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Kramer, A.

Publications and source records attributed to Kramer, A..

33 records · Page 2Linked to original sources

Transposable elements drive intron gain in diverse eukaryotes

There is massive variation in intron numbers across eukaryotic genomes, yet the major drivers of intron content during evolution remain elusive. Rapid intron loss and gain in some lineages contrasts with long term evolutionary stasis in others. Episodic intron gain could be explained by recently discovered specialized transposons called Introners, but so far introners are only known from a handful of species. Here, we performed a systematic search across 3,325 eukaryotic genomes and identified 27,563 Introner-derived introns in 175 genomes (5.2%). Species with introners span remarkable phylogenetic diversity, from animals to basal protists, representing lineages whose last common ancestor dates to over 1.7 billion years ago. Marine organisms were 6.5 times more likely to contain Introners than their terrestrial counterparts. Introners exhibit mechanistic diversity but most are consistent with DNA transposition, indicating that Introners have evolved convergently hundreds of times from autonomous transposable elements. Transposable elements and marine taxa are associated with high rates of horizontal gene transfer, suggesting that this combination of factors may explain the punctuated and biased diversity of species containing Introners. More generally our data suggest that Introners may explain the episodic nature of intron gain across the eukaryotic tree of life. These results illuminate the major source of ongoing intron creation in eukaryotic genomes.

genomics↗

Inter-layer and inter-subject variability of circadian gene expression in human skin

The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports circadian gene expression in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep schedules. 10% of the expressed genes showed rhythms at the population level, of which only a third differed between the two layers. Broadly, expression magnitudes of circadian genes were consistent across subjects in each layer. Amplitude and phases of circadian gene expression, however, varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in the their variability across subjects. We used this data to identify small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin to realize the potential of circadian medicine as well as a novel methodology to quantify sources of variability in human circadian rhythms.

bioinformatics↗

An integrative omics approach reveals posttranscriptional mechanisms underlying circadian temperature compensation

A defining property of circadian clocks is temperature compensation, characterized by the resilience of circadian free-running periods against changes in environmental temperature. As an underlying mechanism, the balance or critical reaction hypothesis have been proposed. While the former supposes a temperature-dependent balancing of reactions with opposite effects on circadian period, the latter assumes an insensitivity of certain critical period determining regulations upon temperature changes. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3-end cleavage and polyadenylation, abolishes circadian temperature compensation in U-2 OS cells. We apply a combination of 3-End-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3 UTR length as well as gene and protein expression between wild type and CPSF6 knock-down cells and their dependency on temperature. Analyzing differential responses upon temperature changes in wild type and CPSF6 knockdown cells reveals candidate genes underlying circadian temperature compensation. We identify that eukaryotic translation initiation factor 2 subunit 1 (EIF2S1) is among these candidates. EIF2S1 is known as a master regulator of cellular stress responses that additionally regulates circadian rhythms. We show that knockdown of EIF2S1 furthermore impairs temperature compensation, suggesting that the role of CPSF6 in temperature compensation may be mediated by its regulation of EIF2S1.

genomics↗

Insulin-stimulated adiponectin secretion in pregnancy is mediated by inhibition of adiponectin ubiquitination and degradation and is impaired in obesity

In pregnancy, adiponectin serves as an endocrine link between maternal adipose tissue, placental function and fetal growth, with low adiponectin promoting placental function and fetal growth. Circulating adiponectin levels are decreased in obese pregnant women and in gestational diabetes, which is believed to contribute to the insulin resistance and increased risk of fetal overgrowth associated with these conditions. However, the molecular mechanisms governing adiponectin secretion from maternal adipose tissues in pregnancy are poorly understood. Using visceral adipose tissue from lean and obese pregnant mice, we show that obesity in pregnancy is associated with adipose tissue inflammation, ER stress, insulin resistance, increased adiponectin ubiquitination and decreased total abundance of adiponectin. Moreover, adiponectin ubiquitination was increased in visceral fat of obese pregnant women as compared to lean pregnant women. We further observed that insulin prevents, whereas ER stress and inflammation promote, adiponectin ubiquitination and degradation in differentiated 3T3-L1 adipocytes. We have identified key molecular pathways regulating adiponectin secretion in pregnancy. This information will help us better understand the mechanisms controlling maternal insulin resistance and fetal growth in pregnancy and may provide a foundation for the development of strategies aimed at improving adiponectin production in pregnant women with obesity or gestational diabetes.

