso, el valor T2*miocárdico fue menor de 20 ms. Se observó una alta correlación para los valores T2*hepático/CHH (r: -0,912). La correlación fue estadísticamente significativa para T2* hepático/ferritina (r: -0,541). La correlación T2*miocárdico/ferritina, T2*miocárdico/CHH y T2*miocárdico/T2*hepático no fue estadísticamente significativa. Conclusión: Los valores T2* hepático muestran una alta correlación con la CHH y una correlación estadísticamente significativa con la ferritina. No se observó correlación entre los valores T2* miocárdico y la ferritina en sangre, la CHH, ni con el valor T2* hepático (AU)

Objective: To determine whether there is iron overload by calculating the T2* value in the liver and myocardium in patients with secondary haemochromatosis. To analyse the correlation of the values obtained with the iron levels in blood, with the liver iron concentration (LIC) calculated using magnetic resonance (MR) imaging, and the correlation between them. Material and methods: A total of 16 patients (13 males, 3 females), with a mean age of 61 years, were included and evaluated in the years 2008 and 2009. Fifteen of them had received multiple transfusions, and one was diagnosed with hereditary sideroblastic anaemia. The measurements included, blood ferritin, LIC by MRI, cardiac function using MRI and the T2* value by means of multi-echo sequences in the liver (TR/TE1/ TE/No of echos/ : 21/1,18/1.0/20/35◦) and myocardium (26/1.04/0.8/30/60◦). A correlation-regression analysis was performed by comparing the cardiac and liver T2* values with the ferritin, LIC and between each of them. Results: A total of 13 patients had ferritin values greater than 1000 ng/ml (median/minimum/maximum: 1762/294/3785 ng/ml). An increased LIC greater than 80 mol/g (median/minimum/maximum: 125.4/41.2/241.5 mol/g) was observed in 13 patients. In all cases cardiac function was conserved, and in 15 cases the liver T2* value was less than 6.3 ms. The myocardium T2* value was less than 20 ms. in only one case. A high correlation was observed between the liver T2* values and the LIC (r: -0.912). The correlation was statistically significant between the liver T2* value and ferritin (r: -0.541). The correlations between myocardium T2* and ferritin, myocardium T2* and LIC, and myocardium T2* and liver T2* were not statistically significant. Conclusions: The liver T2* showed a high correlation with LIC and a statistically significant correlation with ferritin. No association was observed between the myocardium T2* values and ferritin in blood, the LIC or the liver T2* value (AU)

Landscape heterogeneity shapes host-parasite interactions and results in apparent plant-virus codivergence.

Knowledge on how landscape heterogeneity shapes host-parasite interactions is central to understand the emergence, dynamics and evolution of infectious diseases. However, this is an underexplored subject, particularly for plant-virus systems. Here, we analyse how landscape heterogeneity influences the prevalence, spatial genetic structure, and temporal dynamics of Pepper golden mosaic and Pepper huasteco yellow vein begomoviruses infecting populations of the wild pepper Capsicum annuum glabriusculum (chiltepin) in Mexico. Environmental heterogeneity occurred at different nested spatial scales (host populations within biogeographical provinces), with levels of human management varying among host population within a province. Results indicate that landscape heterogeneity affects the epidemiology and genetic structure of chiltepin-infecting begomoviruses in a scale-specific manner, probably related to conditions favouring the viruses' whitefly vector and its dispersion. Increased levels of human management of the host populations were associated with higher virus prevalence and erased the spatial genetic structure of the virus populations. Also, environmental heterogeneity similarly shaped the spatial genetic structures of host and viruses. This resulted in the congruence between host and virus phylogenies, which does not seem to be due to host-virus co-evolution. Thus, results provide evidence of the key role of landscape heterogeneity in determining plant-virus interactions.

[Computed tomography characteristics of isolated caecal ischaemia]. / Características en tomografía computarizada de la isquemia cecal aislada.

