Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

Altered levels of tissue and urinary prostacyclin in rats subjected to pancreas transplantation

Significant increases of TXB2 and PGE2 are reported to occur in pancreas transplantation. These increases are prevented with scavengers of oxygen-free radicals. In this communication, we report on changes of prostacyclin metabolites such as tissue 6-keto prostaglandin F1 alpha and urinary 2,3-dinor 6-keto prostaglandin F1 alpha in rats subjected to pancreas transplantation after different periods of organ cold preservation ischemia as well as the effect of superoxide dismutase (SOD) on these changes. For this purpose, male Lewis rats were classified as follows: Group I, Control; Group II, syngenic pancreas transplantation after 15 min of organ preservation in Collins solution at 4 degrees C; Group III, same as II but with 12 hours of organ preservation; Group IV, same as III, but with SOD pretreatment. Results have shown significant posttransplantation increases of both tissue 6-keto PGF1 alpha and urinary 2, 3 dinor 6-keto PGF1 alpha, the latter being a useful marker to evaluate systemic prostacyclin (PGI2) production by rat pancreas. This effect was prevented when the organ had been exposed to SOD during the period of cold preservation ischemia. These results confirm the implication of oxygen-free radicals (OFR) in the ischemia-reperfusion process associated to rat pancreas transplantation leading to enhanced arachidonic acid metabolism.     

Hypertrophic cardiomyopathy associated with left ventricular aneurysm and normal coronary arteries: Case study indicating genetic tendencies of cardiomyopathy

A 50-year-old man presented with hypertrophic obstructive cardiomyopathy (HOC) associated with a left ventricular aneurysm and normal coronary arteries. His history revealed no evidence of myocardial infarction or atypical angina. Physical examination disclosed HOC but did not suggest the presence of an aneurysm. Although the patient was treated medically, heart failure ensued, and he died suddenly while working his farm. Subsequent investigation of the patient's family revealed that three of his five children were also affected by cardiomyopathy, which was especially pronounced in the eldest, a 22-year-old man. The possible hemodynamic relationship between HOC and left ventricular aneurysm is discussed, along with probable indications. The role of left ventricular aneurysm is also presented in relation to the natural history of the disease.     

Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.     

Aflatoxin B1, zearalenone and deoxynivalenol production on irradiated corn kernels: Influence of inoculum size

The influence of inoculum size on aflatoxin B₁ (AFB₁), zearalenone (ZEN) and deoxynivalenol (DON) production was examined on irradiated corn kernels. Spore concentrations were determined in serial dilutions and adjusted to 10,10²,10³,10⁵ and 10⁶ spores/ml. Aflatoxin B₁ production was dependent on the inoculum size. The high levels of aflatoxin B₁ produced byA. parasiticus (21 and 30 mg/kg) were obtained with 10² and 10³ spores/ml after 35 and 20 days incubation. There was no spore concentration influence on zearalenone and deoxynivalenol production after 10, 20 and 35 days incubation. At 28°C and 0.97 water activity (aw), the mean levels of zearalenone production were 382, 267 and 520 μg/kg and the mean levels on deoxynivalenol production were 697,465 and 782 μg/kg.     

Structural properties of recombinant nonrepetitive and repetitive parts of major ampullate spidroin 1 from Euprosthenops australis: implications for fiber formation

Spider dragline silk proteins, spidroins, have a tripartite composition; a nonrepetitive N-terminal domain, a central repetitive region built up from many iterated poly-Ala and Gly rich blocks, and a C-terminal nonrepetitive domain. It is generally believed that the repetitive region forms intermolecular contacts in the silk fibers, while precise functions of the terminal domains have not been established. Herein, thermal, pH, and salt effects on the structure and aggregation and/or polymerization of recombinant N- and C-terminal domains, a repetitive segment containing four poly-Ala and Gly rich coblocks, and combinations thereof were studied. The N- and C-terminal domains have mainly alpha-helical structure, and interestingly, both form homodimers. Dimerization of the end domains allows spidroin multimerization independent of the repetitive part. Reduction of an intersubunit disulfide in the C-terminal domain lowers the thermal stability but does not affect dimerization. The repetitive region shows helical secondary structure but appears to lack stable folded structure. A protein composed of this repetitive region linked to the C-terminal domain has a mainly alpha-helical folded structure but shows an abrupt transition to beta-sheet structures upon heating. At room temperature, this protein self-assembles into macroscopic fibers within minutes. The secondary structures of none of the domains are altered by pH or salt. However, high concentrations of phosphate affect the tertiary structure and accelerate the aggregation propensity of the repetitive region. Implications of these results for dragline spidroin behavior in solution and silk fiber formation are discussed.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens

