SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization

Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2' hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem-loops, extensive long-range interactions (LRIs) and a small, palindromic stem-loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem-loops are static structures, the 5' and 3' regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem-loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs.     

Cushions of Thylacospermum caespitosum (Caryophyllaceae) do not facilitate other plants under extreme altitude and dry conditions in the north-west Himalayas

Background

Cushion plants are commonly considered as keystone nurse species that ameliorate the harsh conditions they inhabit in alpine ecosystems, thus facilitating other species and increasing alpine plant biodiversity. A literature search resulted in 25 key studies showing overwhelming facilitative effects of different cushion plants and hypothesizing greater facilitation with increased environmental severity (i.e. higher altitude and/or lower rainfall). At the same time, emerging ecological theory alongside the cushion-specific literature suggests that facilitation might not always occur under extreme environmental conditions, and especially under high altitude and dryness.

Methods

To assess these hypotheses, possible nursing effects of Thylacospermum caespitosum (Caryophyllaceae) were examined at extremely high altitude (5900 m a.s.l.) and in dry conditions (precipitation <100 mm year⁻¹) in Eastern Ladakh, Trans-Himalaya. This is, by far, the highest site, and the second driest, at which the effects of cushions have been studied so far.

Key Results

In accordance with the theoretical predictions, no nursing effects of T. caespitosum on other alpine plants were detected. The number and abundance of species were greater outside cushions than within and on the edge of cushions. None of the 13 species detected was positively associated with cushions, while nine of them were negatively associated. Plant diversity increased with the size of the area sampled outside cushions, but no species–area relationship was found within cushions.

Conclusions

The results support the emerging theoretical prediction of restricted facilitative effects under extreme combinations of cold and dryness, integrating these ideas in the context of the ecology of cushion plants. This evidence suggests that cases of missing strong facilitation are likely to be found in other extreme alpine conditions.     

α1-Antichymotrypsin inactivates staphylococcal cysteine protease in cross-class inhibition

Staphylococcal cysteine proteases are implicated as virulence factors in human and avian infections. Human strains of Staphylococcus aureus secrete two cysteine proteases (staphopains A and B), whereas avian strains express staphopain C (ScpA2), which is distinct from both human homologues. Here, we describe probable reasons why the horizontal transfer of a plasmid encoding staphopain C between avian and human strains has never been observed. The human plasma serine protease inhibitor α₁-antichymotrypsin (ACHT) inhibits ScpA2. Together with the lack of ScpA2 inhibition by chicken plasma, these data may explain the exclusively avian occurrence of ScpA2. We also clarify the mechanistic details of this unusual cross-class inhibition. Analysis of mutated ACHT variants revealed that the cleavage of the Leu383-Ser384 peptide bond results in ScpA2 inhibition, whereas hydrolysis of the preceding peptide bond leads to ACHT inactivation. This evidence is consistent with the suicide-substrate-like mechanism of inhibition.     

Induction of hyperglycemia in adult intrauterine growth-restricted rats: effects on renal function

Intrauterine growth restriction (IUGR) leads to a reduction in nephron endowment at birth and is linked to renal dysfunction in adulthood. The aim of the present study was to determine whether kidneys of IUGR rat offspring are more vulnerable to a secondary insult of hyperglycemia. IUGR was induced in Wistar-Kyoto rats by maternal protein restriction. At 24 wk of age, diabetes was induced in male IUGR and non-IUGR offspring by streptozotocin injection; insulin was injected daily to maintain blood glucose levels at either a mild (7-10 mmol/l; n=8/group) or a moderate (10-15 mmol/l; n=8/group) level. At 32 wk of age, renal function was assessed using ultrasound and [(3)H]inulin and [(14)C]para-aminohippurate clearance techniques. Conscious mean arterial blood pressure and heart rate were unchanged in IUGR offspring. Relative kidney length was increased significantly in IUGR offspring, and renal function was altered significantly; of importance, there was a significant increase in filtration fraction, indicative of glomerular hyperfiltration. Induction of hyperglycemia led to marked impairment of renal function. However, the response to hyperglycemia was not different between IUGR and non-IUGR offspring. Maintaining blood glucose levels at a mild hyperglycemic level led to marked improvement in all measures of renal function in IUGR and non-IUGR offspring. In conclusion, while the IUGR offspring showed evidence of hyperfiltration, the response to hyperglycemia was similar in IUGR and non-IUGR kidneys in adulthood. Importantly, maintaining blood glucose levels at a mild hyperglycemic level markedly attenuated the renal dysfunction associated with diabetes, even in IUGR offspring.     

