Germline loss‐of‐function variants in MBD4 are rare in Finnish patients with uveal melanoma

Uveal melanoma (UM) is a rare intraocular cancer with the highest incidence in northern latitudes. Metastases develop in approximately 50% of patients, whereafter the median survival is 13 months. Generally, the mutation burden of these tumors is low. Germline variants predisposing to UM have been previously described in BRCA1‐associated protein 1 (BAP1). Recently, germline and somatic loss‐of‐function (LOF) variants in the methyl‐CpG‐binding domain 4 (MBD4) gene have been found to cause a hypermutated UM, and MBD4 also has been put forward as a gene predisposing to UM. We sequenced for MBD4 germline variants in 440 Finnish patients with UM and identified seven rare exonic missense variants in 16 (3.6%) patients, of which one likely alters MBD4 function. The frequency of likely pathogenic variants in our cohort is 0.23% (1/432; 95% CI, 0.01–1.28). We identified no LOF variants though their frequency in the Finnish population is 0.052%. Thus, our data do not support the suggestion that MBD4 germline variants predispose to UM. Somatic loss of MBD4 might modify the mutational burden in UM and change its response to immune checkpoint inhibitors.     

Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer

High androgen receptor (AR) level in primary tumour predicts increased prostate cancer-specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR-binding sites (ARBs) on LNCaP-1F5 cell chromatin that was commensurate with changes in androgen-dependent gene expression signature. We identified three distinct classes of ARBs and androgen-responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid-dependent signalling in LNCaP-1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.     

Different response of two reindeer forage plants to enhanced UV-B radiation: modification of the phenolic composition

The long-term effects of enhanced UV-B radiation on the content and composition of leaf phenolics in Epilobium angustifolium L. and Eriophorum russeolum Fries ex Hartman were studied in northern Finland (68°N) using two UV-B enhancement experiments, both simulating UV-B_CIE radiation and corresponding to a 20% loss of ozone layer. High proportions of hydrolyzable tannins (69%) and condensed tannins (66%) characterized both Epilobium and Eriophorum leaves, respectively. No UV treatment effect was detected in the content or composition of Epilobium leaf soluble phenolics, whereas significant UV effects were detected in Eriophorum leaves in a developmental-specific manner. At the end of the growing season, the proportion of total soluble phenolics was higher in leaves exposed to enhanced UV-A and UV-B radiation than in the control leaves, but the phenolic composition was not significantly modified. This study introduces a new example on plants’ phenolic response to UV radiation being species-specific and detectable only at certain developmental stages. Possible consequences of increased phenolic content in forage plants for selection and digestibility by reindeer are, however, not yet known.     

Association analysis of the AIRE and insulin genes in Finnish type 1 diabetic patients

Mutations in the autoimmune regulator (AIRE) gene cause a recessive Mendelian disorder autoimmune polyendocrinopathy syndrome type 1 (APS-1 or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy). APS-1 patients develop multiorgan autoimmune diseases including type 1 diabetes (prevalence 12%). The AIRE protein controls the central tolerance induction in the thymus by regulating the expression levels of tissue-specific peripheral antigens, such as insulin. We hypothesized that the insulin gene (INS) polymorphisms together with the AIRE variations may predispose individuals to diabetes. The role of the AIRE gene was tested both independently and on the condition of the INS risk genotype in the Finnish type 1 diabetes sample. A total of 733 type 1 diabetic cases and 735 age- and sex-matched healthy controls were used in the analysis. Five common single nucleotide polymorphisms (SNPs) in the AIRE gene were selected from the public database (dbSNP). The −23HphI polymorphism was used as a surrogate marker for the INS gene promoter repeat. The five genotyped SNPs in the AIRE gene showed no evidence of association with type 1 diabetes. As expected, the INS gene polymorphism −23HphI was significantly associated with susceptibility to type 1 diabetes (P=6.8×10⁻¹², χ² test). When the subclass of patients carrying the homozygote genotype of the INS gene was used in the analysis, the AIRE polymorphisms showed no association with the disease. In conclusion, the AIRE gene does not seem to contribute to disease susceptibility in Finnish type 1 diabetic patients, whereas the insulin gene represents a notable risk factor for disease in this population.     

