Head Of Embryos Research Articles

DanioCTC: Analysis of Circulating Tumor Cells from Metastatic Breast Cancer Patients in Zebrafish Xenografts.

Circulating tumor cells (CTCs) serve as crucial metastatic precursor cells, but their study in animal models has been hindered by their low numbers. To address this challenge, we present DanioCTC, an innovative xenograft workflow that overcomes the scarcity of patient-derived CTCs in animal models. By combining diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the CellCelector setup, DanioCTC effectively enriches and isolates CTCs from metastatic breast cancer (MBC) patients for injection into zebrafish embryos. Validation experiments confirmed that MDA-MB-231 cells, transplanted following the standard protocol, localized frequently in the head and blood-forming regions of the zebrafish host. Notably, when MDA-MB-231 cells spiked (i.e., supplemented) into DLA aliquots were processed using DanioCTC, the cell dissemination patterns remained consistent. Successful xenografting of CTCs from a MBC patient revealed their primary localization in the head and trunk regions of zebrafish embryos. DanioCTC represents a major step forward in the endeavors to study the dissemination of individual and rare patient-derived CTCs, thereby enhancing our understanding of metastatic breast cancer biology and facilitating the development of targeted interventions in MBC. Summary statement: DanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies of the capacity of these rare tumor cells to induce metastases.

A revised terminology for the pharyngeal arches and the arch arteries.

The pharyngeal arches are a series of bulges found on the lateral surface of the head of vertebrate embryos. In humans, and other amniotes, there are five pharyngeal arches and traditionally these have been labelled from cranial to caudal-1, 2, 3, 4 and 6. This numbering is odd-there is no '5'. Two reasons have been given for this. One is that during development, a 'fifth' arch forms transiently but is not fully realised. The second is that this numbering fits with the evolutionary history of the pharyngeal arches. Recent studies, however, have shown that neither of these justifications have basis. The traditional labelling is problematic as it causes confusion to those trying to understand the development of the pharyngeal arches. In particular, it creates difficulties in the field of congenital cardiac malformations, where it is common to find congenital cardiac lesions interpreted on the basis of persistence of the postulated arteries of the fifth arch. To resolve these problems and to take account of the recent studies that have clarified pharyngeal arch development, we propose a new terminology for the pharyngeal arches. In this revised scheme, the pharyngeal arches are to be labelled as follows-the first, most cranial, the mandibular (M), the second, the hyoid (H), the third, the carotid (C), the fourth, the aortic (A) and the last, most caudal, the pulmonary (P).

Chronic intake of an enriched diet with spirulina (Arthrospira maxima) alleviates the embryotoxic effects produced by realistic concentrations of tetracycline in Danio rerio

Tetracycline (TC) is one of the most consumed antibiotics worldwide. Due to its high consumption, recent studies have reported its presence in aquatic environments and have assessed its effects on fish, algae, and daphniids. However, in most of those works, authors have tested TC toxicity at concentrations higher than the ones reported in the water matrix. Herein, we aimed to assess the likely embryotoxic and oxidative damage induced by environmentally relevant concentrations of TC in embryos of Danio rerio. Moreover, we seek to determine whether or not an enriched diet with spirulina can alleviate the embryotoxic damage produced by TC. Our findings indicated that TC at concentrations of 50 to 500 ng/L induced pericardial edema, tail deformities, and absence of head and fin in embryos after 96 h of exposure. Moreover, this antibiotic prompted the death of embryos in a concentration-dependent manner. According to our integrated biomarker response index, TC induced oxidative damage on Danio rerio embryos, as star plots showed a tendency to lipoperoxidation, hydroperoxides, and protein carbonyl content. Spirulina reduced the toxicity of TC by diminishing the levels of oxidative damage biomarkers, which resulted in a decrease in the rate of death and malformed embryos. Overall, TC at concentrations of ng/L prompted oxidative stress and embryotoxicity in the early life stages of Danio rerio; nonetheless, the algae spirulina was able to reduce the severity of those effects.

