Cis-regulatory evolution spotlights species differences in the adaptive potential of gene expression plasticity.

Phenotypic plasticity is the variation in phenotype that a single genotype can produce in different environments and, as such, is an important component of individual fitness. However, whether the effect of new mutations, and hence evolution, depends on the direction of plasticity remains controversial. Here, we identify the cis-acting modifications that have reshaped gene expression in response to dehydration stress in three Arabidopsis species. Our study shows that the direction of effects of most cis-regulatory variants differentiating the response between A. thaliana and the sister species A. lyrata and A. halleri depends on the direction of pre-existing plasticity in gene expression. A comparison of the rate of cis-acting variant accumulation in each lineage indicates that the selective forces driving adaptive evolution in gene expression favors regulatory changes that magnify the stress response in A. lyrata. The evolutionary constraints measured on the amino-acid sequence of these genes support this interpretation. In contrast, regulatory changes that mitigate the plastic response to stress evolved more frequently in A. halleri. Our results demonstrate that pre-existing plasticity may be a stepping stone for adaptation, but its selective remodeling differs between lineages.

Chicken eggshell powder more effectively alleviates bone loss comparted to inorganic calcium carbonate: an animal study performed on ovariectomized rats.

This study aimed to evaluate the effects of chicken eggshell powder rich in calcium and the inorganic form of calcium carbonate on osteoporotic bone structure using an animal model of ovariectomized rats. Animals were divided into four groups: sham-operated rats (SHAM group); ovariectomized rats untreated (OVX group); OVX rats treated with eggshell powder (Biomin H® OVX + ECa group; total Ca content 1.5%); and OVX rats receiving inorganic calcium carbonate (calcium carbonate precipitated, total Ca content 1.5%; OVX + ICa group) during 8 weeks. Ovariectomy increased total body weight, plasma alkaline phosphatase activity, cortical bone thickness, periosteal bone apposition, and considerably worsened the trabecular bone microarchitecture. Calcium supplementation in both OVX + ECa and OVX + ICa groups elevated blood supply in the cortical bone when compared to OVX rats. Treatment with eggshell powder, unlike the OVX + ICa group, significantly lowered bone resorption, increased plasma calcium level, relative volume of trabecular bone, and secondary osteon population density, which together contributes to the enhanced bone strength. In addition, sizes of Haversian canals and secondary osteons in OVX + ECa group reached the values of SHAM group. Our results suggest that chicken eggshell powder more effectively ameliorates bone loss in ovariectomized rats than precipitated calcium carbonate.

Subacute exposure to amygdalin influences compact bone remodeling of rabbits.

Amygdalin is most commonly occurring cyanogenic glycoside. It is found in seeds of many plant species. Our study was aimed to reveal whether pure intramuscularly injected amygdalin or apricot seeds peroral exposure cause changes in bone microstructure of rabbits. Twenty clinically healthy 5 months-old male rabbits were segregated into five groups. Animals from groups A1 and A2 were intramuscularly injected with amygdalin at doses of 0.6 and 3 mg/kg b.w. daily for 28 days. The groups S1 and S2 received commercial feed for rabbits mixed with crushed bitter apricot seeds at doses of 60 and 300 mg/kg b.w. during 28 days. The control (C) group did not receive any amygdalin. Intramuscular and peroral amygdalin administration did not affect total body weight, femoral length and femoral weight of rabbits. Similarly, microcomputed tomography (3D analysis) has shown that amygdalin had insignificant effect on relative bone volume, bone mineral density, cortical bone thickness, bone surface, trabecular thickness, trabecular number, trabecular separation. However, histological (2D analysis) revealed evident changes in compact bone microstructure of amygdalin-exposed rabbits consistent with a different vascularization and changed biomechanical properties. We can conclude that subacute exposure to amygdalin (both intramuscular and peroral) at the doses used in our study influenced compact bone remodeling.

Subacute exposure to alcohol in relation to bone microstructure of mice.

Our study aimed to investigate subacute exposure to alcohol in relation to bone microstructure of mice. Animals from experimental (E) group drank a solution composed of 15 % ethanol and water for 14 days (one remodeling cycle), while those from control (C) group drank only water. In the compact bone of E group, decreased bone formation and increased porosity were observed which corresponds with lower levels of serum alkaline phosphatase and glutathione. Alcohol significantly increased sizes of primary osteon's vascular canals and decreased those of secondary osteons, Haversian canals. Relative bone volume, bone mineral density (BMD), relative bone volume without marrow cavity were also lower in E group. On the contrary, trabecular bone microstructure did not differ significantly between E and C groups. Liver function test showed higher levels of alanine aminotransferase, aspartate aminotransferase in alcohol-fed mice. Serum calcium, phosphate were significantly lower in E group. According to our study, only changes in compact bone microstructure of mice following one remodeling cycle were observed due to both direct and indirect effects of alcohol.