In previous work, UEA1 and UCSC1, two geographically distinct, powdery mildew isolates, were recognized for their ability to infect Arabidopsis thaliana. We have clarified the identity of these isolates by determining their host ranges, reexamining their morphology, and comparing their DNA sequences for the 5.8S ribosomal RNA and two flanking internal transcribed spacer sequences. These experiments confirm that UEA1 is a member of Erysiphe cruciferarum and that UCSC1 belongs to E. cichoracearum. Interactions of the two Erysiphe isolates with 360 A. thaliana accessions were examined to provide a comprehensive profile of naturally occurring powdery mildew resistance in this weedy species. The majority of A. thaliana accessions (213) were susceptible to both isolates. Among the accessions exhibiting some degree of resistance, most (84) responded differentially to UEA1 and UCSC1 and the remainder were resistant to both isolates. Notably, resistance to UCSC1 cosegregated with RPW7, a locus previously demonstrated to confer resistance to UEA1 in Ms-0 x Landsberg (erecta) crosses. With this large collection of resistant accessions, questions about species specificity, genetic diversity and the evolution of resistance to powdery mildews can be addressed.     

Functional analysis of RPS27 mutations and expression in melanoma

Next‐generation sequencing has enabled genetic and genomic characterization of melanoma to an unprecedent depth. However, the high mutational background plus the limited depth of coverage of whole‐genome sequencing performed on cutaneous melanoma samples make the identification of novel driver mutations difficult. We sought to explore the somatic mutation portfolio in exonic and gene regulatory regions in human melanoma samples, for which we performed targeted sequencing of tumors and matched germline DNA samples from 89 melanoma patients, identifying known and novel recurrent mutations. Two recurrent mutations found in the RPS27 promoter associated with decreased RPS27 mRNA levels in vitro. Data mining and IHC analyses revealed a bimodal pattern of RPS27 expression in melanoma, with RPS27‐low patients displaying worse prognosis. In vitro characterization of RPS27‐high and RPS27‐low melanoma cell lines, as well as loss‐of‐function experiments, demonstrated that high RPS27 status provides increased proliferative and invasive capacities, while low RPS27 confers survival advantage in low attachment and resistance to therapy. Additionally, we demonstrate that 10 other cancer types harbor bimodal RPS27 expression, and in those, similarly to melanoma, RPS27‐low expression associates with worse clinical outcomes. RPS27 promoter mutation could thus represent a mechanism of gene expression modulation in melanoma patients, which may have prognostic and predictive implications.     

Intra- and interannual variation in the diet of the Magellanic penguin (Spheniscus magellanicus) at Martillo Island,Beagle Channel

Knowledge of seabirds’ diet at each breeding site and its temporal variation is key to understanding and evaluating how changes in marine resources affect each seabird population. In this study, we determined the diet of Magellanic penguins (MP, Spheniscus magellanicus) at Martillo Island, accounting for sex, breeding stage and year. We analyzed a total of 144 stomach contents during three consecutive breeding seasons (2006–2007, 2007–2008 and 2008–2009) and stages (incubation, early and late chick-rearing). MP fed mainly on fuegian sprat (Sprattus fuegensis), which represented 75 % of the biomass consumed by birds during the entire study. The next important prey was squat lobster (Munida gregaria), followed by Patagonian squid (Loligo gahi). Both sexes consumed similar prey items. We observed variation in diet relative composition among breeding years and stages. Fuegian sprat consumption decreased throughout the years whereas squat lobster increased. Penguins consumed a higher proportion of squat lobster and Patagonian squid during the incubation stage than in the chick-rearing stages, whereas fuegian sprat was almost the only prey item consumed during the late chick-rearing stage. MPs show certain flexibility in the use of resources probably as a response to changes in prey populations. Variability in the diet among different reproductive stages could be related to changes in the distribution and abundance of their main prey near the colony during the breeding season together with changes in the energy requirements of seabirds.