Interpretation of fossil embryos requires reasonable assessment of developmental age

AbstractDinosaur embryos cause a lot of excitement in the scientific literature and are often widely reported because of the general public's interest in dinosaur biology. Well-preserved, articulated oviraptorosaur embryos in eggs are usually interpreted as representing a stage of development close to hatching because of their large size and good level of skeletal ossification. Based on this evidence, a recent report suggested that the position of the one embryo's head was reminiscent of an avian-like hatching position. Here we explore how the developmental stage of well-preserved oviraptorosaur embryos can be estimated, rather than assumed. This will help in our understanding of their developmental biology and its evolutionary consequences. Using quantitative methods and comparison with modern crocodilian embryos, we show that all articulated oviraptorosaur embryos are small relative to the egg and most likely at a stage of development equivalent to around 50%–60% of the developmental period, that is, not even close to hatching. This conclusion is supported by the fact that many elements of the crocodilian skeleton are well ossified many weeks before hatching and the position of oviraptorosaur embryos’ heads was also comparable to a crocodilian embryo many days before hatching. Misunderstandings about the stage of the developmental biology of these well-preserved oviraptorosaur embryos hampers our understanding of the true nature of their reproductive biology. We urge a more conservative approach to their interpretation. This is important, because misunderstandings in the minds of the public about dinosaur biology are hard to counter once poorly evidenced ideas have been reported around the world.

Toxicological Effects of Sewage Water on Chick Embryonic Development.

For toxicity research, a total of 100 fertilized nonincubated eggs were used for this study. There were two trials in this experiment which were further divided into 2 phases based on a different days of sewage water treatment and observation days. In each trial, 50 eggs were used and divided into 5 groups. Group A, B, and C were treated with three different concentrations of pure and diluted sewage water (100%, 70%, and 30%), respectively. Control group D was given 0.3 ml saline solution (0.9% NaCl) and group E was uninjected. Different parameters such as the embryo's body weight, body length, forelimb length, hindlimb length, and head diameter were determined. In trial 1, eggs were treated with sewage water on 7th day of incubation and opened on 8th day (phase I) and 9th day (phase II). When the trial 1 (phase I) findings were compared to the control groups, it was observed that body weight, body length, forelimb length, and hindlimb length were highly statistically significant differences (p < 0.01), but the head diameter was not significant (p > 0.05). Phase II result showed embryo's head diameter was a highly statistically significant difference (p < 0.01), whereas forelimb length was significant (p < 0.05), and body weight, body length, and hindlimb length were nonsignificant (p > 0.05). In trial 2, eggs were treated with sewage water on 14th day of incubation and opened on 15th day (phase I) and 16th day (phase II). Results of 15th day showed a highly statistically significant (p < 0.01) difference in hindlimb length, while body weight, body length, forelimb length, and head diameter were nonsignificant (p > 0.05). Phase II of trial 2 showed that on 16th day, body weight, body length, forelimb length, hindlimb length, and head diameter showed a nonsignificant (p > 0.05) difference between experimental and control groups. Embryos were observed to be deforming on the 9th day (after 48 hours of exposure to sewage water). Other phases showed no signs of deformation. Except on 8th day of incubation, dose-related mortalities were present in experimental groups, while the control group showed no mortality.

