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In vivo liquid biopsy, especially using the photoacoustic (PA) method, demonstrated high clinical potential for early diagnosis of deadly diseases such as cancer, infections, and cardiovascular disorders through the detection of rare circulating tumor cells (CTCs), bacteria, and clots in the blood background. However, little progress has been made in terms of standardization of these techniques, which is crucial to validate their high sensitivity, accuracy, and reproducibility. In the present study, we addressed this important demand by introducing a dynamic blood vessel phantom with flowing mimic normal and abnormal cells. The light transparent silica microspheres were used as white blood cells and platelets phantoms, while hollow polymeric capsules, filled with hemoglobin and melanin, reproduced red blood cells and melanoma CTCs, respectively. These phantoms were successfully used for calibration of the PA flow cytometry platform with high-speed signal processing. The results suggest that these dynamic cell flow phantoms with appropriate biochemical, optical, thermal, and acoustic properties can be promising for the establishment of standardization tool for calibration of PA, fluorescent, Raman, and other detection methods of in vivo flow cytometry and liquid biopsy. [ABSTRACT FROM AUTHOR]

Label-free characterization of cell subpopulations is a very promising biomedical approach. Nowadays, there are several label-free methods based on different physical properties such as size, density, stiffness, etc. allowing the characterization of biological objects. However, fluorescence properties are the most suitable feature for the label-free study of tissue and cells. Understanding the autofluorescence level peculiarities of normal and pathological / live and dead cells can become a helpful tool for cells' metabolic activity, viability evaluation, and diagnostics of a number of diseases. In this study, we applied a series of mouse cell lines (RAW 264.7 - macrophages, L929 - fibroblasts, C2C12 – myoblasts, and B16-F10 – melanoma) to compare cell autofluorescence of live and dead cells under 488 nm laser excitation and found the difference between their autofluorescence depending on a cell state and type. [ABSTRACT FROM AUTHOR]

Objective: To analyze the expression, regulation, and role of microRNA‐125b (miR‐125b) in systemic sclerosis (SSc). Methods: MiR‐125b expression was assessed by quantitative polymerase chain reaction (qPCR) of RNA from dermal fibroblasts and whole skin biopsy specimens from healthy controls and SSc patients. To identify downstream effectors, RNA from healthy control fibroblasts was sequenced after miR‐125b knockdown and further validated using qPCR and Western blotting. Fibrosis, apoptosis, and proliferation were assessed by Caspase‐Glo 3/7 assay, Western blotting, immunofluorescence staining for cleaved caspase 3, and annexin V real‐time assay in dermal fibroblasts. Results: Expression of miR‐125b was significantly down‐regulated in SSc skin biopsy specimens by 53% (median fold change 0.47 [interquartile range 0.35–0.69]; P < 0.001) and in SSc dermal fibroblasts by 47% (median fold change 0.53 [interquartile range 0.36–0.58]; P < 0.001) compared to healthy control skin biopsy specimens and fibroblasts, respectively (n = 10 samples per group). Treatment with the histone deacetylase inhibitors trichostatin A and tubastatin A significantly decreased the expression of miR‐125b in dermal fibroblasts. MiR‐125b knockdown significantly reduced cell proliferation and α‐smooth muscle actin (α‐SMA) expression at the messenger RNA (mRNA) and protein levels. RNA‐Seq identified BAK1, BMF, and BBC3 as potential targets of miR‐125b. Quantitative PCR confirmed that knockdown of miR‐125b up‐regulated these genes (P < 0.01; n = 12). Bcl‐2 homologous antagonist killer 1 showed the strongest induction confirmed at the protein level (P < 0.01; n = 10). Consequently, miR‐125b knockdown increased apoptosis compared to scrambled control. Accordingly, miR‐125b overexpression decreased apoptosis. Conclusion: Our findings indicate that miR‐125b is down‐regulated in SSc skin and primary dermal fibroblasts. MiR‐125b down‐regulation increases apoptosis and decreases proliferation and α‐SMA expression in dermal fibroblasts, indicating that its compensatory, antifibrotic mechanism may be a potential novel therapeutic option. [ABSTRACT FROM AUTHOR]

Abstract A novel type of microcontainers based on hollow silver alginate microspheres and magnetite nanoparticles is reported as development of recently published technology. Magnetite nanoparticles were incorporated by two methods - co-precipitation with porous calcium carbonate during template formation and adsorption onto CaCO 3 particles or microcontainers' shell. Amount of magnetite loaded and microshells size (4.6 to 6.9 μm) were found to depend on the chosen method for magnetite nanoparticles incorporation. Stability of hollow microshells in saline, phosphate buffer and culturing media was studied. Microcontainers' susceptibility to magnetic field was investigated in solutions of varied viscosity, and their group movement velocity under constant magnetic field was evaluated by sequential optical microscopy imaging. Cell viability tests with prepared microshells were performed that demonstrated negligible cytotoxicity effect on human dermal fibroblasts cells. With HeLa cells moderate viability inhibition was found at high carriers:cells ratio at early time points which is attributed to more active and receptor-mediated endocytosis of carriers as well as known cytotoxicity of magnetite in some cancer cells. At 24 and 48 h time points HeLa cells proliferation fully recovers. Reported data opens perspectives for further biomedical-oriented studies and application of this novel kind of microcontainers with a number of techniques applicable for imaging, control and triggered cargo release provided by presence of silver and magnetite nanoparticles in the carriers and their suitability for further versatile functionalization by traditional LbL approach if needed. Highlights • In-situ loading of recently reported novel type of microcarriers with controlled amount and location of Ag and Fe 3 O 4 ; • Susceptibility of carriers to magnetic field in viscous media (resembling biological liquids such as blood, lymph) is shown; • No or moderate cytotoxic effect of microshells during first 12 h of incubation with full later recovery. [ABSTRACT FROM AUTHOR]

