Lightsheet-based flow cytometer for whole blood with the ability for the magnetic retrieval of objects from the blood flow

Detection and extraction of circulating tumor cells and other rare objects in the bloodstream are of great interest for modern diagnostics, but devices that can solve this problem for the whole blood volume of laboratory animals are still rare. Here we have developed SPIM-based lightsheet flow cytometer for the detection of fluorescently-labeled objects in whole blood. The bypass channel between two blood vessels connected with the external flow cell was used to visualize, detect, and magnetically separate fluorescently-labeled objects without hydrodynamic focusing. Carriers for targeted drug delivery were used as model objects to test the device performance. They were injected into the bloodstream of the rat, detected fluorescently, and then captured from the bloodstream by a magnetic separator prior to filtration in organs. Carriers extracted from the whole blood were studied by a number of in vitro methods

Investigation of CaMoO4 single crystals with low residual absorption

Calcium molybdate enriched with the 100Mo isotope (40Ca100MoO4) is a promising material for use in cryogenic scintillation detectors. The main requirements of crystalline elements of the detector are absence of color and the attenuation coefficient (μ) not higher than 0.01 cm−1 at 520 nm wavelength. 40Ca100MoO4 and 40Ca100MoO4:Nb5+ single crystals have been investigated. The influence of isothermal annealing on the attenuation spectra in the 350 to 700 nm wavelength range has been studied. A broad absorption band with a maximum at λ=460 nm is observed in the attenuation spectra of the sample. The dichroism phenomenon which is associated with anisotropy of the color centers in the crystals is observed along directions perpendicular to the optical axis. Annealing of the enriched samples at 1250 °C in an O2 atmosphere leads to a substantial reduction of the intensity of the band near 460 nm. The attenuation coefficient of the 40Ca100MoO4:Nb5+ crystals meets the requirement, which is μ≪0.01 cm−1 at λ=520 nm. It is determined that absorption band near 460 nm and dichroism are absent

Detection of Rare Objects by Flow Cytometry: Imaging, Cell Sorting, and Deep Learning Approaches

Flow cytometry nowadays is among the main working instruments in modern biology paving the way for clinics to provide early, quick, and reliable diagnostics of many blood-related diseases. The major problem for clinical applications is the detection of rare pathogenic objects in patient blood. These objects can be circulating tumor cells, very rare during the early stages of cancer development, various microorganisms and parasites in the blood during acute blood infections. All of these rare diagnostic objects can be detected and identified very rapidly to save a patient’s life. This review outlines the main techniques of visualization of rare objects in the blood flow, methods for extraction of such objects from the blood flow for further investigations and new approaches to identify the objects automatically with the modern deep learning methods

The etiology of cleft palate formation in BMP7-deficient mice

Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis

The Influence of Magnetic Composite Capsule Structure and Size on Their Trapping Efficiency in the Flow

A promising approach to targeted drug delivery is the remote control of magnetically sensitive objects using an external magnetic field source. This method can assist in the accumulation of magnetic carriers in the affected area for local drug delivery, thus providing magnetic nanoparticles for MRI contrast and magnetic hyperthermia, as well as the magnetic separation of objects of interest from the bloodstream and liquid biopsy samples. The possibility of magnetic objects’ capture in the flow is determined by the ratio of the magnetic field strength and the force of viscous resistance. Thus, the capturing ability is limited by the objects’ magnetic properties, size, and flow rate. Despite the importance of a thorough investigation of this process to prove the concept of magnetically controlled drug delivery, it has not been sufficiently investigated. Here, we studied the efficiency of polyelectrolyte capsules’ capture by the external magnetic field source depending on their size, the magnetic nanoparticle payload, and the suspension’s flow rate. Additionally, we estimated the possibility of magnetically trapping cells containing magnetic capsules in flow and evaluated cells’ membrane integrity after that. These results are required to prove the possibility of the magnetically controlled delivery of the encapsulated medicine to the affected area with its subsequent retention, as well as the capability to capture magnetically labeled cells in flow

Transdermal platform for the delivery of the antifungal drug naftifine hydrochloride based on porous vaterite particles

Development of a skin-targeted particulate delivery system providing an extended or sustained release of the payload and a localized therapeutic effect is one of the main challenges in the treatment of fungal skin infections. In the topical administration of antifungals, the drug should penetrate into the stratum corneum and lower layers of the skin in effective concentrations. Here, we introduce biodegradable calcium carbonate carriers containing 4.9% (w/w) of naftifine hydrochloride antimycotic allowing the efficient accumulation into the skin appendages. The proposed particulate formulation ensures the enhancement of the local drug concentration, prolongation of the payload release, and control over its rate. Furthermore, it provides a highly efficient cellular uptake and excellent bioavailability in vitro and enables a deep penetration during transfollicular delivery in vivo. The enhanced fungi growth inhibition efficiency of naftifine-loaded calcium carbonate carriers compared to naftifine solution makes them a promising alternative to creams and gels currently existing on the market

Bmp7 expression in <i>Bmp7-lacZ</i> mouse embryos at stage E13.5.

