Biology subjects

Fedotova, A.

Publications and source records attributed to Fedotova, A..

9 recordsLinked to original sources

bioRxiv2025

Four phases and temporal threshold of population calcium response during locomotion in cortical astrocytes

Calcium activity is a major form of astrocyte excitability essential to their function and plasticity. Astrocytic calcium events arise both spontaneously and in response to behaviour 1. In this study, we examined how these unitary events shape population-level calcium activity during quiescence and locomotion. Using two-photon imaging of astrocytic calcium in the primary somatosensory cortex (S1) of awake mice running on a freely rotating disk, we found that spontaneous fluctuations during quiescence were driven mainly by the co-occurrence of new calcium events rather than by their enlargement. During locomotion, population responses progressed through four phases: the emergence of new events, their merging into a superevent, fragmentation of this superevent, and an afterburst. New events appeared immediately at locomotion onset, indicating a rapid astrocytic response. The response also exhibited a temporal threshold of [~]5 seconds: when locomotion was shorter than this, the superevent phase failed to develop, and the population response was markedly reduced. In suprathreshold responses, the superevent displayed a {Delta}F/F intensity pattern that reproduced reliably across locomotion episodes. Hotspots within this pattern had shorter latencies and were also more active during quiescence, suggesting that the response is at least partly deterministic. Consistent with this, we were able to reproduce both the response phases and the superevent pattern using a dynamic mode decomposition with control (DMDc) model driven by synthetic or real locomotion inputs. Together, these findings dissect the population calcium response into quantifiable properties of unitary calcium events and reveal the formation and reproducibility of the superevent pattern, offering a potential framework for the astrocytic component of the engram 2,3.

neuroscience↗

bioRxiv2025

Computational modeling of necrosis in neural organoids

Neural organoids (NOs), also known as brain organoids, are derived from human-induced pluripotent stem cells and are Microphysiological Systems (MPS) of the brain that can recapitulate key aspects of neurodevelopment. They enable in vitro studies of brain development and disease mechanisms, providing disease models for various neurodegenerative or neurodevelopmental/degenerative disorders like Alzheimers disease, microcephaly, and autism. There are many protocols to generate NOs with different complexities and sizes, varying from 400 m to several mm in diameter, with a starvation-induced necrotic core eventually forming depending on the diameter and culture conditions. Thus, they can benefit from vascularization and more optimal culture conditions. There have been several attempts to decrease necrosis while growing larger NOs, such as by using orbital shaking or 2D/3D microfluidic chips, but only with limited success. In this study, we describe a 3D finite element model to simulate O2 starvation-induced necrosis in NOs using the Damkohler Number (Da) and the Michaelis-Menten kinetics. We measured the necrotic areas in NOs using fluorescent imaging and used them to calibrate the model with a specific Da. Using these calibrated values, we systematically compared simulations of different NO culture methods--static, orbital shaking, and microfluidic flow around organoids--highlighting their relative impacts on nutrient diffusion and necrosis. We observed that these culture strategies cannot prevent necrosis beyond a diameter of [~]800 m. Based on these findings, we propose that 3D spatial perfusion, achieved through uniformly distributed fluidic capillaries within the NO, could significantly reduce necrosis. We conducted parametric studies on capillary spacing, density, and layout. Our calibrated model offers insights for designing next-generation microfabricated bioreactors and culture devices, not just for NOs but also for all 3D tissue engineering and organoid research.

bioengineering↗

bioRxiv2024

Cerebral perfusion and metabolic response of astrocytes and neurons during locomotion

The hemodynamic response links neural oxidative metabolism changes to increased cerebral blood flow during brain activity1. Although Roy and Sherrington introduced this concept over a century ago (1890), the exact cellular mechanisms remain unclear. This study demonstrates how local blood supply increases correlate with the metabolic response of individual brain cells during locomotion. Using Raman microspectroscopy, we observed an elevation in oxygen saturation levels (sO2) in cortical venules but not in arterioles, which were already near saturation. The increased sO2 in the venules was accompanied by vasodilation, indicating blood oversupply in the local brain area, a phenomenon known as functional hyperemia. We then analyzed the metabolic response of individual neurons and astrocytes to locomotion. In neurons, the levels of reduced cytochromes of c and b types [cyt c,b (Fe2+)] rapidly decreased at the onset of locomotion. This suggests an increase in the activity of the mitochondrial respiratory chain (electron transport chain, ETC) in response to heightened energy demands in these cells2. In contrast, because astrocytes rely less on oxidative phosphorylation for their energy metabolism than neurons3, we did not observe an initial decrease in reduced cytochromes in these cells. However, as locomotion continued, the cyt c,b (Fe2+) levels steadily increased in both cell types. In neurons, this led to a slow recovery from the initial drop, while in astrocytes, the increase exceeded baseline levels. Consequently, we observed an overload of electrons in the astrocytic ETC. The distinct responses of astrocytic and neuronal mitochondria to locomotion may reflect differences in the organization of the ETC in the two cell types4. Furthermore, astrocytes may shift to glycolysis under increased neuronal activity5,6. This difference also resulted in hydrogen peroxide (H202) production in astrocytic mitochondria but not neuronal mitochondria. Since H202 is a signaling molecule critical for cognitive function in the brain7, we speculate that astrocytic mitochondria act as signaling hubs during exercise.