OBJECTIVE: The aims of this review are to describe the main characteristics for the CT diagnosis of isolated caecal ischaemia (ICA) and give details of the differential diagnosis with other conditions with a similar clinical picture. MATERIAL AND METHODS: A retrospective study was conducted to review the CT findings of 4 patients diagnosed with ICA in our hospital. The parameters recorded to analyse their characteristics in the CT were: maximum thickness of the caecum wall, the appearance of the peri-caecum fat, presence of free fluid, signs of caecal or portal pneumatosis, the appearance of the caecal appendix, and general signs of the presence of vasculopathy. RESULTS: In all cases it was recorded that there was a thickening of the walls of the blind loop with an abrupt transition between the caecal wall and the walls of the ascending colon wall. In all cases the caecal thickening had a characteristic image in the central area. Signs of caecal pneumatosis were observed in two cases. All of them had an appendix with normal characteristics. CONCLUSIONS: The combination of caecal wall thickening with oedematous characteristics, with no changes in the appendix, ileum and colon, suggest the diagnosis of caecal ischaemia, particularly with the presence of pneumatosis.

Rotura accidental de catéter caudal en el espacio paravertebral / No disponible

No disponible

Brain edema and inflammatory activation in bile duct ligated rats with diet-induced hyperammonemia: A model of hepatic encephalopathy in cirrhosis.

Studies of the pathogenesis of hepatic encephalopathy are hampered by the lack of a satisfactory animal model. We examined the neurological features of rats after bile duct ligation fed a hyperammonemic diet (BDL+HD). Six groups were studied: sham, sham pair-fed, hyperammonemic, bile duct ligation (BDL), BDL pair fed, and BDL+HD. The BDL+HD rats were made hyperammonemic via an ammonia-containing diet that began 2 weeks after operation. One week later, the animals were sacrificed. BDL+HD rats displayed an increased level of cerebral ammonia and neuroanatomical characteristics of hepatic encephalopathy (HE), including the presence of type II Alzheimer astrocytes. Both BDL and BDL+HD rats showed activation of the inflammatory system. BDL+HD rats showed an increased amount of brain glutamine, a decreased amount of brain myo-inositol, and a significant increase in the level of brain water. In coordination tests, BDL+HD rats showed severe impairment of motor activity and performance as opposed to BDL rats, whose results seemed only mildly affected. In conclusion, the BDL+HD rats displayed similar neuroanatomical and neurochemical characteristics to human HE in liver cirrhosis. Brain edema and inflammatory activation can be detected under these circumstances.

Reelin expression and glycosylation patterns are altered in Alzheimer's disease.

Reelin is a glycoprotein that is essential for the correct cytoarchitectonic organization of the developing CNS. Its function in the adult brain is less understood, although it has been proposed that Reelin is involved in signaling pathways linked to neurodegeneration. Here we analyzed Reelin expression in brains and cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients and nondemented controls. We found a 40% increase in the Reelin protein levels in the cortex of AD patients compared with controls. Similar increases were detected at the Reelin mRNA transcriptional level. This expression correlates with parallel increases in CSF but not in plasma samples. Next, we examined whether CSF Reelin levels were also altered in neurological diseases, including frontotemporal dementia, progressive supranuclear palsy, and Parkinson's disease. The Reelin 180-kDa band increased in all of the neurodegenerative disorders analyzed. Moreover, the 180-kDa Reelin levels correlated positively with Tau protein in CSF. Finally, we studied the pattern of Reelin glycosylation by using several lectins and the anti-HNK-1 antibody. Glycosylation differed in plasma and CSF. Furthermore, the pattern of Reelin lectin binding differed between the CSF of controls and in AD. Our results show that Reelin is up-regulated in the brain and CSF in several neurodegenerative diseases and that CSF and plasma Reelin have distinct cellular origins, thereby supporting that Reelin is involved in the pathogenesis of a number of neurodegenerative diseases.

Changes in liver and plasma acetylcholinesterase in rats with cirrhosis induced by bile duct ligation.