<p>Widespread transcriptional activity of <i>Bmp7</i> is revealed by lacZ staining in the developing palate (A), the tongue (B, C), and the lower lip (D). Note the strong lacZ staining in the developing palatal rugae, the gustatory follicles and the midline of the lower lip (arrowhead). Weak <i>Bmp7</i> expression is observed in Meckel's cartilage (arrows). (E–I) <i>Bmp7</i> transcriptional activity appeared to be dynamic with some regional anteroposterior differences and could be detected both in palatal epithelium and mesenchyme (Fig. 2E–I; E anterior, I posterior). <i>Scale bar, 50 </i><i>μm.</i></p

Expression of genes involved in palatal shelf adhesion and fusion.

<p>In situ hybridization for <i>Tgfβ3</i>, <i>Jagged2</i> and <i>fibronectin (Fn)</i>. (A) The expression of <i>Tgfβ3</i> at E14.5 is similar in both <i>Bmp7^wt/wt</i> and <i>Bmp7^Δ/Δ</i> embryos and limited to the presumptive medial edge epithelium. Expression of <i>Fn</i> is evident in the medial edge epithelium of the palatal shelves in <i>Bmp7^wt/wt</i> embryos, but absent from corresponding regions of the <i>Bmp7^Δ/Δ</i> palatal shelves at the same developmental stage. (B) In the <i>Bmp7^Δ/Δ</i> embryos expression of <i>Tgfβ3</i> at the medial edge epithelium of the palatal shelves is still strong at E15.5 and starts disappearing at E16.5. In the <i>Bmp7^Δ/Δ</i> embryos a specific down-regulation of <i>Jagged2</i> expression at the presumptive MEE is noted at stages E15.5 and E16.5. <i>Scale bar: 100 </i><i>μm.</i></p

Histological analysis of secondary palate development and morphological features of <i>Bmp7-</i>deficient mouse embryos.

<p>(A) Coronal sections from the middle region of the palate at E13.5-E15.5. Whereas the palatal shelves in both <i>Bmp7^wt/wt</i> and <i>Bmp7^Δ/Δ</i> embryos were similar in shape at E13.5, the shelves of the <i>Bmp7^Δ/Δ</i> embryos at E14.5 remained vertically oriented when compared to <i>Bmp7^wt/wt</i> littermate embryos. Note the increased tongue height in <i>Bmp7^Δ/Δ</i> embryos. At E15.5 the palatal shelves of <i>Bmp7^Δ/Δ</i> embryos were in the process of reorientation, while the shelves of <i>Bmp7^wt/wt</i> littermates already had started to fuse along their medial edges (arrow). (B) Coronal sections along the A-P axis of the developing palates at E15.5 stained with Alcian Blue and Fast Red demonstrate regional differences in palatal shelf reorientation in the <i>Bmp7^Δ/Δ</i> embryos. The delay in reorientation is most evident in the anterior region. The mid-palatal region was defined by the presence of the 1^st molar tooth bud (*) on the sections. n; nose, p; palatal shelf, t; tongue, <i>Scale bar: 100 </i><i>μm.</i> (C) Ventral views of Meckeĺs cartilage (stained with Alcian Blue) at E14.5 reveal that its halves are significantly shorter and fail to fuse in the anterior region in <i>Bmp7^Δ/Δ</i> embryos, resulting in a shorter mandible (white dotted lines) with a protruding tongue (yellow dotted line). Upper lips are marked (red dotted lines). (D) Side views of <i>Bmp7^Δ/Δ</i> and <i>Bmp7^wt/wt</i> littermate heads show that the growth of the nasal prominence is also stunted in the mutant in addition to the shorter mandible. Yellow dotted outline in image of mutant embryo corresponds to wt littermate.</p

<i>In vitro</i> cultures of palatal explants from <i>Bmp7</i> show more <i>^Δ</i>^{<b><i>/</i></b><i>Δ</i>} embryos.

<p>(A) Pairs of palatal shelves dissected from developmental stages E14.5 to E16.5 were placed in apposition <i>in vitro and</i> cultured. The results showed that <i>Bmp7^Δ/Δ</i> palatal shelves retain the ability to fuse even at E16.5. (B) An example of a <i>Bmp7^Δ/Δ</i> palatal shelf from E14.5 is shown at the start (0 h), after 20 h and 84 h of culture. Fusion at the midline is visually evident at 84 h and the formation of palatal rugae indicates continuing palatal development. <i>Scale bar: 500 </i><i>μm</i> (C) Explants were sectioned coronally and stained for Keratin14. Continuity of the epithelial layer and disappearance of the medial epithelial seam indicate successful fusion.</p