Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.      

Evaluation of APOE polymorphisms and the risk for age-related macular degeneration in a Southeastern Brazilian population

This study aimed to evaluate the role of APOE polymorphisms (rs429358 and rs7412) in the risk of age-related macular degeneration in a sample of the Southeastern Brazilian population. Seven hundred and five unrelated individuals were analyzed, 334 with age-related macular degeneration (case group), and 371 without the disease (control group). In the case group, patients were further stratified according to disease phenotypes, divided into dry and wet age-related macular degeneration, and non-advanced and advanced age-related macular degeneration. APOE polymorphisms (rs429358 and rs7412) were evaluated through polymerase chain reaction and direct sequencing. In the comparison of cases vs. controls, none of the associations reached statistical significance, considering the Bonferroni-adjusted P-value, although there was a suggestive protection for the E3/E4 genotype (OR = 0.626; P-value = 0.037) and E4 carriers (OR = 0.6515; P-value = 0.047). Statistically significant protection for both the E3/E4 genotype and E4 carriers was observed in the comparisons: advanced age-related macular degeneration vs. controls (OR = 0.3665, P-value = 0.491 × 10⁻³ and OR = 0.4031, P-value = 0.814 × 10⁻³, respectively), advanced age-related macular degeneration vs. non-advanced age-related macular degeneration (OR = 0.2529, P-value = 0.659 × 10⁻⁴ and OR = 0.2692, P-value = 0.631 × 10⁻⁴, respectively). In the comparison of wet age-related macular degeneration vs. control, protection was statistically significant only for E3/E4 (OR = 0.4052, P-value = 0.001). None of the comparisons demonstrated any significant association for E2 genotypes or E2 carriers in age-related macular degeneration risk in this study. Findings suggest a protective role of the E4 haplotype in the APOE gene in the risk for advanced and wet forms of age-related macular degeneration, in a sample of the Brazilian population. To our knowledge, this is the first Brazilian study to show the association between APOE polymorphisms and age-related macular degeneration.     

Pregnancies following long luteal phases in southern white rhinoceros (Ceratotherium simum simum)

All extant species in the Rhinocerotidae family are experiencing escalating threats in the wild, making self-sustaining captive populations essential genetic reservoirs for species survival. Assisted reproductive technologies (ARTs) will become increasingly important for achieving and maintaining ex situ population sustainability and genetic diversity. Previous reports have shown that a large proportion of captive southern white rhinoceros (SWR) females are irregularly cyclic or acyclic, and that cycling females display two different estrous cycle lengths of approximately 30 or 70 days. It has been suggested that the longer estrous cycle length is infertile or subfertile, as no term pregnancies have been observed following long cycles. Here we report the achievement of two pregnancies following long luteal phases, using ovulation induction and artificial insemination with either fresh or frozen-thawed semen. One female SWR conceived on the first insemination attempt and gave birth to a live offspring. A second female conceived twice in consecutive long cycles although the first embryo was resorbed by 33 days post-insemination. A pregnancy from this female's second insemination is ongoing with expected parturition in November 2019. Whether prolonged estrous cycles in SWR are subfertile or infertile in natural breeding situations remains unclear. However, our findings demonstrate that the application of ARTs following prolonged cycles can result the successful establishment of pregnancies in SWR. Therefore, with ARTs, female SWR otherwise considered nonreproductive due to long estrous cycles may still have the potential for representation and contribution to the ex situ population.     

1‐Undecene from Pseudomonas aeruginosa is an olfactory signal for flight‐or‐fight response in Caenorhabditis elegans

Animals possess conserved mechanisms to detect pathogens and to improve survival in their presence by altering their own behavior and physiology. Here, we utilize Caenorhabditis elegans as a model host to ask whether bacterial volatiles constitute microbe‐associated molecular patterns. Using gas chromatography–mass spectrometry, we identify six prominent volatiles released by the bacterium Pseudomonas aeruginosa. We show that a specific volatile, 1‐undecene, activates nematode odor sensory neurons inducing both flight and fight responses in worms. Using behavioral assays, we show that worms are repelled by 1‐undecene and that this aversion response is driven by the detection of this volatile through AWB odor sensory neurons. Furthermore, we find that 1‐undecene odor can induce immune effectors specific to P. aeruginosa via AWB neurons and that brief pre‐exposure of worms to the odor enhances their survival upon subsequent bacterial infection. These results show that 1‐undecene derived from P. aeruginosa serves as a pathogen‐associated molecular pattern for the induction of protective responses in C. elegans.     