neuroscience↗

bioRxiv2024

Yttrium-90-doped metal-organic frameworks (MOFs) for low-dose rate intratumoral radiotherapy

Brachytherapy, or intratumoral radiation therapy, is a highly effective treatment option for localized tumors. Herein, we engineered injectable and biodegradable metal-organic frameworks (MOFs) to deliver the therapeutic radioisotope yttrium-90 (90Y). Particles of bimetallic MIL-100(Fe,Y) and Y-BTC, doped with 90Y and 88Y, were synthesized in a single step and retained radioyttrium in various buffer solutions. Tumor injectability and radioisotope retention were evaluated using tumor-bearing mice. In vivo analysis and calculations showed that radiolabeled MIL-100(Fe,Y) emitted more than 38% of its radioactivity, while Y-BTC emitted greater than 75% of its radioactivity, through 7 days at the tumor site upon intratumoral injection, without significant yttrium accumulation in off-target tissues. The anticancer effects of MIL-100(Fe,Y,90Y) and 90Y,Y-BTC particles were assessed using 3D multicellular tumor spheroids and a tumor-bearing mouse model, respectively. 90Y-doped MIL-100(Fe,Y) particles penetrated A549 tumor spheroids and caused superior cytotoxic effects compared to non-radioactive particles or 90YCl3, added at the same dose. Brachytherapy with 90Y-doped Y-BTC MOFs induced inhibition of B16F1 melanoma tumor growth and resulted in an increased median survival of 8.5 days compared to 4.5 days in untreated mice. This study exhibits the feasibility to prepare radioactive 90Y-containing biodegradable, non-toxic MOF particles that are advantageous for low-dose rate internal radiotherapy.

biophysics↗

bioRxiv2024

The first chicken oocyte nucleus whole transcriptomic profile defines the spectrum of maternal mRNA and non-coding RNA genes transcribed by the lampbrush chromosomes

Lampbrush chromosomes, with their unusually high rate of nascent RNA synthesis, provide a valuable model for studying the mechanisms of global transcriptome up-regulation. Here, we aimed to establish a full set of sequences transcribed on the lateral loops of chicken lampbrush chromosomes. For the first time, a whole-genomic profile of transcription along the entire length of all lampbrush chromosomes in the chicken karyotype including sex chromosomes and dot chromosomes was obtained. For that, we performed RNA-seq of oocyte nuclear and cytoplasmic total RNA, poly(A) RNA and small RNA libraries and aligned expressed RNA sequences against the chicken genome. This led to the identification of a full spectrum of maternal RNAs that accumulate in the nucleus and cytoplasm of chicken oocytes at the lampbrush chromosome stage, are then transferred to the zygote and can be used during the early stages of embryogenesis, including transcripts of protein-coding genes, long non-coding RNAs and small housekeeping non-coding RNAs. We also present the first high-throughput transcriptome characterisation of miRNAs and piRNAs in chicken oocytes at the lampbrush chromosome stage. Major targets of predicted piRNAs include CR1 and LTR containing retrotransposable elements. Transcription of tandem repeat arrays including 41bp higher-order repeats as well as unique centromere sequences was demonstrated by alignment against the whole telomere-to-telomere chromosome assemblies. We show that transcription of telomere-derived RNAs, including telomeric repeat-containing RNA (TERRA) and subtelomeric repeat-containing RNA, is initiated at adjacent LTR elements. With nuclear RNA-seq, we obtained information about a wider set of transcripts, including long non-coding RNAs retained in the nucleus and stable intronic sequence RNAs (sisRNAs). We identified 5 UTR sisRNAs that may potentially support host gene transcription both during oogenesis and after activation of the embryonic genome. For a number of protein-coding genes, we visualised nascent transcripts and demonstrated their co-transcriptional splicing on the lateral loops of lampbrush chromosomes by RNA-FISH. The nuclear RNA-seq profile predicted the chromomere-loop organisation of the genomic regions. During oocyte maturation, transcriptional unit boundaries were maintained, while transcriptional output tended to decrease. We conclude that cytoplasmic and nuclear transcripts emerge from nascent transcripts on the lateral loops of lampbrush chromosomes. Most of gene transcripts are initiated at promoters, normally spliced, terminated and polyadenylated. The set of genes transcribed on the lampbrush chromosomes is required for basic cellular processes, is characterised by a broad expression pattern and is similar to sets of genes expressed in other hypertranscriptional systems. We conclude that hypertranscription on the lateral loops of giant lampbrush chromosomes is the main mechanism for synthesising large amounts of transferred to the embryo maternal RNA for thousands of genes.

