First insights into female sperm storage duration in tardigrades

Female sperm storage is ubiquitous in the animal kingdom and it has been shown to be linked to several evolutionary processes, from postcopulatory sexual selection to dispersal. Here we report, for the first time, long‐term sperm storage in females of the tardigrade Macrobiotus polonicus. Females, isolated after a short contact with a male, were able to use the stored sperm for up to 5 weeks (mean of 2 weeks), which translates to a considerable proportion of female post‐mating longevity under controlled laboratory conditions (60% on average). Our study provides the first insights into the duration of sperm storage, an underexplored feature of the reproductive biology of tardigrades. Additionally, we discuss important considerations for reproductive studies on these non‐model animals.     

Intensity of nest defence is not related to degree of paternity in the willow tit Parus montanus

We asked whether willow tit Parus montanus males adjust their parental care according to their paternity in current brood. The origin of the nestlings was determined by using molecular technique, and the studied broods were assigned into extra-pair paternity (EPP) broods, if at least one nestling was fathered by another male, and truly monogamous broods. Over 3  years, 14 of 40  broods (35%) included EP-offspring, and 29 of 273  nestlings (11%) were EP-young. Intensity of parental care was measured with risk-taking against a potential predator, mounted stoat Mustela erminea . The results showed that risk-taking by EPP males did not differ from that by monogamous males. Neither was the sexual difference in risk-taking different at EPP and monogamous broods. Our results are consistent with the hypothesis that males do not adjust their level of care to paternity, perhaps because they have no reliable cues for assessing their paternity. This may be related to the success of mate-guarding in their breeding environment, closed forests. Guarding is seemingly successful as the EPP levels are rather low, but it is not totally sure making the potential costs, rejection of own young, too high. We also discuss other population characteristics which may further prevent the evolution of paternity assessment in northern willow tits.     

The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket

OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, K(d)=9.7x10(-8) M; ORP1S, K(d)=8.4 x10(-8) M), while the affinity of GST-ORP2 for 25OH was lower (K(d)=3.9x10(-6) M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand-binding domains of the ORPs or the full-length proteins, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.     

Serum, but not monocyte macrophage foam cells derived from low HDL-C subjects, displays reduced cholesterol efflux capacity

The main antiatherogenic function of HDL is to promote the efflux of cholesterol from peripheral cells and transport it to the liver for excretion in a process termed reverse cholesterol transport. The aim of this study was to evaluate the cholesterol efflux capacity in low- and high-HDL subjects by utilizing monocytes and serum from 18 low-HDL and 15 high-HDL subjects. Low and high HDL levels were defined, respectively, as HDL < or =10(th) and HDL > or =90(th) Finnish age/sex-specific percentile. Cholesterol efflux from [(3)H]cholesterol-oleate-acetyl-LDL-loaded monocyte-derived macrophages to standard apolipoprotein A-I (apoA-I), HDL(2), and serum was measured. In addition, cholesterol efflux from acetyl-LDL-loaded human THP-1 macrophages to individual sera (0.5%) derived from the study subjects was evaluated. Cholesterol efflux to apoA-I, HDL(2), and serum from macrophage foam cells derived from low- and high-HDL subjects was similar. The relative ABCA1 and ABCG1 mRNA expression levels in unloaded macrophages, as well as their protein levels in loaded macrophage foam cells, were similar in the two study groups. Cholesterol efflux from THP-1 foam cells to serum recovered from high-HDL subjects was slightly higher than that to serum from low-HDL subjects (P = 0.046). Cholesterol efflux from THP-1 macrophages to serum from study subjects correlated with serum apoB (P = 0.033), apoA-I (P = 0.004), apoA-II (P < 0.0001), and the percentage of apoA-I present in the form of prebeta-HDL (P = 0.0001). Our data reveal that macrophages isolated from either low- or high-HDL subjects display similar cholesterol efflux capacity to exogenous acceptors. However, sera from low-HDL subjects have poorer cholesterol acceptor ability as compared with sera from high-HDL subjects.     

