Automated Image Analysis of Transmission Electron Micrographs: Nanoscale Evaluation of Radiation-Induced DNA Damage in the Context of Chromatin

Background: Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. Methods: Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. Results: Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. Conclusions: The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns

The Ontogeny of Osmoregulation in the Nile Tilapia (Oreochromis niloticus L.)

Abstract In recent times, diminishing freshwater resources, due to the rapidly increasing drain of urban, industrial and agricultural activities in combination with the impact of climate change, has led to an urgent need to manage marine and brackish water environments more efficiently. Therefore the diversification of aquacultural practices, either by the introduction of new candidate species or by the adaptation of culture methods for existing species, is vital at a time when innovation and adaptability of the aquaculture industry is fundamental in order to maintain its sustainability. The Nile tilapia (Oreochromis niloticus, Linnaeus, 1758), which has now been spread well beyond its natural range, dominates tilapia aquaculture because of its adaptability and fast growth rate. Although not considered to be amongst the most salt tolerant of the cultured tilapia species, the Nile tilapia still offers considerable potential for culture in low-salinity water. An increase in knowledge of the limits and basis of salinity tolerance of Nile tilapia during the sensitive early life stages and the ability to predict responses of critical life-history stages to environmental change could prove invaluable in improving larval rearing techniques and extend the scope of this globally important fish species. The capability of early life stages of the Nile tilapia to withstand variations in salinity is due to their ability to osmoregulate, therefore the ontogeny of osmoregulation in the Nile tilapia was studied from spawning to yolk-sac absorption after exposure to different experimental conditions ranging from freshwater to 25 ppt. Eggs were able to withstand elevated rearing salinities up to 20 ppt, but transfer to 25 ppt induced 100% mortality by 48 h post-fertilisation. At all stages embryos and larvae hyper-regulated at lower salinities and hypo-regulated at higher salinities, relative to the salinity of the external media. Osmoregulatory capacity increased during development and from 2 days post-hatch onwards remained constant until yolk-sac absorption. Adjustments to larval osmolality, following abrupt transfer from freshwater to experimental salinities (12.5 and 20 ppt), appeared to follow a pattern of crisis and regulation, with whole-body osmolality for larvae stabilising at c. 48 h post-transfer for all treatments, regardless of age at time of transfer. Age at transfer to experimental salinities (7.5 – 20 ppt) had a significant positive effect on larval ability to osmoregulate; larvae transferred at 8 dph maintained a more constant range of whole body osmolality over the experimental salinities tested than larvae at hatch. Concomitantly, survival following transfer to experimental salinities increased with age. There was a significant effect (GLM; p < 0.05) of salinity of incubation and rearing media on the incidence of gross larval malformation that was seen to decline over the developmental period studied. It is well established that salinity exerts a strong influence on development and growth in early life stages of fishes therefore the effects of varying low salinities (0 - 25 ppt) on hatchability, survival, growth and energetic parameters were examined in the Nile tilapia during early life stages. Salinity up to 20 ppt was tolerable, although reduced hatching rates at 15 and 20 ppt suggest that these salinites may be less than optimal. Optimum timing of transfer of eggs from freshwater to elevated salinities was 3-4 h post-fertilisation, following manual stripping and fertilisation of eggs, however increasing incubation salinity lengthened the time taken to hatch. Salinity was related to dry body weight, with larvae in salinities greater than 15 ppt displaying, at hatch, a significantly (GLM: p < 0.05) lower body weight but containing greater yolk reserves than those in freshwater or lower salinities. Survival at yolk-sac absorption displayed a significant (GLM; p < 0.05) inverse relationship with increasing salinity and mortalities were particularly heavy in the higher salinities of 15, 20 and 25 ppt. Mortalities occurred primarily during early yolk-sac development yet stabilised from 5 dph onwards. Salinity had a negative effect on yolk absorption efficiency (YAE). Salinity-related differences in oxygen consumption rates were not detectable until 3 days post-hatch; oxygen consumption rates of larvae in freshwater between days 3 – 6 post-hatch were always significantly higher (GLM p < 0.05) than those in 7.5, 15, 20 and 25 ppt, however, on day 9 post-hatch this pattern was reversed and freshwater larvae had a significantly lower QO2 than those in elevated salinities. Salinity had a significant inverse effect on larval standard length, with elevated salinities producing shorter larvae from hatch until 6 dph, after which time there was no significant differences between treatments. Salinity had a significant effect on whole larval dry weight, with heavier larvae in elevated salinities throughout the yolk-sac period (GLM; p < 0.05). The ability of the Nile tilapia to withstand elevated salinity during early life stages is due to morphological and ultrastructural modifications of extrabranchial mitochondria-rich cells (MRCs) that