Emx2is Required for Nephron Segment Development in the Zebrafish Pronephros

The empty spiracles homeobox gene 2 (emx2)encodes a transcription factor that is expressed during neuronal, auditory, olfactory, and renal development, but its role in the latter has not yet been fully elucidated. Here, our objective is to utilize the zebrafish animal model to investigate the functions of emx2 during the development of nephrons, which are the structural and functional units of the kidney. Using whole mount in situ hybridization (WISH), we detected emx2transcript beginning at the 6 somite‐stage in renal progenitors and this expression continued throughout the process of forming discrete proximal convoluted tubule (PCT) and proximal straight tubule (PST) segment regions at 24 hours post fertilization (hpf). Based on this spatiotemporal pattern of emx2 expression in renal progenitors, we hypothesized that emx2is essential for segmentation of the nephron into discrete proximal and distal regions. To investigate this, we generated emx2‐deficient embryos and compared them to wild‐type (WT) controls at various time points from 24‐96 hpf. Knockdown of emx2resulted in head necrosis and diminished yolk sac extension at 24 hpf, as well as development of smaller head, eye and body, pericardial edema, abnormal fin growth, yolk ball inflammation and vascular hemorrhage in the central nervous system from 2‐5 days post fertilization. We used WISH to stain for each nephron segment in fixed animals and then quantified segment length and cell number. WISH analysis revealed a significantly reduced podocyte area, diminished PCT and reduced multiciliated cell numbers in emx2‐deficient embryos compared to WT embryos. WISH results also revealed secondary phenotypes, such as reduced fin bud area and somite in emx2‐deficient embryos. Additionally, Alcian Blue staining at 96 hpf revealed jaw abnormalities and pharyngeal arch malformation in emx2‐deficient embryos. O‐Dianisidine staining showcased vascular hemorrhage in the eyes and head of emx2deficient embryos. We concluded that emx2is essential for kidney segmentation in zebrafish, especially in podocyte and PCT formation. Additionally, emx2deficiency causes secondary jaw, fin, cartilage blood accumulation and muscle phenotypes. Future studies will delineate the cellular defects in nephron development in emx2 deficient embryos and determine the relationship between Emx2 and pathways which are essential for proximal segment ontogeny, such as retinoic acid signaling.

Mapping the Multi‐Modal Distribution of Craniofacial Phenotypes in NOSIP Mutants

Nitric Oxide Synthase Interacting Protein (NOSIP) is an E3 mono-ubiquitin ligase that regulates the activity of protein phosphatase 2A. Deletion of NOSIP leads to severe and variable malformations of the craniofacial complex. Our goal was to use NOSIP mutant mice to understand genetic and cellular mechanisms of phenotypic variation similar to human disease. The phenotype of an individual embryo is the result of the developmental trajectory that it previously traversed. We propose that this concept of canalization is essential for understanding the etiology of complex phenotypic outcomes. We tested this by creating a 3D morphospace of NOSIP heterozygous and homozygous mutants to determine if embryos occupy unique morphospace regions based on affected craniofacial structures. Embryos between embryonic day 10.5 (E10.5) to E17 were collected, fixed in PaxGene, and imaged (mCT). Anatomical features were reconstructed using Avizo. RNAscope was used to assess Nosip expression, and RNA was the extracted from the head of embryos and prepared for sequencing. While some heterozygous embryos appear completely unaffected, both homozygous null and heterozygous embryos presented with a mild to severe spectrum of phenotypic malformations, with no homozygous null embryos appearing unaffected. Embryos on the more severe side of the spectrum has a single telencephalic lobe with severe facial malformations, including loss of maxillary processes, micrognathia and obstructed airway. Embryos on the milder spectrum had normal telencephalic vesicle separation and had less severe facial phenotypes, including mild left-right asymmetry and clefting. NOSIP expression between E10.5 and E12.5 appears to fairly ubiquitously, with high expression in the neuroepithelium, lower jaw mesenchyme, nasal tissue. Expression was markedly reduced in normal NOSIP heterozygotes. Mutant transcriptomes at E10.5 cluster based on genotype, but by E12.5, transcriptomes cluster based on phenotype severity, suggesting that transcriptional profiles cannot be solely predicted based on genotype alone. Pathways most affected include protein function, SHH and canonical WNT signaling. Six3 and Shh appear to be negatively affected in NOSIP mutants, and correlates with more severe brain defects. Overall, it appears that Nosip-KO creates a multi-modal distribution of craniofacial phenotypes along the developmental timeline by affecting early brain development, suggesting that understanding canalization may provide insight into the complexity of genotype-phenotype map. Future work will focus on identifying specific dysregulations in protein function of mutants and normalizing early signaling pathways to rescue more normal phenotypes.