physiology↗

Studies overestimate the extent of circadian rhythm reprogramming in response to dietary and genetic changes

The circadian clock modulates key physiological processes in many organisms. This widespread role of circadian rhythms is typically characterized at the molecular level by profiling the transcriptome at multiple time points. Subsequent analysis identifies transcripts with altered rhythms between control and perturbed conditions, i.e., are differentially rhythmic (DiffR). Commonly, Venn Diagram analysis (VDA) compares lists of rhythmic transcripts to catalog transcripts with rhythms in both conditions or have gained or lost rhythms. However, unavoidable errors in the rhythmicity detection propagate to the final DiffR classification resulting in overestimated DiffR. We show using artificial experiments constructed from biological data that VDA indeed produces excessive false DiffR hits both in the presence and absence of true DiffR transcripts. We present a hypothesis testing and a model selection approaches in an R-package compareRhythms that instead compare circadian amplitude and phase of transcripts between the two conditions. These methods identify transcripts with gain, loss, change or have the same rhythms; the third category is missed by VDA. We reanalyzed three studies on the interplay between metabolism and the clock in the mouse liver that used VDA. We found not only fewer DiffR transcripts than originally reported, but VDA overlooked many relevant DiffR transcripts. Our analyses confirmed some and contradicted other conclusions in the original studies and also generated novel hypotheses. Our insights also generalize easily to studies using other -omics technologies. We trust that avoiding Venn Diagrams and using our R-package will contribute to improved reproducibility in chronobiology.

bioinformatics↗

A Modular Workflow for Model Building, Analysis, and Parameter Estimation in Systems Biology and Neuroscience

Neuroscience incorporates knowledge from a range of scales, from molecular dynamics to neural networks. Modeling is a valuable tool in understanding processes at a single scale or the interactions between two adjacent scales and researchers use a variety of different software tools in the model building and analysis process. While systems biology is among the more standardized fields, conversion between different model formats and interoperability between various tools is still somewhat problematic. To offer our take on tackling these shortcomings and by keeping in mind the FAIR (findability, accessibility, interoperability, reusability) data principles, we have developed a workflow for building and analyzing biochemical pathway models, using pre-existing tools that could be utilized for the storage and refinement of models in all phases of development. We have chosen the SBtab format which allows the storage of biochemical models and associated data in a single file and provides a human readable set of syntax rules. Next, we implemented custom-made MATLAB(R) scripts to perform parameter estimation and global sensitivity analysis used in model refinement. Additionally, we have developed a web-based application for biochemical models that allows simulations with either a network free solver or stochastic solvers and incorporating geometry. Finally, we illustrate convertibility and use of a biochemical model in a biophysically detailed single neuron model by running multiscale simulations in NEURON. Using this workflow, we can simulate the same model in three different simulators, with a smooth conversion between the different model formats, enhancing the characterization of different aspects of the model. Information Sharing StatementBoth the source code and documentation of the Subcellular Workflow are available at https://github.com/jpgsantos/Subcellular_Workflow and licensed under GNU General Public License v3.0. The model is stored in the SBtab format (Lubitz et al. 2016). Model reduction, parameter estimation and global sensitivity analysis tools are written in MATLAB(R) (RRID:SCR_001622) and require the SimBiology(R) toolbox. Conversion script to VFGEN (Weckesser 2008), MOD and SBML (RRID:SCR_007422) is written in R (RRID:SCR_001905). Conversion to SBML requires the use of libSBML (RRID:SCR_014134). Validations are run in COPASI (RRID:SCR_014260; Hoops et al. 2006), NEURON (RRID:SCR_005393; Hines and Carnevale 1997) and with the subcellular simulation setup application (RRID:SCR_018790; available at https://subcellular.humanbrainproject.eu/model/simulations) that uses a spatial solver provided by STEPS (RRID:SCR_008742; Hepburn et al. 2012) and network-free solver NFsim (available at http://michaelsneddon.net/nfsim/). The medium spiny neuron model (Lindroos et al. 2018) used in NEURON simulations is available in ModelDB database (RRID:SCR_007271) with access code 237653. The FindSim use case model is available in https://github.com/BhallaLab/FindSim (Viswan et al. 2018).

