RT-qPCR reveals opsin gene upregulation associated with age and sex in guppies (Poecilia reticulata) - a species with color-based sexual selection and 11 visual-opsin genes

Background  

PCR-based surveys have shown that guppies (Poecilia reticulata) have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system.     

Behavioural adjustment in response to increased predation risk: a study in three duck species

Predation directly triggers behavioural decisions designed to increase immediate survival. However, these behavioural modifications can have long term costs. There is therefore a trade-off between antipredator behaviours and other activities. This trade-off is generally considered between vigilance and only one other behaviour, thus neglecting potential compensations. In this study, we considered the effect of an increase in predation risk on the diurnal time-budget of three captive duck species during the wintering period. We artificially increased predation risk by disturbing two groups of 14 mallard and teals at different frequencies, and one group of 14 tufted ducks with a radio-controlled stressor. We recorded foraging, vigilance, preening and sleeping durations the week before, during and after disturbance sessions. Disturbed groups were compared to an undisturbed control group. We showed that in all three species, the increase in predation risk resulted in a decrease in foraging and preening and led to an increase in sleeping. It is worth noting that contrary to common observations, vigilance did not increase. However, ducks are known to be vigilant while sleeping. This complex behavioural adjustment therefore seems to be optimal as it may allow ducks to reduce their predation risk. Our results highlight the fact that it is necessary to encompass the whole individual time-budget when studying behavioural modifications under predation risk. Finally, we propose that studies of behavioural time-budget changes under predation risk should be included in the more general framework of the starvation-predation risk trade-off.     

Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

Background

Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.

Methods

We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).

Results

Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.

Conclusions

Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.     

Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens

We compared the NaOH-N-acetyl cysteine (NaOH-NALC) and the sulfuric acid decontamination procedure in the detection of mycobacteria using the Mycobacteria Growth Indicator Tube (MGIT). In total 219 sputum specimens were collected from 142 Zambian patients and subjected to mycobacterial culture. One half of the specimen was decontaminated with NaOH-NALC and the other half was decontaminated with sulfuric acid. From the 438 samples a total of 261 (60%) cultures yielded growth of mycobacteria, consisting of 22 different species. The sulfuric acid method was more successful than the NaOH-NALC method in recovering mycobacteria in MGITs (146 versus 115 respectively, p = 0.001). Of the 146 positive mycobacterial cultures recovered after sulfuric acid decontamination 28 were Mycobacterium tuberculosis, 84 nontuberculous mycobacteria (NTM) and 34 acid fast bacterial isolates which could not be identified to the species level. The 115 mycobacteria recovered by the NaOH-NALC method consisted of 34 M. tuberculosis strains, 55 NTM and 26 acid fast bacteria that could not be identified. The most frequently isolated NTM were Mycobacterium lentiflavum and Mycobacterium intracellulare. Comparing the two decontamination methods the recovery of NTM in the sulfuric acid group was significant higher than in the NaOH-NALC group (p = 0.001). In contrast, no significant difference was found for the recovery of M. tuberculosis. These results show that the decontamination method used affects the recovery of nontuberculous mycobacteria in particular.     

Covert human immunodeficiency virus replication in dendritic cells and in DC-SIGN-expressing cells promotes long-term transmission to lymphocytes

HIV-1 virions are efficiently captured by monocyte-derived immature dendritic cells (iDCs), as well as by cell lines expressing the lectin DC-SIGN. Viral infectivity can be retained for several days, and even enhanced, before transmission to CD4+ lymphocytes. The role of DC-SIGN in viral retention and enhancement of infection is not fully understood and varies according to the cell line expressing the lectin. We studied here the mechanisms underlying this process. We focused our study on X4-tropic human immunodeficiency virus (HIV) strains, since they were widely believed not to replicate in iDCs. However, we first show that X4 HIV replicates covertly and slowly in iDCs. This is also the case in Raji-DC-SIGN cells, which are classically used to study HIV transmission. We used either single-cycle or replicative HIV and measured viral RT and replication to further demonstrate that transfer of incoming virions from iDCs or DC-SIGN+ cells occurs only on the short-term (i.e., a few hours after viral exposure). There is no long-term storage of original HIV particles in these cells. A few days after viral exposure, replicative viruses, and not single-cycle virions, are transmitted to CD4+ cells. The cell-type-dependent activity of DC-SIGN reflects the ability of HIV to replicate covertly in some cells, and not in others.     

A universal primer set for PCR amplification of nuclear histone H4 genes from all animal species

To control the quality of genomic DNA of samples from a wide variety of animals, a heminested PCR assay specifically targeting a nuclear gene has been developed. The histone H4 gene family comprises a small number of genes considered among the most conserved genes in living organisms. Tissue samples from necropsies and from cells belonging to 43 different species were studied, eight samples from invertebrates and 35 samples from vertebrates covering all classes. Ancient DNA samples from three Siberian woolly mammoths (Mammuthus primigenius) dating between 40,000 and 49,000 years before present were also tested for PCR amplification. Performance of HIST2H4 amplification were also compared with those of previously published universal PCRs (28S rRNA, 18S rRNA, and cytochrome b). Overall, 95% of species studied yielded an amplification product, including some old samples from gorilla and chimpanzees. The data indicate that the HIST2H4 amplimers are, thus, suitable for both DNA quality testing as well as species identification in the animal kingdom.     

Role of cardiolipin on tBid and tBid/Bax synergistic effects on yeast mitochondria

The apoptotic effector Bid regulates cell death at the level of mitochondria. Under its native state, Bid is a soluble cytosolic protein that undergoes proteolysis and yields a 15 kDa-activated form tBid (truncated Bid). tBid translocates to mitochondria and participates in cytochrome c efflux by a still unclear mechanism, some of them at least mediated by Bax. Using mitochondria isolated from wild-type and cardiolipin (CL)-synthase-less yeast strains, we observed that tBid perturbs mitochondrial bioenergetics by inhibiting state-3 respiration and ATP synthesis and that this effect was strictly dependent on the presence of CL. In a second set of experiments, heterologous coexpression of tBid and Bax in wild-type and CL-less yeast strains showed that (i) tBid binding and the subsequent alteration of mitochondrial bioenergetics increased Bax-induced cytochrome c release and (ii) the absence of CL favors Bax effects independently of the presence of t-Bid. These data support recent views suggesting a dual function of CL in mitochondria-dependent apoptosis.     

tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.