Silver nanocolloid affects hindbrain vascular formation during medaka embryogenesis.

Angiogenesis is essential for the normal development of an embryo. Silver nanocolloid (SNC) is known to induce vascular malformation in the medaka embryo. We focused on the development of the central arteries (CtAs) in the hindbrain of Japanese medaka. The CtAs and the basilar artery from which they branch are essential for transporting the blood and nutrients necessary to support the hindbrain parenchyma and the development of the pons and cerebellum from the hindbrain. We exposed medaka embryos at developmental stage 21 (6 somite stage), to 0, 0.5, 5, or 10 mg/L SNC and evaluated hatching rate, number of thrombi per embryo, head size (length and width), body length, and angiogenesis. Although all SNC-exposed embryos hatched, their head size and body length were small in comparison to controls; in addition, the number of thrombi in the head increased and head size and body length decreased as the SNC concentration increased. To evaluate vasculogenic abnormalities, we performed whole-mount in situ hybridization using a vascular marker (eg, fl7) and visualized the CtAs in medaka embryos. In control embryos, CtAs started to sprout at stage 32 (somite completion stage) and their extension was complete by stage 35 (pectoral fin blood circulation stage). In contrast, CtAs failed to sprout in SNC-exposed embryos, and thrombi were present. Furthermore, qRT-PCR analysis showed that SNC significantly suppressed the egfl7 expression level at stage 35. Together, our findings suggest that SNC induced decreased developments of head and body in medaka embryos due to insufficient angiogenesis and hindbrain vascular formation.

Evolutionary and Developmental Associations of Neural Crest and Placodes in the Vertebrate Head: Insights From Jawless Vertebrates

Neural crest and placodes are key innovations of the vertebrate clade. These cells arise within the dorsal ectoderm of all vertebrate embryos and have the developmental potential to form many of the morphological novelties within the vertebrate head. Each cell population has its own distinct developmental features and generates unique cell types. However, it is essential that neural crest and placodes associate together throughout embryonic development to coordinate the emergence of several features in the head, including almost all of the cranial peripheral sensory nervous system and organs of special sense. Despite the significance of this developmental feat, its evolutionary origins have remained unclear, owing largely to the fact that there has been little comparative (evolutionary) work done on this topic between the jawed vertebrates and cyclostomes—the jawless lampreys and hagfishes. In this review, we briefly summarize the developmental mechanisms and genetics of neural crest and placodes in both jawed and jawless vertebrates. We then discuss recent studies on the role of neural crest and placodes—and their developmental association—in the head of lamprey embryos, and how comparisons with jawed vertebrates can provide insights into the causes and consequences of this event in early vertebrate evolution.

Mutation of Eftud2 in Mouse Neural Crest Cells Models MFDM and Reveals Contribution of P53 in Abnormal Craniofacial Development

