Floristic turnover in Iceland from 15 to 6 Ma – extracting biogeographical signals from fossil floral assemblages

Aim This study aims to document the floristic changes that occurred in Iceland between 15 and 6 Ma and to establish the dispersal mechanisms for the plant taxa encountered. Using changing patterns of dispersal, two factors controlling floristic changes are tested. Possible factors are (1) climate change, and (2) the changing biogeography of Iceland over the time interval studied; that is, the presence or absence of a Miocene North Atlantic Land Bridge. Location The North Atlantic. Methods Species lists of fossil plants from Iceland in the time period 15 to 6 Ma were compiled using published data and new data. Closest living analogues were used to establish dispersal properties for the fossil taxa. Dispersal mechanisms of fossil plants were then used to reconstruct how Iceland was colonized during various periods. Results Miocene floras of Iceland (15–6 Ma) show relatively high floristic turnover from the oldest floras towards the youngest; and few taxa from the oldest floras persist in the younger floras. The frequencies of the various dispersal mechanisms seen in the 15‐Ma floras are quite different from those recorded in the 6‐Ma floras, and there is a gradual change in the prevailing mode of dispersal from short‐distance anemochory and dyschory to long‐distance anemochory. Two mechanisms can be used to explain changing floral composition: (1) climate change, and (2) the interaction between the dispersal mechanisms of plants and the increasing isolation of proto‐Iceland during the Miocene. Main conclusions Dispersal mechanisms can be used to extract palaeogeographic signals from fossil floras. The composition of floras and dispersal mechanisms indicate that Iceland was connected both to Greenland and to Europe in the early Middle Miocene, allowing transcontinental migration. The change in prevalence of dispersal modes from 15 to 6 Ma appears to reflect the break‐up of a land bridge and the increasing isolation of Iceland after 12 Ma. Concurrent gradual cooling and isolation caused changes in species composition. Specifically, the widening of the North Atlantic Ocean prevented taxa with limited dispersal capability from colonizing Iceland, while climate cooling led to the extinction of thermophilous taxa.     

Hepatic organelle interaction. IV. Mechanism of succinate enhancement of formaldehyde accumulation from endoplasmic reticulum N-dealkylations

Further evidence for organelle interaction during drug metabolism by the liver is presented. The apparent stimulation by succinate of formaldehyde accumulation in the medium, which was reported to occur with liver slices and homogenates as well as with mitochondria plus microsomes, has been shown to be the result of succinate inhibition of mitochondrial aldehyde dehydrogenase. The mechanism of succinate inhibition is shown to be by reverse electron transport, and an increase in the NADH to NAD+ ratio in the mitochondria; the aldehyde dehydrogenase requires the oxidized form of the pyridine nucleotide as its cofactor. Studies on in vitro N-demethylation by liver microsomes and endoplasmic reticulum segments which cosediment with the mitochondria indicate that formaldehyde produced by the mixed function oxidase is handled differently from formaldehyde added to the medium. The latter is mainly retained in the medium containing 5 mM semicarbazide, while the generated formaldehyde is more than 50% consumed by the mitochondria. Electron microscopy has indicated that the microsomes and the endoplasmic reticulum fragments have a tendency to align themselves close to the mitochondria when present in the same medium. Consequently, it is possible that formaldehyde released to the medium adjacent to the mitochondria, as by N-demethylation, would be exposed to semicarbazide for shorter periods than that added directly to the medium. In agreement with this suggestion, complexing of formaldehyde with semicarbazide was observed spectroscopically not to be an extremely rapid reaction even at 37 degrees C. This is believed to be the reason for the greater extent of consumption of formaldehyde generated by the endoplasmic reticulum.     

A dendrite-autonomous mechanism for direction selectivity in retinal starburst amacrine cells

