The MlotiK1 channel transports ions along the canonical conduction pore

Although the cyclic nucleotide-modulated potassium channel from Mesorhizobium loti, MlotiK1, is easily studied using a 86Rb+ flux assay, its comparatively low activity raises serious concerns about the integrity of the purified protein. We investigated the pathway of uptake using a multi-pronged approach. First, we probed the conduction pathway using quaternary ammonium compounds known to block conduction in eukaryotic K+ channels. Second, we examined the effect of chemical modification of putative pore-lining residues. Our results are consistent with ions traversing MlotiK1 along a conduction pathway like that of the eukaryotic channels, but at a much slower rate.     

Sequencing complete mitochondrial and plastid genomes

Organelle genomics has become an increasingly important research field, with applications in molecular modeling, phylogeny, taxonomy, population genetics and biodiversity. Typically, research projects involve the determination and comparative analysis of complete mitochondrial and plastid genome sequences, either from closely related species or from a taxonomically broad range of organisms. Here, we describe two alternative organelle genome sequencing protocols. The "random genome sequencing" protocol is suited for the large majority of organelle genomes irrespective of their size. It involves DNA fragmentation by shearing (nebulization) and blunt-end cloning of the resulting fragments into pUC or BlueScript-type vectors. This protocol excels in randomness of clone libraries as well as in time and cost-effectiveness. The "long-PCR-based genome sequencing" protocol is specifically adapted for DNAs of low purity and quantity, and is particularly effective for small organelle genomes. Library construction by either protocol can be completed within 1 week.     

Prognostic molecular markers in pediatric liver disease – Are there any?

Pediatric liver disease (PLD) is a major cause of severe morbidity and prolonged hospitalizations in children. Stratifying patients in terms of prognosis remains challenging. The limited knowledge about molecular mechanisms causing and accompanying PLD remains the main obstacle in a search for reliable prognostic biomarkers. A systematic search of MEDLINE via PubMed and Embase via OVID was conducted on studies published between August 2007 and August 2017. Molecular markers with a prognostic potential in terms of survival, need for liver transplantation or disease progression/regression were selected. In general, identified studies were single center smaller case-control studies or case series with a low level of evidence and a high risk of bias. Only 23 studies comprising 898 patients could be included, mostly focusing on biliary atresia, non-alcoholic fatty liver disease, viral hepatitis, and LT; and markers related to morphogenesis and fibrosis. Furthermore, molecular markers in metabolic pathways and inflammation shown to be relevant, however requiring further validation. Hence, further biological and clinical studies are needed to gain greater molecular insight into PLD.     

Aboveground impacts of a belowground invader: how invasive earthworms alter aboveground arthropod communities in a northern North American forest

Declining arthropod communities have recently gained a lot of attention, with climate and land-use change among the most frequently discussed drivers. Here, we focus on a seemingly underrepresented driver of arthropod community decline: biological invasions. For approximately 12 000 years, earthworms have been absent from wide parts of northern North America, but they have been re-introduced with dramatic consequences. Most studies investigating earthworm-invasion impacts focus on the belowground world, resulting in limited knowledge on aboveground-community changes. We present observational data on earthworm, plant and aboveground arthropod communities in 60 plots, distributed across areas with increasing invasion status (low, medium and high) in a Canadian forest. We analysed how earthworm-invasion status and biomass impact aboveground arthropod community abundance, biomass and species richness, and how earthworm impacts cascade across trophic levels. We sampled approximately 13 000 arthropods, dominated by Hemiptera, Diptera, Araneae, Thysanoptera and Hymenoptera. Total arthropod abundance, biomass and species richness declined significantly from areas of low to those with high invasion status, with reductions of 61, 27 and 18%, respectively. Structural equation models suggest that earthworms directly and indirectly impact arthropods across trophic levels. We show that earthworm invasion can alter aboveground multi-trophic arthropod communities and suggest that belowground invasions might be underappreciated drivers of aboveground arthropod decline.     

Neuronal postdevelopmentally acting SAX-7S/L1CAM can function as cleaved fragments to maintain neuronal architecture in Caenorhabditis elegans

Whereas remarkable advances have uncovered mechanisms that drive nervous system assembly, the processes responsible for the lifelong maintenance of nervous system architecture remain poorly understood. Subsequent to its establishment during embryogenesis, neuronal architecture is maintained throughout life in the face of the animal’s growth, maturation processes, the addition of new neurons, body movements, and aging. The Caenorhabditis elegans protein SAX-7, homologous to the vertebrate L1 protein family of neural adhesion molecules, is required for maintaining the organization of neuronal ganglia and fascicles after their successful initial embryonic development. To dissect the function of sax-7 in neuronal maintenance, we generated a null allele and sax-7S-isoform-specific alleles. We find that the null sax-7(qv30) is, in some contexts, more severe than previously described mutant alleles and that the loss of sax-7S largely phenocopies the null, consistent with sax-7S being the key isoform in neuronal maintenance. Using a sfGFP::SAX-7S knock-in, we observe sax-7S to be predominantly expressed across the nervous system, from embryogenesis to adulthood. Yet, its role in maintaining neuronal organization is ensured by postdevelopmentally acting SAX-7S, as larval transgenic sax-7S(+) expression alone is sufficient to profoundly rescue the null mutants’ neuronal maintenance defects. Moreover, the majority of the protein SAX-7 appears to be cleaved, and we show that these cleaved SAX-7S fragments together, not individually, can fully support neuronal maintenance. These findings contribute to our understanding of the role of the conserved protein SAX-7/L1CAM in long-term neuronal maintenance and may help decipher processes that go awry in some neurodegenerative conditions.     

