Various hypertensive stimuli lead to exuberant adventitial collagen deposition in large arteries, exacerbating blood pressure elevation and end-organ damage. Collagen production is generally ...attributed to resident fibroblasts; however, other cells, including resident and bone marrow-derived stem cell antigen positive (Sca-1(+)) cells and endothelial and vascular smooth muscle cells, can produce collagen and contribute to vascular stiffening. Using flow cytometry and immunofluorescence, we found that adventitial Sca-1(+) progenitor cells begin to produce collagen and acquire a fibroblast-like phenotype in hypertension. We also found that bone marrow-derived cells represent more than half of the matrix-producing cells in hypertension, and that one-third of these are Sca-1(+). Cell sorting and lineage-tracing studies showed that cells of endothelial origin contribute to no more than one fourth of adventitial collagen I(+) cells, whereas those of vascular smooth muscle lineage do not contribute. Our findings indicate that Sca-1(+) progenitor cells and bone marrow-derived infiltrating fibrocytes are major sources of arterial fibrosis in hypertension. Endothelial to mesenchymal transition likely also contributes, albeit to a lesser extent and pre-existing resident fibroblasts represent a minority of aortic collagen-producing cells in hypertension. This study shows that vascular stiffening represents a complex process involving recruitment and transformation of multiple cells types that ultimately elaborate adventitial extracellular matrix.
The objective of the current research work was to fabricate a fosfestrol (FST)-loaded self-nanoemulsifying drug delivery system (SNEDDS) to escalate the oral solubility and bioavailability and ...thereby the effectiveness of FST against prostate cancer.
3
full factorial design was employed, and the effect of lipid and surfactant mixtures on percentage transmittance, time required for self-emulsification, and drug release were studied. The optimized solid FST-loaded SNEDDS (FSTNE) was characterized for in vitro anticancer activity and Caco-2 cell permeability, and in vivo pharmacokinetic parameters.
Using different ratios of surfactant and co-surfactant (Km) a pseudo ternary phase diagram was constructed. Thirteen liquid nano emulsion formulations (LNE-1 to LNE-13) were formulated at Km = 3:1. LNE-9 exhibited a higher % transmittance (99.25 ± 1.82 %) and a lower self-emulsification time (24 ± 0.32 s). No incompatibility was observed in FT-IR analysis. Within 20 min the solidified FST loaded LNE-9 (FSTNE) formulation showed almost complete drug release (98.20 ± 1.30 %) when compared to marketed formulation (40.36 ± 2.8 %), and pure FST (32 ± 3.3 %) in 0.1 N HCl. In pH 6.8 phosphate buffer, the release profiles are found moderately higher than in 0.1 N HCl. FSTNE significantly (P < 0.001) inhibited the PC-3 prostate cell proliferation and also caused apoptosis (P < 0.001) compared to FST. The in vitro Caco-2 cell permeability study results revealed 4.68-fold higher cell permeability of FSTNE than FST. Remarkably, 4.5-fold rise in bioavailability was observed after oral administration of FSTNE than plain FST.
FSTNE remarkably enhanced the in vitro anticancer activity and Caco-2 cell permeability, and in vivo bioavailability of FST. Thus, FST-SNEDDS could be utilized as a potential carrier for effective oral treatment of prostate cancer.
In the paper, we present an integrated flow cytometer with a 2D array of magnetic sensors based on dual-frequency oscillators in a 65-nm CMOS process, with the chip packaged with microfluidic ...controls. The sensor architecture and the presented array signal processing allows uninhibited flow of the sample for high throughput without the need for hydrodynamic focusing to a single sensor. To overcome the challenge of sensitivity and specificity that comes as a trade off with high throughout, we perform two levels of signal processing. First, utilizing the fact that a magnetically tagged cell is expected to excite sequentially an array of sensors in a time-delayed fashion, we perform inter-site cross-correlation of the sensor spectrograms that allows us to suppress the probability of false detection drastically, allowing theoretical sensitivity reaching towards sub-ppM levels that is needed for rare cell or circulating tumor cell detection. In addition, we implement two distinct methods to suppress correlated low frequency drifts of singular sensors-one with an on-chip sensor reference and one that utilizes the frequency dependence of the susceptibility of super-paramagnetic magnetic beads that we deploy as tags. We demonstrate these techniques on a 7<inline-formula><tex-math notation="LaTeX">\times</tex-math></inline-formula>7 sensor array in 65 nm CMOS technology packaged with microfluidics with magnetically tagged dielectric particles and cultu lymphoma cancer cells.