systems biology↗

Regular Tai Chi Practice Is Associated with Improved Memory as well as Structural and Functional Integrity of the Hippocampal Formation in the Elderly

ObjectiveThe current study aimed at determining effects of Tai Chi as an example of a combined motor-cognitive exercise relative to regular walking as an example of an exercise without cognitive demands on cognitive functioning and the functional and structural integrity of the brain in the elderly. MethodsHealthy elderly women with at least 6 years of regular Tai Chi or brisk walking exercise were recruited and underwent cognitive assessment via the Montreal Cognitive Assessment and brain structural and resting state functional MRI assessments. ResultsEpisodic memory in Tai Chi group was superior to that of the walking group; (2) higher gray matter density in inferior and medial temporal regions, including the hippocampal formation; (3) higher ReHo in temporal regions, specifically the fusiform gyrus and hippocampal formation (4) significant partial correlations were found between the gray matter density of the left hippocampus and episodic memory in the whole sample (5) significant partial correlations were observed between the ReHo in left hippocampus, left parahippocampal, left fusiform and delayed memory task was observed among all subjects. ConclusionThe present study suggest that long-term Tai Chi practice may improve memory performance via remodeling structure and the function of the hippocampal formation.

neuroscience↗

A Novel Therapeutic Approach to Corneal Alkaline Burn Model by Targeting Fidgetin-like 2, a Microtubule Regulator

Purpose To determine the efficacy of nanoparticle-encapsulated FL2 siRNA (FL2-NPsi), a novel therapeutic agent targeting the Fidgetin-like 2 (FL2) gene, for the treatment of corneal alkaline chemical injury.Methods Eighty 12-week-old, male Sprague-Dawley rats were divided evenly into 8 treatment groups: prednisolone, empty nanoparticles, control-NPsi (1 μM, 10 μM, 20 μM) and FL2-NPsi (1 μM, 10 μM, 20 μM). An alkaline burn was induced onto the cornea of each rat, which was then treated for 14 days according to group assignment. Clinical (N=10 per group), histopathologic (N=6 per group), and immunohistochemical (N=4 per group) analyses were conducted to assess for wound healing. FL2-NPsi-mediated knockdown of FL2 was confirmed by in vitro qPCR. Toxicity assays were performed to assess for apoptosis (TUNEL assay, N=3 per group) and nerve damage (whole mount immunochemical staining, N=2 per group). Statistical analyses were performed using student’s t-test and ANOVA.Results Compared to controls, FL2-NPsi-treated groups demonstrated enhanced corneal wound healing, with the 10 and 20 μM FL2-NPsi-treated groups demonstrating maximum rates of corneal re-epithelialization (p=0.0003 at Day 4 and p<0.0001 at Day 8) as assessed by ImageJ software, enhanced corneal transparency, and improved stromal organization on histology. Immunohistochemical analysis of vascular endothelial cells, macrophages, and neutrophils did not show significant differences between treatment groups. FL2-NPsi was not found to be toxic to nerves or induce apoptosis (p=0.917).Conclusion Dose-response studies found both 10 and 20 μM FL2-NPsi to be efficacious in this rat model. FL2-NPsi may offer a novel treatment for corneal alkaline chemical injuries.Competing Interest StatementDr. Wang, Dr. Kramer, Dr. Baker, and Dr. Dey are employees of MicroCures, Inc. Dr. Sharp is co-founder and Chief Scientific Officer for MicroCures, Inc and is the inventor of U.S. Patent #20130022667 entitled "Fidgetin-like 2 as a target to enhance wound healing,” which has been licensed by MicroCures. Dr. Chuck is a scientific advisor for MicroCures, Inc. Dr. Friedman holds stock in MicroCures. No other author has a conflict of interest.View Full Text

physiology↗

Fidgetin-like 2 is a novel negative regulator of axonal growth and can be targeted to promote functional nerve regeneration after injury.