Daily impact score in long-term acceleration measurements of exercise

Mechanical loading increases and maintains bone mass and strength. Daily stress stimulus and osteogenic index theories have been suggested to describe the osteogenic potential of exercise, using exponential or logarithmic relationships, respectively, between loading numbers and magnitude. Inspired by these theories, the aim of this study was to develop and test a daily impact score (DIS) using long-term continuous acceleration measurements of exercise.Acceleration data were collected during a previous exercise trial, in which the subjects (healthy women, 35–40 years, N=34 in the high-impact exercise group and N=30 in the control group) wore a body movement monitor on their waist during the 12-month study. DIS was calculated from the 12-month average daily acceleration distributions in two ways: DIS_Exp adopted from the daily stress stimulus and DIS_Log simplified from the osteogenic index. Areal bone mineral density (aBMD) at the proximal femur and cortical bone geometry at the mid-femur were measured at baseline and 12 months.DIS calculated in either of the ways was significantly higher in the exercise group than in the control group. DIS_Exp and DIS_Log were strongly correlated (R=0.982). Both DIS_Exp and DIS_Log were significantly associated with 12-month aBMD changes at the hip (R up to 0.550, p<0.01) and geometry changes at the mid-femur (R up to 0.472, p<0.05) in the exercise group.DIS calculated either from exponential or logarithmic relationship can be used in acceleration-based measurements of daily exercise. DIS was positively related with changes in hip aBMD and mid-femur bone geometry after 12 months of exercise.     

Glutamine synthetase and glucose-6-phosphate isomerase are adhesive moonlighting proteins of Lactobacillus crispatus released by epithelial cathelicidin LL-37

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.     

The shift in plant species composition in a subarctic mountain birch forest floor due to climate change would modify the biogenic volatile organic compound emission profile

Background and aims

Mountain birch forests dominate in the Subarctic but little is known of their non-methane biogenic volatile organic compound (BVOC) emissions. The dwarf shrubs Empetrum hermaphroditum, Vaccinium myrtillus and Vaccinium uliginosum co-dominate in the forest floors of these forests. The abundance of these three dwarf shrubs relative to each other could be affected by climate warming expected to increase nutrient availability by accelerating litter decomposition and nutrient mineralization. We 1) compared the BVOC emission profiles of vegetation covers dominated by E. hermaphroditum and V. myrtillus plus V. uliginosum in a subarctic mountain birch forest floor, 2) distinguished the BVOCs emitted from plants and soil and 3) measured how the BVOC emissions from the different vegetation covers differed under darkness.

Methods

BVOCs were sampled during two growing seasons using a conventional ecosystem chamber-based method, collected on adsorbent and analyzed with gas chromatography–mass spectrometry.

Results

High abundance of E. hermaphroditum increased the sesquiterpene emissions. Soil released fewer different BVOCs than controls (i.e. natural vegetation) but the total emission rates were similar. Darkness did not affect the emissions. Carbon emitted as BVOCs was less than 0.2% of the CO₂ exchange.

Conclusions

Our results suggest that sesquiterpene emissions from subarctic mountain birch forest floors would be reduced following an increased abundance of V. myrtillus and V. uliginosum with climate change because these species respond rapidly to increased nutrient availability.     

Origin, structure, and biological activities of peroxidases in human saliva

Human whole saliva contains two peroxidases, salivary peroxidase (hSPO) and myeloperoxidase (hMPO), which are part of the innate host defence in oral cavity. Both hSPO as well as human milk lactoperoxidase (hLPO) are coded by the same gene, but to what extent the different producing glands, salivary and mammary glands, affect the final conformation of the enzymes is not known. In human saliva the major function of hSPO and hMPO is to catalyze the oxidation of thiocyanate (SCN(-)) in the presence of hydrogen peroxide (H(2)O(2)) resulting in end products of wide antimicrobial potential. In addition cytotoxic H(2)O(2) is degraded. Similar peroxidation reactions inactivate some mutagenic and carcinogenic compounds, which suggests another protective mechanism of peroxidases in human saliva. Although being target of an active antimicrobial research, the structure-function relationships of hSPO are poorly known. However, recently published method for recombinant hSPO production offers new tools for those investigations.     

OSBP-related protein 2 is a sterol receptor on lipid droplets that regulates the metabolism of neutral lipids

Oxysterol binding protein-related protein 2 (ORP2) is a member of the oxysterol binding protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism. We show here that ORP2 also binds 22(R)-hydroxycholesterol [22(R)OHC], 7-ketocholesterol, and cholesterol, with 22(R)OHC being the highest affinity ligand of ORP2 (K_d 1.4 × 10⁻⁸ M). We report the localization of ORP2 on cytoplasmic lipid droplets (LDs) and its function in neutral lipid metabolism using the human A431 cell line as a model. The ORP2 LD association depends on sterol binding: Treatment with 5 μM 22(R)OHC inhibits the LD association, while a mutant defective in sterol binding is constitutively LD bound. Silencing of ORP2 using RNA interference slows down cellular triglyceride hydrolysis. Furthermore, ORP2 silencing increases the amount of [¹⁴C]cholesteryl esters but only under conditions in which lipogenesis and LD formation are enhanced by treatment with oleic acid. The results identify ORP2 as a sterol receptor present on LD and provide evidence for its role in the regulation of neutral lipid metabolism, possibly as a factor that integrates the cellular metabolism of triglycerides with that of cholesterol.