We develop a high-throughput technique to relate positions of individual cells to their three-dimensional (3D) imaging features with single-cell resolution. The technique is particularly suitable for ...nonadherent cells where existing spatial biology methodologies relating cell properties to their positions in a solid tissue do not apply. Our design consists of two parts, as follows: recording 3D cell images at high throughput (500 to 1,000 cells/s) using a custom 3D imaging flow cytometer (3D-IFC) and dispensing cells in a first-in-first-out (FIFO) manner using a robotic cell placement platform (CPP). To prevent errors due to violations of the FIFO principle, we invented a method that uses marker beads and DNA sequencing software to detect errors. Experiments with human cancer cell lines demonstrate the feasibility of mapping 3D side scattering and fluorescent images, as well as two-dimensional (2D) transmission images of cells to their locations on the membrane filter for around 100,000 cells in less than 10 min. While the current work uses our specially designed 3D imaging flow cytometer to produce 3D cell images, our methodology can support other imaging modalities. The technology and method form a bridge between single-cell image analysis and single-cell molecular analysis.
This article presents the development and testing of a low‐cost (<$60), portable, electrical impedance‐based microflow cytometer for single‐cell analysis under a controlled oxygen microenvironment. ...The system is based on an AD5933 impedance analyzer chip, a microfluidic chip, and an Arduino microcontroller operated by a custom Android application. A representative case study on human red blood cells (RBCs) affected by sickle cell disease is conducted to demonstrate the capability of the cytometry system. Impedance values of sickle blood samples exhibit remarkable deviations from the common reference line obtained from two normal blood samples. Such deviation is quantified by a conformity score, which allows for the measurement of intrapatient and interpatient variations of sickle cell disease. A low conformity score under oxygenated conditions or drastically different conformity scores between oxygenated and deoxygenated conditions can be used to differentiate a sickle blood sample from normal. Furthermore, an equivalent circuit model of a suspended biological cell is used to interpret the electrical impedance of single flowing RBCs. In response to hypoxia treatment, all samples, regardless of disease state, exhibit significant changes in at least one single‐cell electrical property, that is, cytoplasmic resistance and membrane capacitance. The overall response to hypoxia is less in normal cells than those affected by sickle cell disease, where the change in membrane capacitance varies from −23% to seven times as compared with −17% in normal cells. The results reported in this article suggest that the developed method of testing demonstrates the potential application for a low‐cost screening technique for sickle cell disease and other diseases in the field and low‐resource settings. The developed system and methodology can be extended to analyze cellular response to hypoxia in other cell types.
A portable electrical impedance‐based microflow cytometer was developed to measure cellular response to hypoxia. The conformity score assessed from the impedance signals of red blood cells showed substantial intra‐ and interpatient variability in sickle cell disease. This development brings affordability to the diagnosis and monitoring of sickle cell disease in the point of care and resource limited settings.
This article presents an imaging and impedance flow cytometer based on a constriction microchannel with corresponding cell-type classification based on deep neural pattern recognition. When an ...incoming cell reached the entrance of the constriction microchannel, the image of the nucleus labeled with fluorescence was captured by a high-speed camera without the concern of losing focus, whereas when the cell deformed through the constriction microchannel by effectively blocking electrical lines, large impedance variations were sampled by an impedance analyzer. Six key biostructural and bioelectrical parameters (e.g., cell diameter, nuclear roundness, and cytoplasmic conductivity) from thousands of single cells were extracted, producing a successful rate of 88.3% in classifying A549 versus Jurkat versus K562 cells based on the feedforward neural network. In addition, multilayer neural networks of deep learning (e.g., VGG16 of CNN and LSTM of RNN) were also used to process fluorescent images and impedance profiles, producing an almost 100% successful rate in cell-type classification. In summary, the microfluidic flow cytometer reported in this article could characterize single-cell biostructural and bioelectrical properties in a high throughput manner, realizing high successful rates of cell-type classification.
The two-step protocol (2 S) is currently used for boar semen cryopreservation. In this method, the cryoprotectant penetrant agents (CPAs) are added at 5 °C to reduce the toxicity of CPAs. An ...alternative is the one-step protocol (1 S), which is easier, cheaper, and reduces the necessity of equipment, but could increase the toxicity of CPAs. Currently, there are no studies that compared both protocols for boar semen cryopreservation. This experiment aimed to study the effect of cryopreservation protocol (1 S vs 2 S) on boar spermatozoa. In the one-step protocol, after centrifugation, the spermatozoa pellet was resuspended at 17 °C in the extender containing CPAs to achieve a concentration of 1 × 109 spermatozoa/mL and then submitted to cryopreservation. For the two-step protocol, the sperm pellet was resuspended in fraction A at 17 °C to achieve a concentration of 1.5 × 109 spermatozoa/ mL, and then allowed to cool to 5º C before fraction B with CPA was added to the sample to achieve a final concentration of 1 × 109 spermatozoa/mL and followed by freezing. The cryopreservation protocol did not impact total motility at 5 °C (1 S: 78.5 % vs 2 S: 79 %, p > 0.05). After thawing, the two-step protocol improved (p < 0.05) total (1 S: 18.2 % vs 2 S: 29.5 %) and progressive motility (1 S: 9 % vs 2 S: 15%). Further, the 2 S protocol increased (p < 0.05) the percentage of rapid spermatozoa (1 S: 8.7 % vs 2 S: 14.6 %) and spermatozoa with intact plasma and acrosomal membrane (IAIP) (1 S: 40.5 % vs 2 S: 61.5 %), and increased (p < 0.05) live sperm cells with high mitochondrial potential (MHIP) (1 S: 42.9 % vs 2 S: 60 %). The boar semen cryopreservation method (TRT) did not (p > 0.05) alter membrane lipid disorder, lipid peroxidation, and superoxide anion. Thus, the best method for boar semen cryopreservation is the two-step protocol.