confer an osmoregulatory capacity before the development of the adult osmoregulatory system. A clearly defined temporal staging of the appearance of these adaptive mechanisms, conferring ability to cope with varying environmental conditions during early development, was evident. Ontogenetic changes in MRC location, 2-dimensional surface area, density and general morphological changes were investigated in larvae incubated and reared in freshwater and brackish water (15 ppt) from hatch until yolk-sac absorption using Na+/K+-ATPase immunohistochemistry with a combination of microscope techniques. The pattern of MRC distribution was seen to change during development under both treatments, with cell density decreasing significantly on the body from hatch to 7 days post-hatch, but appearing on the inner opercular area at 3 days post-hatch and increasing significantly (GLM; p < 0.05) thereafter. Mitochondria-rich cells were always significantly (GLM; p < 0.05) denser in freshwater than in brackish water maintained larvae. In both freshwater and brackish water, MRCs located on the outer operculum and tail showed a marked increase in size with age, however, cells located on the abdominal epithelium of the yolk-sac and the inner operculum showed a significant decrease in size (GLM; p < 0.05) over time. Mitochondria-rich cells from brackish water maintained larvae from 1 day post-hatch onwards were always significantly larger (GLM; p < 0.05) than those maintained in freshwater. Preliminary scanning electron microscopy studies revealed structural differences in chloride cell morphology that varied according to environmental conditions. Mitochondria-rich cell morphology and function are intricately related and the plasticity or adaptive response of this cell to environmental changes is vital in preserving physiological homeostasis and contributes to fishes’ ability to inhabit diverse environments. Yolk-sac larvae were transferred from freshwater at 3 days post-hatch to 12.5 and 20 ppt and sampled at 24 and 48 h post-transfer. The use of scanning electron microscopy allowed a quantification of MRC, based on the appearance and surface area of their apical crypts, resulting in a reclassification of ‘sub-types’ i.e. Type I or absorptive, degenerating form (surface area range 5.2 – 19.6 μm2), Type II or active absorptive form (surface area range 1.1 – 15.7 μm2), Type III or differentiating form (surface area range 0.08 – 4.6 μm2) and Type IV or active secreting form (surface area range 4.1 – 11.7 μm2). In addition, the crypts of mucous cells were discriminated from those of MRCs based on the presence of globular extensions and similarly quantified. Density and frequency of MRCs and mucous cells varied significantly (GLM; p < 0.05) according to the experimental salinity and according to time after transfer; in freshwater adapted larvae all types were present except Type IV but following transfer to elevated salinities, Type I and Type II crypts disappeared and appeared to be replaced by Type IV crypts. The density of Type III crypts remained constant following transfer. Immunogold labelling used in conjunction with transmission electron microscopy, using a novel, pre-fixation technique with anti-Na+/K+-ATPase and anti-CFTR, allowed complementary visualisation of specific localisation of the antibodies on active MRCs at an ultrastructural level, permitting a review of MRC apical morphology and related Na+/K+-ATPase binding sites. Further in depth investigations using immunohistochemistry on whole-mount larvae using Fluoronanogold™ (Nanoprobes, U.S.) as a secondary immunoprobe allowed fluorescent labelling with the high resolution of confocal scanning laser miscroscopy, combined with the detection of immunolabelled target molecules at an ultrastructural level using transmission electron microscopy. Aspects of MRC ontogeny, differentiation and adaptation in Nile tilapia yolk-sac larvae following transfer from freshwater to 12.5 and 20 ppt were revealed. The development of a novel 3-D image analysis technique of confocal stacks, allowing visualisation of MRCs in relation to their spatial location, permitted assessment and classification of active and non-active MRCs based on the distance of the top of the immunopositive cell from the epithelial surface; mean active MRC volume was always significantly larger and displayed a greater staining intensity (GLM; p < 0.05) than non-active MRCs. Following transfer, the percentage of active MRCs was seen to increase as did MRC volume (GLM; p < 0.05). Immunogold labeling with anti-Na+/K+-ATpase allowed the identification of both active and non-active MRCs using transmission electron microscopy. The density of immunogold particles appeared to increase following adaptation to 12.5 and 20 ppt and, similarly, the tubular system appeared denser in elevated salinities. Various developmental stages of MRCs were identified within the epithelium of the tail of yolk-sac larvae, thus contributing towards an understanding of the role of mitochondria-rich cells in the development of osmoregulatory capacity during the critical early hatchery stage, as well as providing valuable information concerning the functional plasticity of iono-regulatory cells. The results of this study have increased our understanding of salinity tolerance of the Nile tilapia during the critical early life stages, which in turn could improve hatchery management practices and extend the scope of this species into brackish water environments. In addition, insights have been made into basic iono-regulatory processes that are fundamental to the understanding of osmoregulatory mechanisms during early life stages of teleosts