Effects of Multidisciplinary Rehabilitation on Chronic Fatigue in Multiple Sclerosis: A Randomized Controlled Trial

Background

Several rehabilitation programmes aim at reducing the impact of fatigue in MS patients. Acute and chronic fatigue should require different management.

Objectives

To assess the effects of individually tailored, multidisciplinary outpatient rehabilitation (MDR) on chronic fatigue.

Methods

Forty-eight ambulatory MS patients with chronic fatigue were randomized to MDR or to MS–nurse consultation. Fatigue was assessed by the Checklist Individual Strength (CIS-20R). Secondary outcomes included the Modified Fatigue Impact Scale, Fatigue Severity Scale, Functional Independence Measure, Disability and Impact Profile (DIP), Multiple Sclerosis Impact Scale and the Impact on Participation and Autonomy (IPA).

Results

The primary outcome measure CIS-20R overall score showed no significant differences between groups at 12 weeks (P = 0.39) and 24 weeks follow-up (P = 0.14), nor for subscales (t = 12 and t = 24, 0.19≤P≤0.88). No significant within-group effects were found for both groups with respect to the primary (0.57≤p≤0.97) and secondary (0.11≤p≤0.92) outcome measures from baseline to 12 or 24 weeks.

Conclusion

Multidisciplinary rehabilitation was not more effective in terms of reducing self-reported fatigue in MS patients compared to MS-nurse consultation. Our results suggest that chronic fatigue in patients with MS may be highly invariant over time, irrespective of interventions.

Trial Registration

controlled-trials.com ISRCTN05017507     

Mutagenicity of the mycotoxin diacetoxyscirpenol on somatic and germ cells of mice

Genotoxicity of diacetoxyscirpenol (DAS) was studied on laboratory mice after intraperitoneal injection with single and repeated doses. DAS was administrated at three different dose levels (0.5, 0.75, and 1.0 mg/kg body weight). The study was conducted on both somatic and germ cells additional to the sperm morphology analysis. DAS treatment resulted in a significant reduction (P<0.01) in mitotic activity at all levels of doses tested, confirming that DAS is a potent protein and DNA synthesis inhibitor. At somatic cells (bone marrow) both structural and numerical chromosome abnormalities were observed. Single dose treatment showed significant abnormalities only with high dose treatment. In contrast, at repeated dose similar abnormalities were also observed with some significance but no systematic relation between the administrated dose and abnormalities ratio could be settled. In germ cells (testicles), structural and numerical abnormalities were also observed. In general, the frequencies of scored abnormalities at germ cells were lower than that the somatic cells. Sperm count test revealed a decrease in the number of released sperm after toxin treatment. Abnormalities of sperm shape (head and tail) were observed, confirming the positive correlation between cytogenetic damage and sperm abnormality. The results also proved that DAS is a very toxic mycotoxin, in addition to inducing chromosomal abnormalities, it causes a severe inhibition of DNA synthesis which subsequently affects the cell cycle and cell division. A good system for good harvesting practice and good food technology can lower the risk for the consumers.     

Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Regulatory effects of eotaxin on acute lung inflammatory injury

Eotaxin, which is a major mediator for eosinophil recruitment into lung, has regulatory effects on neutrophil-dependent acute inflammatory injury triggered by intrapulmonary deposition of IgG immune complexes in rats. In this model, eotaxin mRNA and protein were up-regulated during the inflammatory response, resulting in eotaxin protein expression in alveolar macrophages and in alveolar epithelial cells. Ab-induced blockade of eotaxin in vivo caused enhanced NF-kappaB activation in lung, substantial increases in bronchoalveolar lavage levels of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC), and increased MIP-2 and CINC mRNA expression in alveolar macrophages. In contrast, TNF-alpha levels were unaffected, and IL-10 levels fell. Under these experimental conditions, lung neutrophil accumulation was significantly increased, and vascular injury, as reflected by extravascular leak of (125)I-albumin, was enhanced. Conversely, when recombinant eotaxin was administered in the same inflammatory model of lung injury, bronchoalveolar lavage levels of MIP-2 were reduced, as was neutrophil accumulation and the intensity of lung injury. In vitro stimulation of rat alveolar macrophages with IgG immune complexes greatly increased expression of mRNA and protein for MIP-2, CINC, MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta. In the copresence of eotaxin, the increased levels of MIP-2 and CINC mRNAs were markedly diminished, whereas MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta expression of mRNA and protein was not affected. These data suggest that endogenous eotaxin, which is expressed during the acute lung inflammatory response, plays a regulatory role in neutrophil recruitment into lung and the ensuing inflammatory damage.     

In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

Background

The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.

Results

A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.

Conclusion

In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users.