Inactivation of mouse Twisted gastrulation reveals its role in promoting Bmp4 activity during forebrain development

Twisted gastrulation (Tsg) is a secreted protein that regulates Bmp signaling in the extracellular space through its direct interaction with Bmp/Dpp and Chordin (Chd)/Short gastrulation (Sog). The ternary complex of Tsg/Chd/Bmp is cleaved by the metalloprotease Tolloid (Tld)/Xolloid (Xld). Studies in Drosophila, Xenopus and zebrafish suggest that Tsg can act both as an anti-Bmp and as a pro-Bmp. We have analyzed Tsg loss-of-function in the mouse. Tsg homozygous mutants are viable but of smaller size and display mild vertebral abnormalities and osteoporosis. We provide evidence that Tsg interacts genetically with Bmp4. When only one copy of Bmp4 is present, a requirement of Tsg for embryonic development is revealed. Tsg-/-;Bmp4+/- compound mutants die at birth and display holoprosencephaly, first branchial arch and eye defects. The results show that Tsg functions to promote Bmp4 signaling during mouse head development.     

The tissue-specific distribution of 3H-seocalcitol (EB 1089) and 3H-calcitriol in rats

Seocalcitol (EB 1089) is under development for the treatment of hepato-cellular carcinoma (HCC). The tissue distribution of 3H-seocalcitol was investigated in comparison to 3H-calcitriol in rats. Quantitative whole-body autoradiography was used to quantify the tissue distribution. The greatest difference in distribution between the two compounds was observed in the bloodstream. For most tissues the ratio seocalcitol/calcitriol varied between 0.2 and 3.1. The concentration of radioactivity in the liver was almost the same for the two compounds. For seocalcitol the concentration in the liver was 10 times higher than in serum. Assuming that the liver/serum concentration ratio is the same in rats and humans, the concentration of seocalcitol in the human liver is expected to be higher than the concentration resulting in more than 50% inhibition of cancer cell proliferation, and thus pharmacologically effective in HCC. It is questionable whether calcitriol would be present in the human liver in sufficient concentrations to be effective for the treatment of HCC, as the antiproliferative activity of calcitriol is generally more than 10-fold lower compared to that of seocalcitol and as calcitriol can only be administered at a dose that is ca. three-fold lower than the dose of seocalcitol.     

Genetic heterogeneity of variable number tandem repeats in thymidylate synthase gene in colorectal cancer patients

PURPOSE: To analyze the genetic variability in a variable number of tandem repeats (VNTR) in the thymidylate synthase (TS) enhancer promoter region and assess the influence of functional alterations in mismatch repair genes by analyzing constitutional and tumoral DNA from patients with colorectal adenocarcinoma with a high microsatellite instability (MSI-H) or microsatellite stability (MSS) status. PATIENTS AND METHODS: Patients who underwent surgery for colorectal adenocarcinoma were selected from the colorectal database of our institute and, on the basis of MSI status, assigned to a study group and a control group: group A, MSI-H; group B, MSS. Microsatellite status was investigated using the Bethesda recommended panel (BAT-26, BAT-25, D2S123, D5S346, D17S250). In MSI-H patients an additional analysis was made of the microsatellite loci D18S61 and D18S58, both mapping in the region containing the TS gene (18p11.2-11.32). Based on the number of altered microsatellites (> or = 2, 1, or 0), tumors were considered as having high (MSI-H) or low (MSI-L) instability or microsatellite stability (MSS), respectively. Genotyping for thymidylate synthase promoter polymorphism was carried out on constitutional and tumor DNA of each patient by PCR amplification of the polymorphic region. RESULTS: MSI-H was found in 55 patients (group A) and MSS in 50 patients (group B). In none of the MSI-H patients was microsatellite instability found in the additional D18S61 and D18S58 loci. In five group A and ten group B cases the analysis was not performed because constitutional DNA and/or tumoral DNA were not amplifiable. Homozygotes for the triple repeat variant (3R/3R) displayed only the large PCR product, homozygotes for the double repeat variant (2R/2R) displayed only the smaller PCR product, while heterozygotes (2R/3R) displayed both the larger and smaller PCR products. In 3/50 (6%) group A patients and 5/40 (12%) group B patients repeat variations were found in tumoral DNA. CONCLUSION: Our findings demonstrate that there is genetic homogeneity between constitutional and tumoral DNA but do not support the hypothesis that mismatch repair genes are involved in VNTR recombinant events in TS gene variability.     

Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins

A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and beta-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.