Subcellular labelling of myelin protein (PLP and DM20) in the central nervous system

The Plp1 gene encodes the two proteins DM20 and PLP. Ultrastructural techniques will provide the best resolution to establish the sub-cellular localisation of the two myelin protein isoforms encoded by the Plp1 gene in mice and thus provide insight into the functions for the two isoforms. The gene is expressed primarily by oligodendroglia of the central nervous system (CNS), with some expression (primarily of DM20) in the Schwann cells of the peripheral nervous system. In the CNS, the DM20 isoform is expressed initially prior to myelin initiation and expression maintained throughout life. The function of DM20 is unknown. The expression of the PLP isoform appears to be related to myelination and have a structural role in compact myelin. The objective was to establish an immunolocalisation technique which would allow the hypothesis to be tested that DM20 is expressed in non-compacted regions of the myelinating oligodendrocyte, whilst PLP expression is confined to the compact myelin sheath. In order to differentially label PLP and DM20 at the paranodal region of myelinated axons, two different immunological techniques suitable for subcellular localisation were investigated. The photo-oxidation technique was applied to both cerebellar and cervical spinal cord vibratome sections. A brominated eosin conjugated secondary antibody was created and used to label a primary antibody against PLP and DM20. The photo-oxidation technique, a pre-embedding immunostaining, generated a very fine reaction product that was excellent at light microscopic resolution but only visible at electron microscopic resolution in sections that were not contrast stained. The immunogold technique was applied to sections from ventral columns in cervical spinal cord which has a dense population of medium to large myelinated axons. Assessments were made using anti-PLP antibodies in mature animals (with PLP at its peak of production and the myelin compacted and well established) and in young animals (with PLP in its beginning of production and less compacted). The immunogold technique, a post-embedding immuno-staining, produced a well-defined electron dense particle that was easily visualised in the EM. The technique proved insufficient to differentiate two different PLP antibodies, one labelling both isoforms (i.e. PLP and DM20) and the other specifically labelling only PLP. A substantially higher level of labelling was achieved, uniquely using the freeze substitution method of tissue preservation; however quantitative analysis could not be carried out because preservation was inconsistent both within the same tissue and from sample to sample