cell biology↗

bioRxiv2023

Combining CRISPR/Cas mediated terminal resolution with a novel genetic workflow to achieve high diversity adenoviral libraries

While recombinant Adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated in vivo terminal resolution (CTR) aiding high efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based rAd-libraries employing the efficiency of CTR. We utilized frequent, pre-existing genomic sequences to allow insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In a second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement (HFR) allows introduction of >106 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of [~]2.5x104 modified rAd per cm2 of transfected cell culture. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/566979v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1db61a7org.highwire.dtl.DTLVardef@1e85764org.highwire.dtl.DTLVardef@c17178org.highwire.dtl.DTLVardef@a299_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Global nuclear reorganization during heterochromatin replication in the giant-genome plant Nigella damascena L.

Among flowering plants, genome size varies remarkably, by >2200-fold, and this variation depends on the loss and gain of non-coding DNA sequences that form distinct heterochromatin complexes during interphase. In plants with giant genomes, most chromatin remains condensed during interphase, forming a dense network of heterochromatin threads called interphase chromonemata. Using super-resolution light and electron microscopy, we studied the ultrastructure of chromonemata during and after replication in root meristem nuclei of Nigella damascena L. During S-phase, heterochromatin undergoes transient decondensation locally at DNA replication sites. Due to the abundance of heterochromatin, the replication leads to a robust disassembly of the chromonema meshwork and a general reorganization of the nuclear morphology visible even by conventional light microscopy. After replication, heterochromatin recondenses, restoring the chromonema structure. Thus, we show that heterochromatin replication in interphase nuclei of giant-genome plants induces a global nuclear reorganization.

cell biology↗

bioRxiv2021

A high-fat diet changes astrocytic metabolism to enhance synaptic plasticity and promote exploratory behavior

A high-fat diet (HFD) is generally considered to negatively influence the body, the brain, and cognitive abilities. On the other hand, fat and fatty acids are essential for nourishing and constructing brain tissue. Astrocytes are central for lipolysis and fatty acids metabolism. Here we show that exposure of young mice to one month of HFD elevates lipid content and increases the relative amount of reduced cytochromes in astrocytes but not in neurons. Metabolic changes were paralleled with an enlargement of astrocytic territorial domains due to an increased outgrowth of branches and leaflets. Astrocyte remodeling was associated with an increase in expression of ezrin and with no changes in glial fibrillary acidic protein (GFAP), glutamate transporter-1 (GLT-1), and glutamine synthetase (GS). Such physiological (non-reactive) enlargement of astrocytes in the brain active milieu promoted glutamate clearance and long-term potentiation. These changes translated into improved exploratory behavior. Thus, dietary fat intake is not invariably harmful and might exert beneficial effects depending on the biological context. In BriefA high-fat diet stimulates the metabolism and growth of astrocytes, which improves glutamate clearance, synaptic plasticity, and exploratory behavior in young mice. Thus, dietary fat arguably is an essential component of the diet for children and young adults, supporting the optimal development of the brain. HighlightsO_LIExposure of young mice to a high-fat diet elevated lipid content and increased amount of reduced cytochromes in astrocytes but not in neurons. C_LIO_LIMetabolic changes were paralleled with an enlargement of astrocytic territorial domains due to an increased outgrowth of branches and leaflets. C_LIO_LIAstrocytic enlargement was associated with increased expression of ezrin but not GFAP, hence was not reactive but physiological C_LIO_LIExpansion of astrocytes in the brain active milieu improved glutamate clearance and long-term potentiation. C_LIO_LIThe high-fat diet improved exploratory behavior in young mice. C_LI

neuroscience↗

bioRxiv2018

CLAMP directly interacts with MSL2 to facilitate Drosophila dosage compensation

The binding of Drosophila male-specific lethal (MSL) dosage compensation complex exclusively to male X chromosome provides an excellent model system to understand mechanisms of selective recruitment of protein complexes to chromatin. Previous studies showed that the male-specific organizer of the complex, MSL2, and ubiquitous DNA-binding protein CLAMP are key players in the specificity of X chromosome binding. The CXC domain of MSL2 binds to genomic sites of MSL complex recruitment. Here we demonstrated that MSL2 directly interacts with the N-terminal zinc-finger domain of CLAMP. CLAMP-MSL2 and CXC-DNA interactions are cooperatively involved in recruitment of MSL complex to the X chromosome.

molecular biology↗