Haploinsufficiency of EFTUD2 is associated with MFDM (mandibulofacial dysostosis with microcephaly), but the etiology of this syndrome remains unknown. Our goal is to determine the tissue and temporal requirement for Eftud2 during craniofacial development. We generated a mouse with a conditional mutation of exon 2 of Eftud2 (Eftud2 flox) using the CRISPR/Cas9 editing system. We used the Wnt1‐Cre2 transgenic line to delete exon 2 of Eftud2 specifically in the neural crest cells. Embryos carrying one mutated allele of Eftud2 were normal. However, when both alleles were mutated, all embryos displayed hypoplasia of the midbrain and pharyngeal arches at E9.5. By E11.5, most of these embryos showed an open neural tube and all live embryos exhibited exencephaly at E14.5. At this stage, only 3 out of 12 embryos were still alive. Cartilage preparations revealed an absence of cartilage in the head, reduced/or absence of Meckel’s cartilage, and abnormal inner ear development. Crosses with the Rosa26RlacZ mice reporter line revealed a reduced number of neural crest cells in the pharyngeal region at E9.5 and E10.5, but not at E9.0. We next performed RNA sequencing analysis from the head of Eftud2 neural crest cells mutant embryos at E9.0, before any phenotype was observed. The P53 pathway was the most significantly upregulated in the mutant embryos, compared to their wild‐type littermates. Overexpression of P53 target genes identified in the RNAseq (Ccng1, Phlad3 and Trp53inp1) was validated by qRT‐PCR. Additionally, 373 genes showed increased skipped exons in the mutant embryos. Among them, only 2 genes were also overexpressed, including Mdm2, a major regulator of P53. We found that mutant embryos had significantly increased levels of the Mdm2 transcript lacking exon 3, which was previously reported to produce a truncated MDM2 protein, allowing stabilization of P53. Finally, we evaluated the contribution of P53 in craniofacial malformation of Eftud2 mutants by treating pregnant females with daily i.p. injection of pifithrinα, a P53 inhibitor, from E6.5 to E8.5. 37% of pifithrinα‐treated mutant embryos had normal midbrain development at E9.5. Also, the area perimeter of the first pharyngeal arch of pifithrinα‐treated mutant embryos was significantly higher than the vehicle‐treated ones. Altogether, these data suggest that Eftud2 specifically regulates Mdm2 splicing and leads to neural crest cell death due to overactivation of the P53 pathway. We propose that cranial neural crest cell death is responsible for craniofacial malformations in MFDM patients.Support or Funding InformationCanadian Institutes of Health Research

Lipid Alterations during Zebrafish Embryogenesis Revealed by Dynamic Mass Spectrometry Profiling with C=C Specificity.

Lipids exert substantial influences on vertebrate embryogenesis, but their metabolic dynamics at detailed structural levels remains elusive, primarily owing to the lack of a tool capable of resolving their huge structural diversity. Herein, we present the first large-scale and spatiotemporal monitoring of unsaturated lipids with C=C specificity in single developing zebrafish embryos enabled by photochemical derivatization and tandem mass spectrometry (MS). The lipid isomer composition was found extremely stable in yolk throughout embryogenesis, while notable differences in ratios of C=C location (e.g., PC 16:0_16:1 (7) vs. 16:0_16:1 (9)) and fatty acyl composition isomers (e.g., PC 16:1_18:1 vs. 16:0_18:2) were unveiled between blastomeres and yolk from zygote to 4h post fertilization (hpf). From 24 hpf onwards, lipid isomer compositions in embryo head and tail evolved distinctively with development, suggesting a meticulously regulated lipid remodeling essential for cell division and differentiation. This work has laid the foundation for functional studies of structurally defined lipids in vertebrate embryology.

Clethodim exposure induced development toxicity and behaviour alteration in early stages of zebrafish life

Clethodim is one of the most widely used herbicides in agriculture, however, its potential toxic effects on organisms and the underlying toxicity mechanism are still poorly understood. In this study, zebrafish embryos at 6 h post-fertilization (hpf) were exposed to 10 mg/L, 20 mg/L, and 30 mg/L clethodim for up to 24 hpf, and zebrafish larvae at 6 days post-fertilization (dpf) were exposed to the same density gradient for 24 h. Our results showed that clethodim could cause head and cardiovascular malformations in embryos: blurred brain ventricles, unapparent brain regions, condensation of nucleus and cytoplasm in brain cells, increased intercellular space, developmental malformations of eyes and ears, reduced neonatal neurons, disorder migration of neural ridge cells; morphological aberrations of the vascular ICM, slowing of heart beat and blood flow, reduction of circulating red blood cells, and delayed development of head and tail blood vessels. These defects could be a result of clethodim-induced oxidative stress and decreased acetylcholinesterase (AChE) activity, which in turn affected the expression of neurodevelopmental genes, decreased ATPase activity, and ultimately led to developmental malformations. The swimming behaviour of zebrafish larvae was observed to decrease with increasing concentration of clethodim exposure, but the angular velocity and mobility increased. These could be due to reduced AChE activity and disturbed gene expression of GABA, dopamine and glutamatergic neurotransmitter systems, which thus altered the locomotor behaviour. In summary, we found that clethodim induces developmental toxicity and neurotoxicity in zebrafish embryos and larvae.