Historic and geographic surveillance of Pseudogymnoascus destructans possible from collections of bat parasites.

Specimens archived in wet collections represent valuable material for scientific research. Here, we show that bat fly (Diptera, Nycteribiidae) samples contain DNA of Pseudogymnoascus destructans, a fungus pathogenic to bats. Using dual-probe quantitative PCR, we detected P. destructans DNA on bat flies collected in the Samara, Sverdlovsk and Irkutsk regions of Russia between 2005 and 2017. Fungal load was significantly lower on bat flies from wet collections than on freshly collected mites in the Czech Republic. The bat pathogen was present in the Samara region (European part of Russia) in 2005, that is, a year before recognition of white-nose syndrome in North America. As Samara and Irkutsk regions were identified as new positive locations of P. destructans, our data expand the known geographic distribution of P. destructans. We conclude that ethanol-stored ectoparasites can be used to identify the presence of pathogens in historic bat populations and understudied geographical regions.

Acrylamide-induced changes in femoral bone microstructure of mice.

Acrylamide (AA) is one of the most common toxins in foods. Its effect on bone microstructure has not been investigated. The aim of our study was to analyze the impact of acute exposure to AA on femoral bone microstructure in mice. Adult animals were treated perorally with 2 doses of AA (E1 group, 1 mg/kg b.w.) in a 24-h period and with 3 doses of AA (E2 group, 1 mg/kg b.w.) in a 48-h period. Mice exposed to AA had smaller sizes of primary osteon's vascular canals. Secondary osteons were significantly smaller in mice from E2 group; however their increased number (from 38 % to 77 %) was identified in both E1 and E2 groups. In these groups, a higher number of resorption lacunae (from 100 % to 122 %) was also found. The values for bone volume, trabecular number were increased and that for trabecular separation was decreased in mice administered AA. Significantly higher value of bone surface was observed in mice from E1 group whereas trabecular thickness was increased in E2 group. The effect of AA on microstructure of compact and trabecular bone tissues is different. In our study, one dose of AA was used and acute effects of AA were investigated. Therefore, further studies are needed to study mechanisms by which AA acts on bone.

Pseudogymnoascus destructans: evidence of virulent skin invasion for bats under natural conditions, Europe.

While Pseudogymnoascus destructans has been responsible for mass bat mortalities from white-nose syndrome (WNS) in North America, its virulence in Europe has been questioned. To shed the light on the issue of host-pathogen interaction between European bats and P. destructans, we examined seventeen bats emerging from the fungus-positive underground hibernacula in the Czech Republic during early spring 2013. Dual wing-membrane biopsies were taken from Barbastella barbastellus (1), Myotis daubentonii (1), Myotis emarginatus (1), Myotis myotis (11), Myotis nattereri (1) and Plecotus auritus (2) for standard histopathology and transmission electron microscopy. Non-lethal collection of suspected WNS lesions was guided by trans-illumination of the wing membranes with ultraviolet light. All bats selected for the present study were PCR-positive for P. destructans and showed microscopic findings consistent with the histopathological criteria for WNS diagnosis. Ultramicroscopy revealed oedema of the connective tissue and derangement of the fibroblasts and elastic fibres associated with skin invasion by P. destructans. Extensive fungal infection induced a marked inflammatory infiltration by neutrophils at the interface between the damaged part of the wing membrane replaced by the fungus and membrane tissue not yet invaded by the pathogen. There was no sign of keratinolytic activity in the stratum corneum. Here, we show that lesions pathognomonic for WNS are common in European bats and may also include overwhelming full-thickness fungal growth through the wing membrane equal in severity to reports from North America. Inter-continental differences in the outcome of WNS in bats in terms of morbidity/mortality may therefore not be due to differences in the pathogen itself.

Identification of three novel SEDL mutations, including mutation in the rare, non-canonical splice site of exon 4.