Classical studies of cholinesterase activity during liver dysfunction have focused on butyrylcholinesterase (BuChE), whereas acetylcholinesterase (AChE) has not received much attention. In the current study, liver and plasma AChE levels were investigated in rats with cirrhosis induced after 3 weeks of bile duct ligation (BDL). BDL rats showed a pronounced decrease in liver AChE levels (approximately 50%) compared with sham-operated (non-ligated, NL) controls; whereas liver BuChE appeared unaffected. A selective loss of tetrameric (G4) AChE was detected in BDL rats, an effect also observed in rats with carbon tetrachloride-induced cirrhosis. In accordance, SDS-PAGE analysis showed that the major 55-kd immunoreactive AChE band was decreased in BDL as compared with NL. A 65-kd band, attributed in part to inactive AChE, was increased as became the most abundant AChE subunit in BDL liver. The overall decrease in AChE activity in BDL liver was not accompanied by a reduction of AChE transcripts. The loss of G4 was also reflected by changes observed in AChE glycosylation pattern attributable to different liver AChE forms being differentially glycosylated. BDL affects AChE levels in both hepatocytes and Kupffer cells; however, altered AChE expression was mainly reflected in an alteration in hepatocyte AChE pattern. Plasma from BDL rats had approximately 45% lower AChE activity than controls, displaying decreased G4 levels and altered lectin-binding patterns. In conclusion, the liver is an important source of serum AChE; altered AChE levels may be a useful biomarker for liver cirrhosis.

Evolutionary analysis of genetic variation observed in citrus tristeza virus (CTV) after host passage.

We have studied the genetic variability in two genes (p18 and p20) from two groups of Citrus tristeza virus (CTV) isolates. One group (isolates T385, T317, T318, and T305) was derived from a Spanish source by successive host passages while the other (isolates T388 and T390) was obtained after aphid transmission from a Japanese source. A total of 274 sequences were obtained for gene p18 and 451 for p20. In the corresponding phylogenetic trees, sequences derived from the severe isolates (T318, T305, and T388) clustered together and separately from those derived from mild or moderate isolates (T385, T317, and T390), regardless of their geographic origin. Hierarchical analyses of molecular variance showed that up to 53% of the total genetic variability in p18 and up to 87% of the variation in p20 could be explained by differences in the pathogenicity features of the isolates. Neutrality tests revealed that different selection forces had been acting between isolates and between genes, with purifying selection being suggested for p18 from isolates T385 and T390 and for p20 from isolates T385, T317, and T388, and balancing selection for p18 from isolates T318, T305, and T388 and for p20 from isolates T318 and T390. Furthermore, several models of codon selection were observed, with purifying selection being the most notable one, compatible with low effective population size of the virus populations resulting from transmission bottlenecks. We found no evidence of recombination playing a significant role during p18 and p20 evolution in these isolates. These results suggest that hosts can be an important evolutionary factor for CTV isolates.

Active and inactive ecto-5'-nucleotidase variants in liver of control and dystrophic Lama2dy mice.

The ecto-5'-nucleotidase (eNT) activity and the eNT protein content in liver of normal and merosin-deficient dystrophic Lama2dy mice were studied. After the solubilization procedure, the eNT activity in the final extract was 9.2+/-2.5U/mg (nmol of phosphate released from AMP per min and per mg protein) in normal liver, and it rose to 16.1+/-3.9U/mg (P=0.005) in dystrophic liver. The increase of activity was less pronounced in Lama2dy liver (1.7-fold) than the one reported in muscle (four-fold), which probably reflects the lower content of merosin in liver. Similarly to muscle, liver contained active and inactive eNT, as demonstrated by the higher level of immunoreactive protein in normal than in dystrophic liver in Western blots performed with samples containing the same units of eNT activity. PNGase F digestion decreased the size of liver and muscle eNT from 72 and 69kDa, to 63 and 60kDa. Oligoglycan cleavage did not alter eNT activity or the sedimentation coefficient, revealing that oligosaccharides are not required for catalysis or for maintaining the dimeric structure. The eNT protein content in samples of normal liver decreased by 55 or 80% after the trypsinolysis of native or deglycosylated enzyme, but the activity did not change. Such a high proportion of inactive eNT is unlikely to come from aged enzyme, which suggests the involvement of inactive enzyme in non-catalytic actions.