The Origin of the Little Brain on the Heart and Its Role in the Absence of Brain During Head Transplantation in Adults and Before Formation of Head in Embryos: Testing the Model in Birds and Chick Embryos

Recently, some authors have claimed that head transplantation is possible in human. The question arises that what is the fate of mind and voluntary decision during head transplantation? To response this question, we should accept the claim of other investigators for the existence of little brain on the heart. In this research, we propose some reasons for the emergence of this little brain before formation of head in chick embryos. We take it's radiate wave and show that it controls the process of formation of neural circuits and brain. In fact, the origin of the little brain is the DNA in initial stem cell. This DNA is constructed from various types of genes. For analyzing signals of each gene, one circuit should be emerged that receive signals, analyze and send responses. These circuits are formed from neurons. On the other hand, each of these genes emits several types of waves. To transmit these waves between cells, there should be several terminals or receiver in dendrite and axons of a neuron. Thus, total circuits and neurons of brain are formed by radiated waves from DNAs in initial stem cells. Also, some circuits of neurons are emerged around stem cells that form the little brain. In the absence of neurons in the early stages of chick embryos, most of the initial signals of initial DNA are transformed by blood molecules from heart to other cells and thus, the little brain is formed interior of the heart. We take radiated signals of the little brain on the hearts of birds during head transplantation. We show that after cutting heads of birds, this little brain enters directly in process of decision and manages voluntary behaviour. For this reason, birds without head, could open their wings, fly and jump the barriers. By connecting head to body, the little brain emits more signals to communicate with the brain interior of head.

Diminution of pharyngeal segmentation and the evolution of the amniotes

BackgroundThe pharyngeal arches are a series of bulges found on the lateral surface of the head of vertebrate embryos, and it is within these segments that components of the later anatomy are laid down. In most vertebrates, the post-otic pharyngeal arches will form the branchial apparatus, while in amniotes these segments are believed to generate the larynx. It has been unclear how the development of these segments has been altered with the emergence of the amniotes.ResultsIn this study, we examined the development of pharyngeal arches in amniotes and show that the post-otic pharyngeal arches in this clade are greatly diminished. We find that the post-otic segments do not undergo myogenesis or skeletogenesis, but are remodelled before these processes occur. We also find that nested DLX expression, which is a feature of all the pharyngeal arches in anamniotes, is associated with the anterior segments but less so with the posterior arches in amniotes. We further show that the posterior arches of the mouse embryo fail to properly delineate, which demonstrates the lack of function of these posterior segments in later development.ConclusionIn amniotes, there has been a loss of the ancestral “branchial” developmental programme that is a general feature of gnathostomes; myogenesis and skeletogenesis This is likely to have facilitated the emergence of the larynx as a new structure not constrained by the segmental organisation of the posterior pharyngeal region.

Hollow selenium nanoparticles from potato extract and investigation of its biological properties and developmental toxicity in zebrafish embryos.

A facile synthesis of hollow selenium nanoparticles (hSeNPs) was prepared using potato starch as a reducing and capping agent. The morphological and structural characters of the hSeNPs were characterised by ultraviolet-visible spectroscopy (UV), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDX) and zeta potential analyser. The optical characteristics of hSeNPs were confirmed by UV. The presence of various functional groups in the hSeNPs suspension was confirmed by FTIR. The SEM results suggested that the synthesised hSeNPs were uniformly distributed and circular in shape with a hollow. The average size of the hSeNPs was found to be around 115 nm. The EDX analysis also confirmed the presence of hSeNPs in the sample. The zebrafish embryos were treated with hSeNPs of various concentrations ranging from 10 to 50 µg/ml. Abnormalities such as improper heartbeat, embryo sac oedema, ocular oedema and head oedema were noted at higher concentrations (30-50 µg/ml). A concentration-dependent antioxidant activity of hSeNPs was observed. The hSeNPs showed good antibacterial activity against gram-positive Bacillus subtilis and gram-negative Escherichia coli. The results of this study indicate that potato extract reduces the toxicity of hSeNPs and lower concentrations of hSeNPs could be used for various biomedical applications in near future.

