Maturation of somatic embryos of Pinus strobus is promoted by a high concentration of gellan gum

Application of somatic embryogenesis to Pinus strobus clonal propagation and genetic improvement was hampered by the difficulty in achieving synchronous maturation of a large number of somatic embryos that would germinate and produce plants. Media containing abscisic acid (80 μ M ) and osmotic agents such as sucrose, polyethylene glycol and/or dextran did not sustain development of mature somatic embryos from plated embryonal masses. This indicated that factors other than osmotic agents might be involved in sustaining development of Pinus strobus somatic embryos to maturity. It was subsequently found that media lacking osmotica but containing a high concentration of gellan gum (1%) induced significant improvement in the development of mature somatic embryos in the presence of 80 or 120 μ M abscisic acid. This positive effect was independent of the genotype and all four tested lines displayed similar responses. Media containing gellan gum at concentrations from 0.4 to 1.2% formed gels that varied in their strength. Gel strength was proportional to the concentration of gellan gum in the specific medium but varied depending on the medium formulation. Gel strength increased with the duration of storage of the culture medium by 46% (SD 14) after 14 days of storage. Preliminary results showed that embryos matured on high gellan gum media displayed improved germination frequencies. These results indicate that in Pinus strobus the water status and possibly other medium characteristics that are influenced by increased concentration of gelling agent have stimulatory effects on maturation of somatic embryos.     

PSMC analysis of effective population sizes in molecular ecology and its application to black‐and‐white Ficedula flycatchers

Climatic fluctuations during the Quaternary period governed the demography of species and contributed to population differentiation and ultimately speciation. Studies of these past processes have previously been hindered by a lack of means and genetic data to model changes in effective population size (N_e) through time. However, based on diploid genome sequences of high quality, the recently developed pairwise sequentially Markovian coalescent (PSMC) can estimate trajectories of changes in N_e over considerable time periods. We applied this approach to resequencing data from nearly 200 genomes of four species and several populations of the Ficedula species complex of black‐and‐white flycatchers. N_e curves of Atlas, collared, pied and semicollared flycatcher converged 1–2 million years ago (Ma) at an N_e of ≈ 200 000, likely reflecting the time when all four species last shared a common ancestor. Subsequent separate N_e trajectories are consistent with lineage splitting and speciation. All species showed evidence of population growth up until 100–200 thousand years ago (kya), followed by decline and then start of a new phase of population expansion. However, timing and amplitude of changes in N_e differed among species, and for pied flycatcher, the temporal dynamics of N_e differed between Spanish birds and central/northern European populations. This cautions against extrapolation of demographic inference between lineages and calls for adequate sampling to provide representative pictures of the coalescence process in different species or populations. We also empirically evaluate criteria for proper inference of demographic histories using PSMC and arrive at recommendations of using sequencing data with a mean genome coverage of ≥18X, a per‐site filter of ≥10 reads and no more than 25% of missing data.     

The capacity of UL49.5 proteins to inhibit TAP is widely distributed among members of the genus Varicellovirus

The lifelong infection by varicelloviruses is characterized by a fine balance between the host immune response and immune evasion strategies used by these viruses. Virus-derived peptides are presented to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. The transporter associated with antigen processing (TAP) transports the peptides from the cytosol into the endoplasmic reticulum, where the loading of MHC-I molecules occurs. The varicelloviruses bovine herpesvirus 1 (BoHV-1), pseudorabies virus, and equid herpesviruses 1 and 4 have been found to encode a UL49.5 protein that inhibits TAP-mediated peptide transport. To investigate to what extent UL49.5-mediated TAP inhibition is conserved within the family of Alphaherpesvirinae, the homologs of another five varicelloviruses, one mardivirus, and one iltovirus were studied. The UL49.5 proteins of BoHV-5, bubaline herpesvirus 1, cervid herpesvirus 1, and felid herpesvirus 1 were identified as potent TAP inhibitors. The varicella-zoster virus and simian varicellovirus UL49.5 proteins fail to block TAP; this is not due to the absence of viral cofactors that might assist in this process, since cells infected with these viruses did not show reduced TAP function either. The UL49.5 homologs of the mardivirus Marek's disease virus 1 and the iltovirus infectious laryngotracheitis virus did not block TAP, suggesting that the capacity to inhibit TAP via UL49.5 has been acquired by varicelloviruses only. A phylogenetic analysis of viruses that inhibit TAP through their UL49.5 proteins reveals an interesting hereditary pattern, pointing toward the presence of this capacity in defined clades within the genus Varicellovirus.     