Spondyloepiphyseal dysplasia tarda (SEDT) is an X-linked recessive disorder, characterized by disproportionately short stature and degenerative joint disease, which manifests in the early teens. The gene responsible for SED tarda, SEDL, has been identified in Xp22. We report on three novel SEDL mutations. The first mutation is in the rare, non-canonical 5' splice site of intron 4 (IVS4+4T>C) in an Italian family. Reverse transcription-polymerase chain reaction (RT-PCR) analysis has revealed that this mutation causes alternative splicing of exon 5, and, as a consequence, inclusion of exon 4b sequence. This gives rise to an altered, truncated SEDL protein. We also describe two new deletions: one is a 4-bp deletion in exon 6 [333-336del(GAAT)], identified in a Slovak patient with SEDT, and one is a 1.335-kb deletion (in5/ex6del), found in a Belgian patient. The identification of these novel mutations in SEDL adds to the spectrum of 30 mutations previously identified. A short summary of all currently known SEDL gene mutations is presented.

Membrane mutants: a yeast mutant with a lesion in phosphatidylserine biosynthesis.

A single-gene nuclear choline-requiring mutant of Saccharomyces cerevisiae was studied. Choline as a growth supplement to synthetic media could be substituted by low concentrations of dimethylethanolamine, monomethylethanolamine or ethanolamine. DL-Serine also supported growth, but only at high concentrations: on a molar basis it was approximately one hundred times less effective than choline. When cultured in unsupplemented medium the mutant cells soon ceased to grow. The growth-arrested cells contained less than one fifth of the phosphatidylethanolamine present in wild-type cells and only traces of phosphatidylserine. The relative content of the two phospholipid species was raised by growing the mutant cells in the presence of choline of the other supplements but still remained lower than in wild-type cells. The mutant cells depleted of phosphatidylethanolamine and phosphatidylserine had greatly diminished ability to fuse with other cells in mating and their protoplasts showed increased resistance to hypotonic lysis. Respiration was not substantially affected by the deficit of the two phospholipid species in the mutant. In cell-free preparations, the affinity of the phosphatidylserine synthesizing system for serine was found to be almost two orders of magnitude lower in the mutant than in the wild-type. The impairment of phosphatidylserine synthesis accounts for growth requirement and the abnormal phospholipid composition of the mutant cells.

Mucidin resistance in yeast. Isolation, characterization and genetic analysis of nuclear and mitochondrial mucidin-resistant mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae resistant to the antibiotic mucidin, a specific inhibitor of electron transport between cytochrome b and c, were isolated and divided into three phenotypic groups, as follows. Class 1 mutants were cross-resistant to a variety of mitochondrial inhibitors and exhibited no resistance at the mitochondrial level. Class 2 mutants were specifically resistant to mucidin exhibiting resistance also at the level of isolated mitochondria. Biochemical studies indicated that the mucidin resistance in class 2 mutants involved a modification of mucidin binding of inhibitory sites on the mitochondrial inner membrane without a significance change in the sensitivity of mitochondrial oxygen uptake to antimycin A, 2-heptyl-4-hydroxyquinoline-N-oxide, and 2,3-dimercaptopropanol. Class 3 was represented by a mutant which showed a high degree of resistance to mucidin and was cross-resistant to a variety of mitochondrial inhibitors at the cellular level but exhibited only a resistance to mucidin at the mitochondrial level. Genetic analysis of mucidin-resistant mutants revealed the presence of both nuclear and mitochondrial genes determining mucidin resistance/sensitivity in yeast. Resistance to mucidin in class 1 mutants was due to a single-gene nuclear recessive mutation (mucPR) whereas that in class 2 mutants was caused by mutations of mitochondrial genes. Resistance in class 3 mutant was determined both by single-gene nuclear and mitochondrial mutations. In the mitochondrial mutants the mucidin resistance segregated mitotically and the resistance determinant was lost upon induction of petite mutation by ethidium bromide. Allelism tests indicated that the mucidin resistance mutations fell into two genetic loci (MUC1 and MUC2) which were apparently not closely linked in the mitochondrial genome. Recombination studies showed that the two mitochondrial mucidin loci were not allelic with other mitochondrial loci RIB1, RIB2 and OLI1. An extremely high mucidin resistance at the cellular level was shown to arise from synergistic interaction of the nuclear gene mucPR and the mitochondrial mucidin-resistance gene (MR) in a cell. The results suggest that at least two mitochondrial gene products, responsible for mucidin resistance/sensitivity in yeast, take part in the formation of the cytochrome bc1 region of the mitochondrial respiratory chain.