Content and immunohistochemical localization of hormones during in vitro somatic embryogenesis in long-term proliferating Larix sibirica cultures

We carried out immunoassay of plant hormones [indoleacetic acid (IAA), cytokinins and abscisic acid (ABA)] in the extracts from explants (megagametophytes and embryos) and from four long-term proliferating embryogenic cell lines of Larix sibirica. Immunohistochemical detection of hormones has also been performed at the stage of initiation of somatic embryogenesis and in embryogenic cultures during the process of somatic embryo morphogenesis. During induction of somatic embryogenesis in tissue culture, the first stage of cell development is manifested in their elongation and polarization. Immunohistochemical analysis of hormones showed localization of IAA at one pole of elongated cells of L. sibirica. The polarity of embryogenic structure development persisted later on. The difference has been shown in the content of hormones between the explants and embryogenic cultures. High level of hormones was detected in the cell line (Cl4), distinguished by great number of globular embryos and high capacity for their maturation. Hybrid line Cl5 (L. sibirica × L. sukaszewii) formed very short suspensor, accumulated higher amount of ABA in the head of somatic embryos (SEs) than did other lines, while SEs of this cell line did not maturate. Cl6 was characterized by low plant hormones content in proliferating embryogenic cultures and low yield of mature somatic embryos.

165. Periheral dose in a Cyberknife treatment: The case of a pregnant woman

PurposeWe present peripheral dose measurements in a Cyberknife brain treatment. This dosimetric study was applied to the clinical case of a pregnant woman and the dose to the fetus was estimated in order to verify the safety of the treatment [1]. MethodsDose evaluation at different distances from the target was achieved with the aid of a phantom resembling the human body. Two treatment plans were delivered to the phantom with prescription doses of 25 Gy and 14 Gy respectively. The dose was measured with two different detectors: gafchromic films and Farmer ionization chamber, which were positioned in different part of the phantom at various distances from the zone irradiated. The film measurement put in evidence the propagation of the beams after having hit the target while the ionization chamber gave a punctual detection of the dose. The two detection methods gave the same results at the common point of measurement. A shielding device was designed to further protect the patient and the radiation attenuation was estimated. ResultsThe dose sharply decreased with distance from the target: at a distance of 3 cm the dose was 6 times inferior with respect to the prescribed dose and the application of the shielding determined a reduction of the dose by a factor of three. In the case of a pregnant woman at the third trimester of gestation, the position of the embryo, evaluated with ultrasound imaging, laid from 56 cm (embryo head) to 72 cm (embryo legs) from the lesion. Following our simulation, for the first treatment plan the dose to the embryo was expected to be less than 5 cGy, for the second treatment less than 3 cGy. ConclusionsThe measured peripheral doses were below the threshold of 10 cGy for congenital malformations, mental and growth retardation effect. A brain treatment in pregnancy can therefore be safely administered after a careful evaluation of the dose to the embryo.