Frequency of lactase persistence genotype in a healthy Polish population

The majority of mammals are unable to digest lactose due to post-weaning deactivation of the LCT gene, which is responsible for encoding the enzyme lactase (i.e., adult-type hypolactasia). A substitution of C with T at position −13910 bp upstream of the LCT gene has been linked to the lactase persistence phenotype in European populations. We investigated the frequency of LCT-13910C>T polymorphism in 223 blood donors from central Poland. All samples were genotyped by polymerase chain reaction and direct sequencing. The LCT-13910 T allele (lactase persistence) was present in 51% of individuals sampled from the Polish population. We did not find any non-European variants associated with lactase persistence (LCT-13907C>G, LCT-13913T>C, LCT-13915T>G), or any new polymorphisms within the sequenced region. Allele frequencies obtained are in agreement with results from other countries and confirm the unique pattern of distribution of the LCT-13910C>T genotype in Europe.     

A regulatory role for NBS1 in strand-specific mutagenesis during somatic hypermutation

Activation-induced cytidine deaminase (AID) is believed to initiate somatic hypermutation (SHM) by deamination of deoxycytidines to deoxyuridines within the immunoglobulin variable regions genes. The deaminated bases can subsequently be replicated over, processed by base excision repair or mismatch repair, leading to introduction of different types of point mutations (G/C transitions, G/C transversions and A/T mutations). It is evident that the base excision repair pathway is largely dependent on uracil-DNA glycosylase (UNG) through its uracil excision activity. It is not known, however, which endonuclease acts in the step immediately downstream of UNG, i.e. that cleaves at the abasic sites generated by the latter. Two candidates have been proposed, an apurinic/apyrimidinic endonuclease (APE) and the Mre11-Rad50-NBS1 complex. The latter is intriguing as this might explain how the mutagenic pathway is primed during SHM. We have investigated the latter possibility by studying the in vivo SHM pattern in B cells from ataxia-telangiectasia-like disorder (Mre11 deficient) and Nijmegen breakage syndrome (NBS1 deficient) patients. Our results show that, although the pattern of mutations in the variable heavy chain (V(H)) genes was altered in NBS1 deficient patients, with a significantly increased number of G (but not C) transversions occurring in the SHM and/or AID targeting hotspots, the general pattern of mutations in the V(H) genes in Mre11 deficient patients was only slightly altered, with an increased frequency of A to C transversions. The Mre11-Rad50-NBS1 complex is thus unlikely to be the major nuclease involved in cleavage of the abasic sites during SHM, whereas NBS1 might have a specific role in regulating the strand-biased repair during phase Ib mutagenesis.     

Hemoglobin adducts and urinary mercapturic acids in rats as biological indicators of butadiene exposure

Binding of 1,2-epoxy-3-butene, the primary metabolite of butadiene, to hemoglobin (Hb) and excretion of its mercapturic acid in urine were studies as potential indicators of butadiene exposure. Four groups of Wistar rats were exposed to butadiene at 0, 250, 500 and 1000 ppm 6 h/day, 5 days/week, during 2 weeks. Blood was collected at the end of exposure and 17 days later for analysis of hemoglobin adducts and adduct stability. Urine was collected each day during exposure (afternoon samples) and in between exposures (morning samples). Adducts of 1,2-epoxy-3-butene to N-terminal valine in Hb were measured using the N-alkyl Edman procedure and GC/MS of the thiohydantoin derivatives. The corresponding mercapturic acid was analysed, after deacetylation, through derivatization with phthaldialdehyde and HPLC with fluorescence detection. The Hb adducts proved to be stable and are therefore useful for dosimetry of long-term exposure to butadiene. The adduct levels increased linearly with exposure dose up to 1000 ppm (3 nmol/g Hb at 1000 ppm). The increase with exposure dose of the mercapturic acid concentration in urine was also compatible with a linear does response up to 1000 ppm. The sensitivity of both analytical methods needs to be improved for their application to human samples.     