Protein release from the larval fat body of the southwestern corn borer, Diatraea grandiosella: An in vitro study

The release of protein from the perivisceral fat body of non-diapausing, pre-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Time course studies showed a selective release of proteins into macromolecule-free Grace's medium. The rate of release of individual proteins differed. The release of some proteins was partially inhibited by the incorporation of potassium cyanide (10^?2 M) and ouabain (5 × 10^?3 M) into the medium. During a 5 min incubation a single major high molecular weight protein fraction was released at a high rate from the fat body of both non-diapausing and diapausing larvae. A low molecular weight protein (the diapause-associated protein) was also released readily from the fat body of diapausing larvae. Although most proteins released from the fat body in vitro appeared to be present in the haemolymph in vivo, one notable exception was the absence of the diapause-associated protein from the haemolymph. The method holds promise for facilitating further studies of protein release from insect fat body.     

The uptake of and structural changes induced by trichloroacetic acid in the needles of Scots pine seedlings

The uptake of trichloroacetic acid (TCA) and structural changesinduced in the needles of Scots pine (Pinus sylvestris L.) seedlingswere studied. Two exposure set-ups, a root route and an atmosphericroute through the surfaces simulating the wet deposition offog, were used. Both set-ups included two dose levels and correspondingcontrol treatments. The temperature and the relative humidityin the climate chambers were adjusted to represent the conditionsof June–July in a subarctic area in central Finland. Theseedlings were exposed three times a week for two months. Theresults showed that the uptake of TCA in needles occurred bothvia roots and via needle surface. However, most of the TCA viathe atmospheric route was absorbed on the surface of the needles.The structural responses in pine needles depended partly onthe treatment method: TCA applied via the atmospheric routedisintegrated the structures of the epicuticular waxes and thatof the stomatal cells, which was not seen in the exposures viaroots. A common feature was the decrease in size of the chloroplastsin concert with the increasing TCA concentrations inside theneedles. Key words: Pinus sylvestris L., climate chamber, effects, microscopic structure, secondary pollutant, TCA, trichloroacetic acid     

Hyperthermostable <Emphasis Type="Italic">Thermotoga maritima</Emphasis> xylanase XYN10B shows high activity at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids

The gene of Thermotoga maritima GH10 xylanase (TmXYN10B) was synthesised to study the extreme limits of this hyperthermostable enzyme at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids (ILs). TmXYN10B expressed from Pichia pastoris showed maximal activity at 100 °C and retained 92 % of maximal activity at 105 °C in a 30-min assay. Although the temperature optimum of activity was lowered by 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), TmXYN10B retained partial activity in 15–35 % hydrophilic ILs, even at 75–90 °C. TmXYN10B retained over 80 % of its activity at 90 °C in 15 % [EMIM]OAc and 15–25 % 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) during 22-h reactions. [EMIM]OAc may rigidify the enzyme and lower V _max. However, only minor changes in kinetic parameter K _m showed that competitive inhibition by [EMIM]OAc of TmXYN10B is minimal. In conclusion, when extended enzymatic reactions under extreme conditions are required, TmXYN10B shows extraordinary potential.     

Synthesis of a tetravalent sialyl Lewis x glycan, a high-affinity inhibitor of L-selectin-mediated lymphocyte binding to endothelium

Kidney transplant rejection is an inflammatory process characterizedby lymphocyte infiltration. Our earlier observations have shownthat peritubular capillary endothelium (PTCE) is the site oflymphocyte entry into the rejecting renal allograft. Duringrejection, PTCE begins to express sialyl Lewis x de novo, andbinds lymphocytes by a mechanism largely dependent on L-selectin.Hence, inhibiting the lymphocyte-endothelial interaction witholigosaccharide ligands of L-selectin offers an attractive possibilityto prevent the inflammation and rejection. Here, we report enzyme-assistedsynthesis of N-acetyllactosamine-based tetra-, deca-, and docosamericsaccharides carrying one, two or four distally located sialylLewis x groups [Neu-NAc     

Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.