Detection of image motion direction begins in the retina, with starburst amacrine cells (SACs) playing a major role. SACs generate larger dendritic Ca²⁺ signals when motion is from their somata towards their dendritic tips than for motion in the opposite direction. To study the mechanisms underlying the computation of direction selectivity (DS) in SAC dendrites, electrical responses to expanding and contracting circular wave visual stimuli were measured via somatic whole-cell recordings and quantified using Fourier analysis. Fundamental and, especially, harmonic frequency components were larger for expanding stimuli. This DS persists in the presence of GABA and glycine receptor antagonists, suggesting that inhibitory network interactions are not essential. The presence of harmonics indicates nonlinearity, which, as the relationship between harmonic amplitudes and holding potential indicates, is likely due to the activation of voltage-gated channels. [Ca²⁺] changes in SAC dendrites evoked by voltage steps and monitored by two-photon microscopy suggest that the distal dendrite is tonically depolarized relative to the soma, due in part to resting currents mediated by tonic glutamatergic synaptic input, and that high-voltage–activated Ca²⁺ channels are active at rest. Supported by compartmental modeling, we conclude that dendritic DS in SACs can be computed by the dendrites themselves, relying on voltage-gated channels and a dendritic voltage gradient, which provides the spatial asymmetry necessary for direction discrimination.     

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.     

Two common nonsynonymous paraoxonase 1 (<Emphasis Type="Italic">PON1</Emphasis>) gene polymorphisms and brain astrocytoma and meningioma

Background  

Human serum paraoxonase 1 (PON1) plays a major role in the metabolism of several organophosphorus compounds. The enzyme is encoded by the polymorphic gene PON1, located on chromosome 7q21.3. Aiming to identify genetic variations related to the risk of developing brain tumors, we investigated the putative association between common nonsynonymous PON1 polymorphisms and the risk of developing astrocytoma and meningioma.     

Differential bone marrow homing capacity of VLA-4 and CD38 high expressing chronic lymphocytic leukemia cells

Background

VLA-4 and CD38 predict a poor clinical outcome in chronic lymphocytic leukemia (CLL). We used CLL samples with discordant VLA-4/CD38 risk to address their individual roles in human bone marrow infiltration (BM), CLL cell homing to murine BM, and in supportive CLL cell-stromal cell interactions.

Methods

VLA-4, CD38, and Ki-67 expression was measured in CLL cells from peripheral blood (PB) and bone marrow (BM) aspirates. CLL BM infiltration rates, routinely determined by Pathology, were correlated to VLA-4 and CD38 expression. Short-term homing capacity of CLL cells was evaluated by adoptive transfer experiments. CLL cell viability and adhesion in stromal cell co-culture was determined.

Results

About 20% of CLL samples in our cohort displayed discordant VLA-4 and CD38 risk, with either high VLA-4 and low CD38 risk or vice versa. Using particularly such samples, we observed that VLA-4, and not CD38, was responsible for recirculation of CLL cells to murine BM. Human BM infiltration was also significantly higher in patients with high VLA-4 risk but not high CD38 risk. However, both molecules acted as independent prognostic markers. While both VLA-4 and CD38 expression were increased in BM-derived CLL cells, and VLA-4+ and CD38+ subpopulations showed enriched Ki-67 expression, VLA-4 did not contribute to CLL cell protection by stromal cells in vitro.

Conclusions

Our data argue for a prominent role of VLA-4 but not CD38 expression in the homing of CLL cells to BM niches and in human BM infiltration,but only a limited role in their protection by stromal cells.     

Haplotype-specific <Emphasis Type="Italic">MAPT</Emphasis> exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders

Background

Genome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson’s disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium.

Methods

In this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay.

Results

Using whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion.

Conclusions

We identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium.

     

High sensitivity of Stark-shift voltage-sensing dyes by one- or two-photon excitation near the red spectral edge

Sensitivity spectra of Stark-shift voltage sensitive dyes, such as ANNINE-6, suggest the use of the extreme red edges of the excitation spectrum to achieve large fractional fluorescence changes with membrane voltage. This was tested in cultured HEK293 cells. Cells were illuminated with light at the very red edge of the dye's excitation spectrum, where the absorption cross section is as much as 100 times smaller than at its peak. The small-signal fractional fluorescence changes were -0.17%/mV, -0.28%/mV, and -0.35%/mV for one-photon excitation at 458 nm, 488 nm, and 514 nm, respectively, and -0.29%/mV, -0.43%/mV, and -0.52%/mV for two-photon excitation at 960 nm, 1000 nm, and 1040 nm, respectively. For large voltage swings the fluorescence changes became nonlinear, reaching 50% and -28% for 100 mV hyper- and depolarization, respectively, at 514 nm and 70% and -40% at 1040 nm. Such fractional sensitivities are approximately 5 times larger than what is commonly found with other voltage-sensing dyes and approach the theoretical limit given by the spectral Boltzmann tail.