The microtubule (MT) cytoskeleton plays a critical role in axon growth and guidance. Here, we identify the MT severing enzyme fidgetin-like 2 (FL2) as a negative regulator of axonal regeneration and a potential therapeutic target for promoting neural regeneration after injury. Genetic knockout of FL2 in cultured adult dorsal root ganglion (DRG) neurons resulted in longer axons and attenuated growth cone retraction in response to inhibitory molecules. Given the axonal growth-promoting effects of FL2 depletion in vitro, we tested whether the enzyme could be targeted to promote regeneration in a rodent model of peripheral nerve injury. In the model used in our experiments, the cavernous nerves (CN) are either crushed or transected, mimicking nerve injury caused by radical prostatectomy (RP). As with patients, CN injury results in erectile dysfunction, for which there are presently poor treatment options. At the time of injury, FL2-siRNA or control-siRNA was applied to the site using nanoparticles or chondroitin sulfate microgels as delivery agents. Treatment significantly enhanced functional nerve recovery, as determined by cavernosometry (measurements of corporal blood pressure in response to electrostimulation of the nerve). Remarkably, following complete bilateral nerve transection, visible and functional nerve regeneration was observed in 7 out of 8 animals treated with FL2-siRNA. In contrast, no control-siRNA treated animals showed regeneration. These observations suggest a novel therapeutic approach to treat peripheral nerve injury, particularly injuries resulting from surgical procedures such as RP, where treatments depleting FL2 could be applied locally at the time of injury.

neuroscience↗

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

The current model of the mammalian circadian oscillator is predominantly based on data from genetics and biochemistry experiments, while the cell biology of circadian clocks is still in its infancy. Here, we describe a new strategy for the efficient generation of knock-in reporter cell lines using CRISPR technology that is particularly useful for lowly or transiently expressed genes, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins, which allowed to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed ([~]5 hours), and CRY1 levels are much higher (>6 times) compared to PER2 questioning the current model of the circadian oscillator. Our knock-in strategy will allow the generation of additional single, double or triple knock-in cells for circadian clock proteins, which should greatly advance our understanding about the cell biology of circadian clocks.

cell biology↗

Susceptibility rhythm to bacterialendotoxin in myeloid clock-knockout mice

Local circadian clocks are active in most cells of our body. However, their impact on circadian physiology is still under debate. Mortality by endotoxic (LPS) shock is highly time-of-day dependent and local circadian immune function such as the cytokine burst after LPS challenge has been assumed to be causal for the large differences in survival. Here, we investigate the roles of light and myeloid clocks on mortality by endotoxic shock. Strikingly, mice in constant darkness (DD) show a three-fold increased susceptibility to LPS as compared to mice in light-dark conditions. Mortality by endotoxic shock as a function of circadian time is independent of light-dark cycles as well as myeloid CLOCK or BMAL1 as demonstrated in conditional knockout mice. Unexpectedly, despite the lack of a myeloid clock these mice still show rhythmic patterns of pro- and anti-inflammatory cytokines such as TNF, MCP-1, IL-18 and IL-10 in peripheral blood as well as time-of-day and site dependent traffic of myeloid cells. We speculate that systemic time-cues are sufficient to orchestrate innate immune response to LPS by driving immune functions such as cell trafficking and cytokine expression.

immunology↗

Circadian period-heterogeneity is governed by clonal inheritance of variable gene expression

A ubiquitous feature of circadian clocks across life forms is its organization as a network of coupled cellular oscillators. Individual cellular oscillators of the network often exhibit a considerable degree of heterogeneity in their intrinsic periods. While the interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clocks entrainability, our knowledge of mechanisms governing network heterogeneity remains elusive. In this study, we aimed to explore the principles that underlie inter-cellular period variation in circadian clock networks (clonal period-heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 h to 28 h. We report that while a single parent clone can produce progeny clones with a wide distribution of circadian periods, heterogeneity is not entirely stochastically driven but has a strong heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our panel, we found that inheritance of different expression patterns in at least three clock genes might govern clonal period-heterogeneity in circadian clock networks. Furthermore, we provide preliminary evidence suggesting that epigenetic variation might underlie such gene expression variation.

cell biology↗

A Robust Model for Circadian Redox Oscillations

The circadian clock is an endogenous oscillator that controls daily rhythms in metabolism, physiology and behavior. Although the timekeeping components differ among species, a common design principle is a transcription-translation negative feedback loop. However, it is becoming clear that other mechanisms can contribute to the generation of 24 h rhythms. In mammalian adrenal gland, heart and brown adipose tissue, peroxiredoxins (Prx) exhibit 24 h rhythms in their redox state. Such rhythms are generated as a result of an inactivating hyperoxidation reaction that is reduced by coordinated import of the sulfiredoxin (Srx) into the mitochondria. However, a quantitative description of the Prx/Srx oscillating system is still missing. We investigate the basic principles that generate mitochondrial Prx/Srx rhythms using computational modeling. We observe that the previously described delay in mitochondrial Srx import, in combination with an appropriate separation of fast and slow reactions is sufficient to generate robust self-sustained relaxation-like oscillations. We find that our conceptual model can be regarded as a series of three consecutive phases and two temporal switches, highlighting the importance of delayed negative feedback and switches in the generation of oscillations.

molecular biology↗

Co-existing feedback loops generate tissue-specific circadian rhythms

Gene regulatory feedback loops generate autonomous circadian rhythms in mammalian tissues. The well-studied core clock network contains many negative and positive regulations. Multiple feedback loops have been discussed as primary rhythm generators but the design principles of the core clock and differences between tissues are still under debate.\n\nHere we use global optimization techniques to fit mathematical models to circadian gene expression profiles for different mammalian tissues. It turns out that for every investigated tissue multiple model parameter sets reproduce the experimental data. We extract for all model versions the most essential feedback loops and find auto-inhibitions of Period and Cryptochrome genes, Bmal1/Rev-erb- loops, and repressilator motifs as possible rhythm generators. Interestingly, the essential feedback loops differ between tissues, pointing to specific design principles within the hierarchy of mammalian tissue clocks. Self-inhibitions of Per and Cry genes are characteristic for models of SCN clocks, while in liver models many loops act in synergy and are connected by a repressilator motif. Tissue-specific use of a network of co-existing synergistic feedback loops could account for functional differences between organs.

systems biology↗

Uncertainty quantification, propagation and characterization by Bayesian analysis combined with global sensitivity analysis applied to dynamical intracellular pathway models

MotivationDynamical models describing intracellular phenomena are increasing in size and complexity as more information is obtained from experiments. These models are often over-parameterized with respect to the quantitative data used for parameter estimation, resulting in uncertainty in the individual parameter estimates as well as in the predictions made from the model. Here we combine Bayesian analysis with global sensitivity analysis in order to give better informed predictions; to point out weaker parts of the model that are important targets for further experiments, as well as give guidance on parameters that are essential in distinguishing different qualitative output behaviours.\n\nResultsWe used approximate Bayesian computation (ABC) to estimate the model parameters from experimental data, as well as to quantify the uncertainty in this estimation (inverse uncertainty quantification), resulting in a posterior distribution for the parameters. This parameter uncertainty was next propagated to a corresponding uncertainty in the predictions (forward uncertainty propagation), and a global sensitivity analysis was performed on the prediction using the posterior distribution as the possible values for the parameters. This methodology was applied on a relatively large and complex model relevant for synaptic plasticity, using experimental data from several sources. We could hereby point out those parameters that by themselves have the largest contribution to the uncertainty of the prediction as well as identify parameters important to separate between qualitatively different predictions.This approach is useful both for experimental design as well as model building.

systems biology↗