•Boar semen cryopreservation in one-step protocol reduces post-thawed total and progressive motility.•Boar semen cryopreservation in one-step protocol reduces viable sperm cells with high mitochondrial potential.•The one-step protocol reduces intact plasma membrane in frozen-thawed semen.•The one-step protocol did not impact motility and membranes integrity at 5 °C.
Antimicrobial susceptibility is slow to determine, taking several days to fully impact treatment. This proof-of-concept study assessed the feasibility of using machine-learning techniques for ...analysis of data produced by the flow cytometer-assisted antimicrobial susceptibility test (FAST) method we developed.
We used machine learning to assess the effect of antimicrobial agents on bacteria, comparing FAST results with broth microdilution (BMD) antimicrobial susceptibility tests (ASTs). We used
(1),
(1) and
(2) strains to develop the machine-learning algorithm, an expanded panel including these plus
(2),
(3),
(1),
(1),
(2) and
(1), tested against FAST and BMD (Sensititre, Oxoid), then two representative isolates directly from blood cultures.
Our data machines defined an antibiotic-unexposed population (AUP) of bacteria, classified the FAST result by antimicrobial concentration range, and determined a concentration-dependent antimicrobial effect (CDE) to establish a predicted inhibitory concentration (PIC). Reference strains of
and
tested with different antimicrobial agents demonstrated concordance between BMD results and machine-learning analysis (CA, categoric agreement of 91 %; EA, essential agreement of 100 %). CA was achieved in 35 (83 %) and EA in 28 (67 %) by machine learning on first pass in a challenge panel of 27 Gram-negative and 15 Gram-positive ASTs. Same-day AST results were obtained from clinical
(1) and
(1) isolates.
The combination of machine learning with the FAST method generated same-day AST results and has the potential to aid early antimicrobial treatment decisions, stewardship and detection of resistance.
A flow cytometer chip fabricated in 65-nm standard CMOS technology embedded into a microfluidic channel is presented. The chip detects cells specifically tagged with magnetic labels by demodulating ...the change in coupling between an excitation coil and a pickup coil of a differential spiral transformer. Operation of the flow cytometer cartridge is verified for the detection of SKBR-3 breast cancer cells tagged with manganese ferrite magnetic labels using EpCAM-biotin-streptavidin chemistry. Magnetic labels of different materials are classified by the difference in the phase of their complex susceptibility.
High dimensional flow cytometry relies on multiple laser sources to excite the wide variety of fluorochromes now available for immunophenotyping. Ultraviolet lasers (usually solid state 355 nm) are a ...critical part of this as they excite the BD Horizon™ Brilliant Ultraviolet (BUV) series of polymer fluorochromes. The BUV dyes have increased the number of simultaneous fluorochromes available for practical high‐dimensional analysis to greater than 40 for spectral cytometry. Immunologists are now seeking to increase this number, requiring both novel fluorochromes and additional laser wavelengths. A laser in the deep ultraviolet (DUV) range (from ca. 260 to 320 nm) has been proposed as an additional excitation source, driven by the on‐going development of additional polymer dyes with DUV excitation. DUV lasers emitting at 280 and 320 nm have been previously validated for flow cytometry but have encountered practical difficulties both in probe excitation behavior and in availability. In this article, we validate an even shorter DUV 266 nm laser source for flow cytometry. This DUV laser provided minimal excitation of the BUV dyes (a desirable characteristic for high‐dimensional analysis) while demonstrating excellent excitation of quantum nanoparticles (Qdots) serving as surrogate fluorochromes for as yet undeveloped DUV excited dyes. DUV 266 nm excitation may therefore be a viable candidate for expanding high‐dimensional flow cytometry into the DUV range and providing an additional incidental excitation wavelength for spectral cytometry. Excitation in a spectral region with strong absorption by nucleic acids and proteins (260–280 nm) did result in strong autofluorescence requiring care in fluorochrome selection. DUV excitation of endogenous molecules may nevertheless have additional utility for label‐free analysis applications.
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