Sturge-Weber syndrome Case report

Sturge-Weber Syndrome (SWS – encephalotrigeminal angiomatosis) is a rare non-hereditary congenital condition. It develops in the early foetal period, at the stage when the embryo head is being formed, as a result of a random somatic mutation of the GNAQ gene due to unknown reasons. The main symptom is the formation of congenital angiomas, especially in the area of capillaries, in the leptomeninges in the brain, and along the course of the trigeminal nerve. Such angiomas may also be present on lips, and in the whole oral cavity: on its bottom, palate and gingivae. It may lead to serious malocclusions and breathing problems. &lt;b&gt;Aim&lt;/b&gt;. This article aims to present problems regarding orthodontic treatment of patients with Sturge-Weber syndrome, using an example of a 7-year-old female patient.&lt;b&gt; Case report&lt;/b&gt;. A 7-year-old girl with all signs of this syndrome, namely glaucoma, epilepsy, neurological disorders, cutis marmorata telangiectatica congenita and after sanitation of the oral cavity, had hygiene procedures and exercises with a vestibul plate introduced. Two attempts to take impressions for appliances failed due to patient’s anxiety reactions. &lt;b&gt;Conclusions&lt;/b&gt;. This case shows difficulties that may be faced by an orthodontist when treating patients with Sturge-Weber syndrome. In this case, namely type I in the Roach scale, due to neurological disorders it was necessary to take special precautions in relation to procedures introduced and to avoid stress in a patient. It makes orthodontic treatment difficult.&lt;b&gt; (Witanowska J, Laskowska M, Zadurska M. Sturge-Weber syndrome. Case report. Orthod Forum 2018; 14: 143-9)&lt;/b&gt;.

Time-lapse imaging of cell death in cell culture and whole living organisms using turn-on deep-red fluorescent probes.

Cell death is a central process in developmental biology and also an important indicator of disease status and treatment efficacy. Two related fluorescent probes are described that are molecular conjugates of one or two zinc dipicolylamine (ZnDPA) coordination complexes with an appended solvatochromic benzothiazolium squaraine dye. The probes were designed to target the anionic phospholipid, phosphatidylserine (PS), that is exposed on the surface of dead and dying cells. A series of spectrometric and microscopy studies using liposomes and red blood cell ghosts as models showed that the probe with two ZnDPA targeting units produced higher affinity, stronger fluorescence "turn-on" effect, and better image contrast than the probe with one ZnDPA. Both fluorescent probes enabled "no-wash" time-lapse microscopic imaging of mammalian cell death within a culture. The probe with two ZnDPA units was used for non-invasive time-lapse imaging of cell death during the development of Xenopus laevis (frog) embryos. In vivo fluorescence micrographs revealed probe accumulation within the embryo tail, head and spine regions that were undergoing regression and apoptosis during growth and maturation. These new fluorescent probes are likely to be useful for time-resolved, non-invasive in vivo imaging of cell death process in range of living organisms. From a broader perspective, it should be possible to utilize the negative solvatochromism exhibited by benzothiazolium squaraine dyes for development of various "turn-on" deep-red fluorescent probes and materials that target cell surface biomarkers for in vitro and in vivo imaging.

Characterization and functional analysis of the Paralichthys olivaceus prdm1 gene promoter

PR domain containing protein 1 (Prdm1) is a transcriptional repressor identified in various species and plays multiple important roles in immune response and embryonic development. However, little is known about the transcriptional regulation of the prdm1 gene. This study aims to characterize the promoter of Paralichthys olivaceus prdm1 (Po-prdm1) gene and determine the regulatory mechanism of Po-prdm1 expression. A 2000bp-long 5'-flanking region (translation initiation site designated as +1) of the Po-prdm1 gene was isolated and characterized. The regulatory elements in this fragment were then investigated and many putative transcription factor (TF) binding sites involved in immunity and multiple tissue development were identified. A 5'-deletion analysis was then conducted, and the ability of the deletion mutants to promote luciferase and green fluorescent protein (GFP) expression in a flounder gill cell line was examined. The results revealed that the minimal promoter is located in the region between -446 and -13bp, and the region between -1415 and -13bp enhanced the promoter activity. Site-directed mutation analysis was subsequently performed on the putative regulatory elements sites, and the results indicated that FOXP1, MSX and BCL6 binding sites play negative functional roles in the regulation of the Po-prdm1 expression in FG cells. In vivo analysis demonstrated that a GFP reporter gene containing 1.4kb-long promoter fragment (-1415/-13) was expressed in the head and trunk muscle fibres of transient transgenic zebrafish embryos. Our study provided the basic information for the exploration of Po-prdm1 regulation and expression.