To understand life-long neurogenesis in the dentate gyrus (DG), characterizing dentate neural stem cells and the signals controlling their development are crucial. In the present study, we show that bone morphogenic protein (Bmp) signaling is a critical regulator of embry-onic dentate development, required for initiating neurogenesis in embryonic DG progenitors and required for the establishment of dentate neural stem cells postnatally. We tested the hypothesis that Bmp signaling regulates dentate development in part by controlling the expression of Lefl, a Wnt responsive transcription factor expressed in dentate stem cells and absolutely required for dentate granule cell production. Bmp activation through the Acvrl receptor induced Lefl expression and neurogenesis in the embryonic DG. Ectopic expression of Bmp7 in the embryonic midline increased DG neurogenesis and inhibition of local Bmp signaling decreased embryonic DG neurogenesis. Mice with selective loss of Bmp expression due to defective meningeal development or with selective conditional deletion of meningeal Bmp7 also have dentate developmental defects. Conditional deletion of Activin receptor type I (Acvrl) or Smad4 (a downstream target nuclear effector of Bmp signaling) in DG neural stem cells resulted in defects in the postnatal subgranular zone and reduced neurogenesis. These results suggest that Acvrl-mediated meningeal Bmp signaling regulates Lefl expression in the dentate, regulating embryonic DG neurogenesis, DG neural stem cell niche formation, and maintenance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Magnetic contrast agents are widely used in magnetic resonance imaging in order to significantly change the signals from the regions of interest in comparison with the surrounding tissue. Despite a high variety of single-mode T1 or T2 contrast agents, there is a need for dual-mode contrast from the one agent. Here, we report on the synthesis of magnetic submicron carriers, containing Fe3O4 nanoparticles in their structure. We show the ability to control magnetic resonance contrast by changing not only the number of magnetite nanoparticles in one carrier or the concentration of magnetite in the suspension but also the structure of the core–shell itself. The obtained data open up the prospects for dual-mode T1/T2 magnetic contrast formation, as well as provides the basis for future investigations in this direction. [ABSTRACT FROM AUTHOR]

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB. Author summary: Gram-negative bacteria possess a highly impermeable outer membrane, which protects against environmental stress and antibiotics. Outer membrane phospholipid transport remained mysterious until YhdP, TamB, and YdbH were recently implicated in phospholipid transport between the inner and outer membranes of E. coli. Similar roles for YhdP and/or TamB have been suggested in both closely and distantly related gram-negative bacteria. Here, given the transporters' apparent partial redundancy, we investigated functional differentiation between YhdP and TamB and its potential link to phospholipid metabolism. Our data demonstrate YhdP and TamB have differential involvement with fatty acid and phospholipid metabolism. Transport of increased levels of cardiolipin and saturated phospholipids in the absence of YhdP and presence of TamB at a non-permissive temperature is lethal. These data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. One possible mechanism for this differentiation would be differential phospholipid transport preference between YhdP and TamB. Diversification of function between YhdP and TamB roles in phospholipid metabolism provides a potential mechanism for regulation of phospholipid composition, and possibly the mechanical strength and permeability of the outer membrane, and so the cell's intrinsic antibiotic resistance, in changing environmental conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Figure Graph: Figure 3 The efficiency of magnetic capture of differently sized polyelectrolyte microcapsules from the flow depending on the amount of loaded magnetite and the flow velocity. The Influence of Magnetic Composite Capsule Structure and Size on Their Trapping Efficiency in the Flow. "The efficiency of magnetic capture of differently sized polyelectrolyte microcapsules from the flow depending on the amount of loaded magnetite and the flow velocity." as published. [Extracted from the article]

Drug carriers based on polyelectrolyte microcapsules remotely controlled with an external magnetic field are a promising drug delivery system. However, the influence of capsule parameters on microcapsules' behavior in vivo is still ambiguous and requires additional study. Here, we discuss how the processes occurring in the blood flow influence the circulation time of magnetic polyelectrolyte microcapsules in mouse blood after injection into the blood circulatory system and their interaction with different blood components, such as WBCs and RBCs. The investigation of microcapsules ranging in diameter 1–5.5 μ m allowed us to reveal the dynamics of their filtration by vital organs, cytotoxicity, and hemotoxicity, which is dependent on their size, alongside the efficiency of their interaction with the magnetic field. Our results show that small capsules have a long circulation time and do not affect blood cells. In contrast, the injection of large 5.5 μ m microcapsules leads to fast filtration from the blood flow, induces the inhibition of macrophage cell line proliferation after 48 h, and causes an increase in hemolysis, depending on the carrier concentration. The obtained results reveal the possible directions of fine-tuning microcapsule parameters, maximizing capsule payload without the side effects for the blood flow or the blood cells. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Vaterite submicron particles provide enhanced loading capacity for naftifine immobilization. • CaCO 3 recrystallization ensures sustained antimycotic release for more than 72 h in model media. • Additional carrier coatings allow sufficient prolongation of this release. • Loaded carriers enable efficient cellular uptake and enhanced fungi growth inhibition. • The system provides deep penetration and gradual degradation in hair follicles in vivo. [ABSTRACT FROM AUTHOR]