In vitro analysis of iron chelating activity of flavonoids

Flavonoids have been demonstrated to possess miscellaneous health benefits which are, at least partly, associated with iron chelation. In this in vitro study, 26 flavonoids from different subclasses were analyzed for their iron chelating activity and stability of the formed complexes in four patho/physiologically relevant pH conditions (4.5, 5.5, 6.8, and 7.5) and compared with clinically used iron chelator deferoxamine. The study demonstrated that the most effective iron binding site of flavonoids represents 6,7-dihydroxy structure. This site is incorporated in baicalein structure which formed, similarly to deferoxamine, the complexes with iron in the stoichiometry 1:1 and was not inferior in all tested pH to deferoxamine. The 3-hydroxy-4-keto conformation together with 2,3-double bond and the catecholic B ring were associated with a substantial iron chelation although the latter did not play an essential role at more acidic conditions. In agreement, quercetin and myricetin possessing all three structural requirements were similarly active to baicalein or deferoxamine at the neutral conditions, but were clearly less active in lower pH. The 5-hydroxy-4-keto site was less efficient and the complexes of iron in this site were not stable at the acidic conditions. Isolated keto, hydroxyl, methoxyl groups or an ortho methoxy-hydroxy groups were not associated with iron chelation at all.     

Changes in intervertebral disc morphology persist 5 mo after 21-day bed rest

As part of the nutrition-countermeasures (NUC) study in Cologne, Germany in 2010, seven healthy male subjects underwent 21 days of head-down tilt bed rest and returned 153 days later to undergo a second bout of 21-day bed rest. As part of this model, we aimed to examine the recovery of the lumbar intervertebral discs and muscle cross-sectional area (CSA) after bed rest using magnetic resonance imaging and conduct a pilot study on the effects of bed rest in lumbar muscle activation, as measured by signal intensity changes in T(2)-weighted images after a standardized isometric spinal extension loading task. The changes in intervertebral disc volume, anterior and posterior disc height, and intervertebral length seen after bed rest did not return to prebed-rest values 153 days later. While recovery of muscle CSA occurred after bed rest, increases (P ≤ 0.016) in multifidus, psoas, and quadratus lumborum muscle CSA were seen 153 days after bed rest. A trend was seen for greater activation of the erector spinae and multifidus muscles in the standardized loading task after bed rest. Greater reductions of multifidus and psoas CSA muscle and greater increases in multifidus signal intensity with loading were associated with incidence of low back pain in the first 28 days after bed rest (P ≤ 0.044). The current study contributes to our understanding of the recovery of the lumbar spine after 21-day bed rest, and the main finding was that a decrease in spinal extensor muscle CSA recovers within 5 mo after bed rest but that changes in the intervertebral discs persist.     

Analysis of the signaling pathways regulating Src-dependent remodeling of the actin cytoskeleton

Cell adhesion to the extracellular matrix is mediated by adhesion receptors, mainly integrins, which upon interaction with the extracellular matrix, bind to the actin cytoskeleton via their cytoplasmic domains. This association is mediated by a variety of scaffold and signaling proteins, which control the mechanical and signaling activities of the adhesion site. Upon transformation of fibroblasts with active forms of Src (e.g., v-Src), focal adhesions are disrupted, and transformed into dot-like contacts known as podosomes, and consisting of a central actin core surrounded by an adhesion ring. To clarify the mechanism underlying Src-dependent modulation of the adhesive phenotype, and its influence on podosome organization, we screened for the effect of siRNA-mediated knockdown of tyrosine kinases, MAP kinases and phosphatases on the reorganization of the adhesion-cytoskeleton complex, induced by a constitutively active Src mutant (SrcY527F). In this screen, we discovered several genes that are involved in Src-induced remodeling of the actin cytoskeleton. We further showed that knockdown of Src in osteoclasts abolishes the formation of the podosome-based rings and impairs cell spreading, without inducing stress fiber development. Our work points to several genes that are involved in this process, and sheds new light on the molecular plasticity of integrin adhesions.     

Pregnancy and periodontal tissues

Periodontitis is today considered to be a serious disease of periodontal tissues, one caused in most cases by bacterial infection which stimulates proteolysis and osteolysis of the tissues. Typical for the disease is formation of periodontal pockets and a chronic destructive inflammation which impacts on the whole organism. Periodontopathic bacteria colonized in a subgingival biofilm cannot be removed by common oral hygiene. Overproduction of bacteria and other pro-inflammatory mediators can increase the total pro-inflammatory state of the organism in pregnant women. Increased levels of some pro-inflammatory cytokines (PGE2) and cells in fetoplacental space can lead to premature rupture of membranes and subsequent delivery of immature babies. An increasing number of studies in this field provide evidence that good professional care and personal oral hygiene can bring benefits through a decreased prevalence of preterm low birth weight infants (PLBWI) in women suffering periodontitis, although definitive conclusions have not yet been reached. Future mothers with periodontitis can run not only an increased risk of PLWBI but often also suffer pre-eclampsia - a state called acute atherosis - which can be ethiopathogenetically associated with high concentrations of various pro-inflammatory mediators. An increased production of female hormones during pregnancy contributes to the development of gingivitis and periodontitis because vascular permeability and possible tissue edema are both increased.