Shelterin dysfunction and p16(INK4a)-mediated growth inhibition in HIV-1-specific CD8 T cells

HIV-1-specific cytotoxic T cell responses are expanded during advanced HIV-1 infection but seem unable to effectively protect the host against disease progression. These cells are able to produce gamma interferon and remain metabolically active but have defective proliferative activities, shortened telomeric DNA, and other signs of accelerated aging. To investigate the molecular mechanisms underlying the premature senescence of HIV-1-specific T cells, we focused here on the expression and function of a group of six nucleoproteins that are responsible for protecting and maintaining the structural integrity of telomeric DNA and are commonly referred to as "shelterin." We show that in progressive HIV-1 infection, the two major shelterin components TRF2 and TPP1 are selectively reduced in HIV-1-specific CD8 T cells, but not in T cells recognizing alternative viral species. This coincided with increased recruitment of 53BP1, a prominent DNA damage response factor, to telomeric DNA sites and was associated with elevated expression of the tumor suppressor p16(INK4a), which causes cellular growth inhibition in response to structural DNA damage. Notably, defective shelterin function and upregulation of p16(INK4a) remained unaffected by experimental blockade of PD-1, indicating a possibly irreversible structural defect in HIV-1-specific CD8 T cells in progressors that cannot be overcome by manipulation of inhibitory cell-signaling pathways. These data suggest that shelterin dysfunction and ensuing upregulation of the tumor suppressor p16(INK4a) promote accelerated aging of HIV-1-specific T cells during progressive HIV-1 infection.     

Mangrove Habitat Use by Juvenile Reef Fish: Meta-Analysis Reveals that Tidal Regime Matters More than Biogeographic Region

Identification of critical life-stage habitats is key to successful conservation efforts. Juveniles of some species show great flexibility in habitat use while other species rely heavily on a restricted number of juvenile habitats for protection and food. Considering the rapid degradation of coastal marine habitats worldwide, it is important to evaluate which species are more susceptible to loss of juvenile nursery habitats and how this differs across large biogeographic regions. Here we used a meta-analysis approach to investigate habitat use by juvenile reef fish species in tropical coastal ecosystems across the globe. Densities of juvenile fish species were compared among mangrove, seagrass and coral reef habitats. In the Caribbean, the majority of species showed significantly higher juvenile densities in mangroves as compared to seagrass beds and coral reefs, while for the Indo-Pacific region seagrass beds harbored the highest overall densities. Further analysis indicated that differences in tidal amplitude, irrespective of biogeographic region, appeared to be the major driver for this phenomenon. In addition, juvenile reef fish use of mangroves increased with increasing water salinity. In the Caribbean, species of specific families (e.g. Lutjanidae, Haemulidae) showed a higher reliance on mangroves or seagrass beds as juvenile habitats than other species, whereas in the Indo-Pacific family-specific trends of juvenile habitat utilization were less apparent. The findings of this study highlight the importance of incorporating region-specific tidal inundation regimes into marine spatial conservation planning and ecosystem based management. Furthermore, the significant role of water salinity and tidal access as drivers of mangrove fish habitat use implies that changes in seawater level and rainfall due to climate change may have important effects on how juvenile reef fish use nearshore seascapes in the future.     

Ecological correlates of species richness and population abundance patterns in the amphibian communities from the Albertine Rift, East Africa

Broad-scale ecological correlates affecting species richness and abundance patterns of amphibians were studied in 37 sampling sites from 15 independent protected areas in the Albertine Rift, East Africa. Amphibians were caught by a combination of sampling techniques, including time-constrained visual searching, pitfalls with drift fences, dip-netting, and opportunistic observations. In total, 73 species of amphibians were recorded, some of them occurring only once in the samples and some more than 1,000 times. Once relative field effort at each study site was taken into account, multivariate regression analyses showed that amphibian species richness was positively influenced by percent canopy cover and altitude. On the other hand, population abundance was influenced by a combination of three different variables: positively by percent canopy cover and rainfall, and negatively by human activities. The rainforest proved to play a very important role in the ecological distribution of species, with about 63% of the species being in one way or the other linked to it either as forest specialists, inhabitants of forest ecotones, or inhabitants of both forests and forest ecotones. These results have wide conservation implications. Indeed, as the mature rainforest habitat (especially at mid altitude-highland) proved to be the key habitat for the amphibian communities of the Albertine Rift, it should be a priority to recommend an accurate management and conservation of mature forests in local and regional protection programs.     

Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine

The peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring Cu(II)-chelating motifs in human serum and cerebrospinal fluid. Here, the sensitive thermodynamic technique isothermal titration calorimetry was used to study the energetics of Cu(II) binding to DAHK and GHK peptides in the presence of the weaker ligand glycine as a competitor. DAHK and GHK bind Cu(II) predominantly in a 1:1 stoichiometry with conditional dissociation constants [i.e., at pH 7.4, in the absence of the competing chelators glycine and 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid buffer] of 2.6 ± 0.4 × 10⁻¹⁴ M and 7.0 ± 1.0 × 10⁻¹⁴ M, respectively. Furthermore, the apparent ΔH values were measured and the number of protons released upon Cu(II) binding was determined by performing experiments in different buffers. This allowed us to determine the conditional ΔG, ΔH, and ΔS, i.e., corrected for the contributions of the weaker ligand glycine and the buffer at pH 7.4. We found that the entropic and enthalpic contributions to the Cu(II) binding to GHK and DAHK are distinct, with a enthalpic contribution for GHK. The thermodynamic parameters obtained correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in isothermal titration calorimetry provides accurate data for Cu(II) binding to high-affinity peptides, which cannot be accurately determined without the use of a competitor ligand.     

Genetic Determinants of Volatile-Thiol Release by Saccharomyces cerevisiae during Wine Fermentation

Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation, Saccharomyces cerevisiae mediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.     

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni²⁺-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering.     

Noninvasive,In Vivo Assessment of Mouse Retinal Structure Using Optical Coherence Tomography

Background

Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration.

Methodology/Principal Findings

We achieved to adapt a commercial 3^rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber''s congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified.

Conclusions/Significance

We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.     

Uncovering the Nutritional Landscape of Food

Recent progresses in data-driven analysis methods, including network-based approaches, are revolutionizing many classical disciplines. These techniques can also be applied to food and nutrition, which must be studied to design healthy diets. Using nutritional information from over 1,000 raw foods, we systematically evaluated the nutrient composition of each food in regards to satisfying daily nutritional requirements. The nutrient balance of a food was quantified and termed nutritional fitness; this measure was based on the food’s frequency of occurrence in nutritionally adequate food combinations. Nutritional fitness offers a way to prioritize recommendable foods within a global network of foods, in which foods are connected based on the similarities of their nutrient compositions. We identified a number of key nutrients, such as choline and α-linolenic acid, whose levels in foods can critically affect the nutritional fitness of the foods. Analogously, pairs of nutrients can have the same effect. In fact, two nutrients can synergistically affect the nutritional fitness, although the individual nutrients alone may not have an impact. This result, involving the tendency among nutrients to exhibit correlations in their abundances across foods, implies a hidden layer of complexity when exploring for foods whose balance of nutrients within pairs holistically helps meet nutritional requirements. Interestingly, foods with high nutritional fitness successfully maintain this nutrient balance. This effect expands our scope to a diverse repertoire of nutrient-nutrient correlations, which are integrated under a common network framework that yields unexpected yet coherent associations between nutrients. Our nutrient-profiling approach combined with a network-based analysis provides a more unbiased, global view of the relationships between foods and nutrients, and can be extended towards nutritional policies, food marketing, and personalized nutrition.     

Real-time pure shift <Superscript>15</Superscript>N HSQC of proteins: a real improvement in resolution and sensitivity

Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t ₂) broadband homodecoupling, suppressing the multiplet structure caused by proton–proton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to ¹⁵N, while the former selects a region of the ¹H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments.