Biology subjects

Winklhofer, M.

Publications and source records attributed to Winklhofer, M..

7 recordsLinked to original sources

bioRxiv2025

Long Non-Coding RNA Transcripts in Crucian Carp Brain - Annotation and Expression Patterns in Anoxia and Reoxygenation

Long non-coding RNA transcripts (lncRNAs) are important regulators of various cellular processes, including gene expression. However, whether they are involved in the regulation of anoxia-induced transcriptomic changes in crucian carp (Carassius carassius), a species that can survive without oxygen for months at low temperatures, has remained unexplored. The existing genome annotation includes mainly protein-coding genes, which are more straightforward to annotate than the lower-expressed lncRNAs. Therefore, this study aimed to annotate lncRNAs in the crucian carp genome, investigate their potential involvement in regulating mRNA abundance in response to anoxia and reoxygenation, and further characterize any differentially regulated lncRNAs and their potential interaction partners (nearby genes). Using next-generation RNA sequencing of brain samples from crucian carp across normoxia, anoxia, and reoxygenation conditions (n=10 per group), 145,264 transcripts were assembled with a reference-guided approach. Using the bioinformatic tools CPC2, CNCI, CPAT, and FEELnc, we identified 6,072 lncRNAs among the assembled transcripts. We observed a substantial transcriptomic response to anoxia, with a total of 56,440 differentially expressed (DE) transcripts (adjusted p-value < 0.05; | log2(fold change)| > 0.38), including 1,321 lncRNAs. The FEELnc classification module identified multiple proximal RNA transcripts that may help to predict lncRNA functions, and due to their proximity to potential interaction partners (IPs), were likely cis-acting. Most (60%) of the interaction sites between DElncRNAs and their targets were located between genes (intergenic), predominantly on the same strand in intergenic subtypes, and primarily nested in the genic context. The log2-fold changes in RNA abundance of DE predicted IPs exhibited a positive correlation with the log2-fold changes of their corresponding DElncRNAs. These findings indicate that lncRNAs are at least co-expressed with and may regulate transcription of specific genes in crucian carp during anoxia and reoxygenation.

bioinformatics↗

bioRxiv2025

Changes in DNA Methylation During Anoxia and Reoxygenation in Crucian Carp Brain

Oxygen is essential for cellular metabolism, and its absence poses a severe risk to vertebrates. However, the crucian carp (Carassius carassius) can survive without oxygen for months due to physiological adaptations. This study investigates the role of epigenomic regulation, specifically DNA methylation, in response to anoxia and subsequent reoxygenation. Brain samples from crucian carp subjected to normoxia, anoxia, and reoxygenation (n = 4 per condition) were analyzed through transcriptome (mRNA) and whole-genome bisulfite sequencing. Results revealed a notable transcriptional response to anoxia, with 1,265 differentially expressed genes (DEGs) identified compared to normoxia. Using the machine-learning-based MethylScore, we found 14 genes with differentially methylated regions (DMRs) during anoxia. These DMR-associated genes did not meet the strict DEGs criteria (adjusted p-value < 0.05; absolute log2fold-change > 0.38), but 17 genes exhibited differences in mRNA abundance when focusing solely on DMRs. DMRs were primarily located within transcriptional start sites, and analysis of protein products revealed that genes involved in transcription regulation, protein trafficking, and signal transduction were differentially methylated and expressed under varying conditions. This study advances our understanding of epigenomic regulation in oxygen deprivation and highlights the potential in exploring alternative epigenomic mechanisms.

physiology↗

bioRxiv2024

Full-length Cryptochrome 1 in the outer segments of the retinal blue cone photoreceptors in humans and great apes suggests a role beyond transcriptional repression

Mammalian cryptochrome 1 (CRY1) is a central player in the circadian transcription-translation feedback loop, crucial for maintaining a roughly 24-hour rhythm. CRY1 was suggested to also function as blue-light photoreceptor in humans and has been found to be expressed at the mRNA level in various cell types of the inner retina. However, attempts to detect CRY1 at the protein level in the human retina have remained unsuccessful so far. Using various C-terminal specific antibodies recognizing full-length CRY1 protein, we consistently detected selective labelling in the outer segments of short wavelength-sensitive (SWS1, "blue") cone photoreceptor cells across human, bonobo, and gorilla retinae. No other retinal cell types were stained, which is in contrast to what would be expected of a ubiquitous clock protein. Subcellular fractionation experiments in transfected HEK cells using a C-terminal specific antibody located full-length CRY1 in the cytosol and membrane fractions. Our findings indicate that human CRY1 has several different functions including at least one non-clock function. Our results also raise the likely possibility that several different versions of CRY1 exists in humans. We suggest that truncation of the C-terminal tail, maybe to different degrees, may affect the localization and function of human CRY1.

cell biology↗

bioRxiv2023

Pseudoreplication and inappropriate statistical tests in the analysis of preference index data from the T-maze group assay for Drosophila behaviour

The binary choice chamber (T-maze) assay has been used as a standard behavioural screen in Drosophila research to test mutants in terms of sensory discrimination skills and synaptic plasticity during memory consolidation and decay. Typically, ca. 100 individuals are tested as a group at the same time and the behavioural readout consists in counting the number of individuals in the testing tube exposed to a stimulus versus the number of flies in the control tube, with the normalized difference in fly count being defined as the batch preference index (PI). Unfortunately, the batch PI has widely been taken as a precise metric of group behaviour, to the point where ANOVA/t-tests have been considered the most powerful statistical tests for analyzing samples in terms of batch PI values, which has led to a hyperinflation of apparently very highly significant effects for small differences in PI values (e.g. -0.1 vs 0.1), leading to problems with replicability. Here it is shown on the basis of a well-established statistical model for binary decisions that application of the t-test to PI data implicitly assumes that each fly in a batch is an independent biological replicate in the extremely strict sense that the decision of each individual was interrogated independently of the other flies. Therefore, t-test analysis of PI data obtained with the intrinsically pseudoreplicative group assay is the cause of extremely optimistic P-values, as suggested recently by Bassetto et al. (2023) on the basis of effect size considerations for proportions. Thus, rather than using inferential statistics, PI data should be assessed on the basis of effect size. A more fundamental problem with the batch PI value is the uncertainty in whether it measures the mean individual preference and if so, with what precision, given the simple readout? This aspect is illustrated here by modelling distributions of flies in the T-maze, which suggest that the effective precision of the PI value is clearly worse than the nominal precision of +/- 0.01 for 100 flies, so that the batch PI can only serve as a rough indicator of group tendencies.

animal behavior and cognition↗

bioRxiv2022

Integration and evaluation of magnetic stimulation in physiology setups

A large number of behavioral experiments have demonstrated the existence of a magnetic sense in many animal species. Further, studies with immediate gene expression markers have identified putative brain regions involved in magnetic information processing. In contrast, very little is known about the physiology of the magnetic sense and how the magnetic field is neuronally encoded. In vivo electrophysiological studies reporting neuronal correlates of the magnetic sense either have turned out to be irreproducible for lack of appropriate artifact controls or still await independent replication. Thus far, the research field of magnetoreception has little exploited the power of ex vivo physiological studies, which hold great promise for enabling stringent controls. However, tight space constraints in a recording setup and the presence of magnetizable materials in setup components and microscope objectives make it demanding to generate well-defined magnetic stimuli at the location of the biological specimen. Here, we present a solution based on a miniature vector magnetometer, a coil driver, and a calibration routine for the coil system to compensate for magnetic distortions in the setup. The magnetometer fits in common physiology recording chambers and has a sufficiently small spatial integration area to allow for probing spatial inhomogeneities. The coil-driver allows for the generation of defined non-stationary fast changing magnetic stimuli. Our ex vivo multielectrode array recordings from avian retinal ganglion cells show that artifacts induced by rapid magnetic stimulus changes can mimic the waveform of biological spikes on single electrodes. However, induction artifacts can be separated clearly from biological responses if the spatio-temporal characteristics of the artifact on multiple electrodes is taken into account. We provide the complete hardware design data and software resources for the integrated magnetic stimulation system.

neuroscience↗

bioRxiv2022

A magnetic pulse does not affect free-flight navigation behaviour of a medium-distance songbird migrant in spring

Current evidence suggests that migratory animals extract map information from the geomagnetic field for true navigation. The sensory basis underlying this feat is elusive, but presumably involves magnetic particles. A common experimental manipulation procedure consists of pre-treating animals with a magnetic pulse. This aims at re-magnetising particles to alter the internal representation of the external field prior to a navigation task. While pulsing provoked deflected bearings in laboratory experiments, analogous studies with free-flying songbirds yielded inconsistent results. Here, we pulsed European robins (Erithacus rubecula), being medium-distance migrants, at an offshore stopover site during spring migration and monitored their free-flight behaviour with a regional-scale tracking system. We found no pulse effect on departure probability, nocturnal departure timing, or departure direction, in agreement with results on a long-distance migrant released at the same site in autumn. This necessitates a reassessment of the importance of geomagnetic maps for migratory decisions for free-flying birds. Summary statementMagnetic pulse pre-treatment disturbs geomagnetic map usage of birds in lab environments. However, our free-flying birds show no effect, suggesting geomagnetic map information is less important in the natural environment.

animal behavior and cognition↗

bioRxiv2016

A magnetic protein compass ?

Qin et al. (A magnetic protein compass, Nature Materials 15, 217-226, 2016) claim that \"MagR is the first known protein that carries an intrinsic magnetic moment at ambient temperature\". We show here that the claim must, unfortunately, be fundamentally wrong.

biophysics↗