Nijmegen breakage syndrome (NBS)

Most inborn errors of metabolism (IEM) are recessive, genetically transmitted diseases and are classified into 3 main groups according to their mechanisms: cellular intoxication, energy deficiency, and defects of complex molecules. They can be associated with endocrine manifestations, which may be complications from a previously diagnosed IEM of childhood onset. More rarely, endocrinopathies can signal an IEM in adulthood, which should be suspected when an endocrine disorder is associated with multisystemic involvement (neurological, muscular, hepatic features, etc.). IEM can affect all glands, but diabetes mellitus, thyroid dysfunction and hypogonadism are the most frequent disorders. A single IEM can present with multiple endocrine dysfunctions, especially those involving energy deficiency (respiratory chain defects), and metal (hemochromatosis) and storage disorders (cystinosis). Non-autoimmune diabetes mellitus, thyroid dysfunction and/or goiter and sometimes hypoparathyroidism should steer the diagnosis towards a respiratory chain defect. Hypogonadotropic hypogonadism is frequent in haemochromatosis (often associated with diabetes), whereas primary hypogonadism is reported in Alstr?m disease and cystinosis (both associated with diabetes, the latter also with thyroid dysfunction) and galactosemia. Hypogonadism is also frequent in X-linked adrenoleukodystrophy (with adrenal failure), congenital disorders of glycosylation, and Fabry and glycogen storage diseases (along with thyroid dysfunction in the first 3 and diabetes in the last). This is a new and growing field and is not yet very well recognized in adulthood despite its consequences on growth, bone metabolism and fertility. For this reason, physicians managing adult patients should be aware of these diagnoses.     

Traces of human impact reflected in the pollen diagram from Tarnowiec mire near Jasło (Jasło-Sanok depression), SE Poland

The pollen diagram from Tarnowiec concentrates on human impact on vegetation, and is supported by the archaeological background. The earliest evidence of human activities dated at ca. 6500 B.P. was probably connected with the Neolithic Linearbandkeramik culture. Further periods of settlement are recorded at ca. 5200 B.P. and at 4800 B.P. The next very distinct period of intensification in human impact at ca. 4200-3800 B.P. was most certainly connected with the presence of the Corded Ware culture. The next period of settlement occurred during the late Bronze Age, in connection with the development of the Lusatian culture from ca. 3200 B.P. but is recorded only faintly in the pollen diagram. The next colonization phase at ca. 2600 B.P. is possibly the result of Scythian invasions. The widest expansion of human impact came during the period of Roman influence. After this, anthropogenic indicators decreased during the Migration period. The youngest part of the pollen diagram reveals an intensification of anthropogenic indicators connected with early Medieval settlement.     

Distance between south-European and south-west Asiatic refugial areas involved morphological differentiation: Pinus sylvestris case study

The phenotypic differentiation of relic P. sylvestris in southern Europe and southwestern Asia was verified using thirty-two populations sampled from the Iberian Peninsula, Massif Central, Balkan Peninsula, Crimea and Anatolia. Twenty-one morphological and anatomical needle traits and 18 cone morphological characteristics were examined to describe the population diversity and differentiation. The needle characters were not correlated to those of cone. The differences between regions were significant based on 12 needle and 9 cone characteristics, suggesting spatial isolation. The differentiation between the Iberian and Anatolian populations was the highest, which indicates the isolation by distance. The high level of morphological differentiation was also found among Iberian populations, supporting the already known complex history of the species in that region. Populations within other regions were differentiated at lower levels; however, the West Anatolian populations differed morphologically from the eastern ones. The described pattern of morphological differentiation supports the idea of the long-lasting existence of P. sylvestris in the south-European and Anatolian mountain regions. To conserve this variation, seed transfer between regions in the forest economy should be restricted.