Microspore embryogenesis: cell wall dynamics and reprogramming of cell fate

[ES] Los dobles haploides son una gran herramienta para la mejora genética de híbridos debido a que se puede alcanzar la homocigosis completa en una sola generación. Entre las técnicas usadas para obtener estas plantas, la inducción de la embriogénesis de microsporas, mediante el cultivo de anteras o de microsporas, es la más eficiente y la más usada. La embriogénesis de microsporas es también un ejemplo de totipotencia de las células vegetales gracias a su habilidad de reprogramar su desarrollo gametofítico hacia una ruta esporofítica, donde las células proliferan de forma organizada para crear un nuevo organismo. Como en muchos otros procesos in vitro, las condiciones de cultivo deben ser optimizadas para incrementar la eficiencia. En la presente Tesis Doctoral, hemos usado dos especies como sistemas experimentales para estudiar y optimizar el cultivo de microsporas. Por un lado, usamos berenjena (Solanum melongena) como un ejemplo de cultivo de importancia económica en el que los protocolos todavía tienen mucho margen de mejora. La optimización de la densidad celular y los reguladores de crecimiento han demostrado ser útiles para modificar la eficiencia del cultivo de microsporas de berenjena. Por otra parte, hemos utilizado el cultivo de microsporas de Brassica napus para estudios básicos puesto que ha sido ampliamente usado como modelo para entender procesos celulares que ocurren durante este cambio en el desarrollo. Se detalla un protocolo estandarizado para el cultivo de microsporas de B. napus, el cual ha sido utilizado en todos los cultivos en esta Tesis para explorar una serie de procesos y estructuras celulares potencialmente implicados en el cambio de desarrollo hacia embriogénesis. Estos procesos incluyen estrés del retículo endoplásmico, muerte celular programada, autofagia y estructura y composición de la pared celular. Estudiamos en paralelo el cultivo de microsporas de dos genotipos de B. napus con diferente respuesta androgénica en condiciones estándar y añadiendo Tricostatina A, un modulador epigenético que ha mostrado ser beneficioso para la respuesta androgénica en algunos casos. En conjunto, esta Tesis representa un avance en la optimización del cultivo de microsporas en estas especies y arroja luz sobre el papel de algunos procesos en el contexto de embriogénesis de microsporas.[CA] Els dobles haploides són una gran eina en millora vegetal per a la producció d'híbrids, a causa de la seua total homozigosi, que es pot aconseguir en només una generació in vitro. Entre les diverses tècniques que s'utilitzen per tal d'obtenir aquestes plantes, la inducció de l'embriogènesi de microspores, mitjançant cultiu d'anteres o microspores, és la més comuna i eficient. L'embriogènesi de microspores també és un exemple de la totipotència de les cèl·lules vegetals, capaços de reprogramar-se d'una via gametofítica a una via esporofítica, on proliferen de manera organitzada per crear un nou organisme. Com en moltes altres tecniques in vitro, s'han d'optimitzar les condicions del cultiu per tal d'augmentar l'eficiència. En la present Tesi Doctoral, hem utilitzat dues espècies de plantes com a sistemes experimentals per estudiar i optimitzar el cultiu de microspores. Per una banda, hem utilitzat l'albergínia (Solanum melongena) com a exemple de cultiu d'importància econòmica on els protocols encara tenen marge per a l'optimització. La optimització de la densitat de cèl·lules en cultiu i la concentració de reguladors de creixement van demostrar ser útils per modificar l'eficiència de la resposta dels cultius de microspores d'albergínia. D'altra banda, hem utilitzat cultius de microspores de Brassica napus principalment per a estudis bàsics, ja que s'utilitza àmpliament com a model per entendre els processos cel·lulars que es produeixen durant aquest canvi de desenvolupament. Es detalla un protocol estandarditzat per al cultiu de microspores de B. napus, que s'ha utilitzat en tots els cultius inclosos en aquesta Tesi per explorar una sèrie de processos i estructures cel·lulars potencialment implicades en el canvi de desenvolupament cap a l'embriogènesi. Aquests inclouen l'estrès del reticle endoplasmàtic, la mort cel·lular programada, l'autofàgia i l'estructura i composició de la paret cel·lular. Vam estudiar en paral·lel cultius de microspores de dos genotips de B. napus amb diferent resposta androgènica, cultivats en condicions estàndard i afegint-hi Tricostatina A, un modulador epigenètic que s¿ha demostrat beneficiós per a la resposta androgènica en alguns casos. En conjunt, aquesta Tesi representa un avanç en l'optimització dels cultius de microsporas en aquestes espècies i aporta llum sobre el paper d'alguns processos en el context de l'embriogènesi de microspores.[EN] Doubled haploids are a great tool for hybrid breeding due to their complete homozygosity achievable in only one in vitro generation. Among the several techniques used to obtain these plants, induction of microspore embryogenesis, via anther or microspore culture, is the most common and efficient approach. Microspore embryogenesis is also an example of totipotency of plant cells due to their ability to reprogram themselves from a gametophytic to a sporophytic pathway, where cells proliferate in an organized way to create a new organism. As in many other in vitro procedures, culture conditions must be optimized in order to increase efficiency. In the present Doctoral Thesis, we used two plant species as experimental systems to study and optimize microspore culture. On one hand, we used eggplant (Solanum melongena) as an example of economically important crop where protocols have still room for optimization. Optimization of cell density and growth regulators demonstrated to be useful to modify the efficiency of eggplant microspore cultures. On the other hand, we used B. napus microspore cultures principally for basic studies since it is widely used as a model to understand cellular processes occurring during this developmental switch. A standardized protocol for Brassica napus microspore culture is detailed, which was used in all the cultures included in this Thesis to explore a series of processes and cellular structures potentially involved in the developmental switch towards embryogenesis. These included endoplasmic reticulum stress, programmed cell death, autophagy, and cell wall structure and composition. We studied in parallel microspore cultures from two B. napus genotypes with different androgenic response cultured in standard conditions and adding Trichostatin A, a epigenetic modulator shown to be beneficial for the androgenic response in some cases. Together, this Thesis represents an advance in the optimization of microspore cultures in these species, and sheds light on the role of some processes within the context of microspore embryogenesis.Thanks are due to the Electron Microscopy Service of Universitat Politècnica de València, Marisol Gascón (IBMCP Microscopy Service). This work was supported by grant AGL2017-88135-R to JMSS from MICINN jointly funded by FEDER and by a Marie Skłodowska-Curie Individual Fellowship (656579) to PC-M This work was supported by grant AGL2017-88135-R to JMSS from MINECO jointly funded by FEDER.Camacho Fernández, C. (2021). Microspore embryogenesis: cell wall dynamics and reprogramming of cell fate [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/163698TESI

Elektron kryo-mikroskopické techniky v biologickém výzkumu a nanotechnologiích

Příprava biologických vzorků pro transmisní elektronovou mikroskopii není triviální úkol. Vzorky musí odolat vakuu přítomném v mikroskopu, a proto je často nutné uplatnit nefyziologické postupy při jejich zpracování. Tyto postupy obvykle zahrnují fixaci na bázi aldehydů, nahrazení vody alkoholem (t.j. dehydrataci/substituci), a zalití do pryskyřice, která vytváří podporu pro následnou přípravu tenkých řezů, které pak mohou být vloženy do mikroskopu. V posledním desetiletí získala dominantní postavení v oblasti výzkumu buněčné biologie metoda kryo-fixace (vitrifikace) za pomoci ultrarychlého vysokotlakého zmrazování a následná kryo-substituce a zalití vzorků do pryskyřice při nízkých teplotách. Tímto způsobem byli úspěšně vitrifikovány různé biologické vzorky s tloušťkou až několik stovek mikrometrů do stavu, který byl srovnatelný s jejich in vivo strukturou. Kryo-fixace izolovaných biologických objektů (s omezenou tloušťkou do několika mikrometrů) je možná i v tenké vrstvě vitrifikované vody za pomoci imerzní kryo-fixace při normálním tlaku. V kombinaci s kryo-elektronovou mikroskopií se tato metoda stala nejefektivnejším a základním principem pro tvorbu elektron kryo-mikroskopických obrázků plně hydratovaných vzorků s velmi vysokým rozlišením na úrovni několika desetin nanometrů. Obě tyto metody...Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...Ústav buněčné biologie a patologie 1. LF UKInstitute of Cell Biology and Pathology First Faculty of Medicine Charles UniversityFirst Faculty of Medicine1. lékařská fakult

Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana

Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN‐FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear. Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze‐fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains. Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell‐wall components as well as connections between the cell wall and the plasma membrane. This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems