Men with diabetes have negative effects on reproduction that causes sexual dysfunction. Medicinal plants are non‐toxic and much safer than synthetic drugs because regular use of synthetic drugs shows ...long‐term side effects. Curcuma amada (Roxb) is a medicinal plant used in Ayurveda and Unani medicinal systems in India. The goal of this study is to rummage the potential efficiency of the most potent solvent fraction of effective extract of hydro‐methanol 60:40 of C. amada rhizome on male gonadal hypofunction in streptozotocin‐induced diabetic rat. Diabetes‐induced testicular hypofunction was evaluated by glycemic, spermiological, biochemical, genomic, flow cytometric, and histology of testicular tissue. The n‐hexane, chloroform, ethyl‐acetate, and n‐butanol solvent fractions of the said extract were administrated for 4 weeks at 10 mg dose/100 g body weight/day. Among all the used fractions, the ethyl‐acetate solvent fraction‐treated group showed maximum recovery in serum insulin (177.42%), sperm count (92.84%), sperm motility (97.15%), and serum testosterone (164.33%). The diabetic rats treated with ethyl‐acetate solvent fraction also exhibited the maximum resettlement in flow cytometric analysis of sperm viability (55.84%) and sperm mitochondrial integrity (149.79%), gene expression patterns of key markers for androgenesis (Δ5, 3β‐HSD 87.50%, and 17β‐HSD 74.66%) and apoptosis (Bax 44.63%, Bcl‐2 54.03%, and Caspase‐3 35.77%) along with testicular histology. The ethyl‐acetate fraction contains alkaloids, flavonoids, and polyphenols where all of these components are not present in other fractions, may be the most effective cause for the recovery of diabetes‐linked oxidative stress‐mediated testicular hypofunctions.
Practical applications
Nowadays worldwide, the use of synthetic drugs are reduced due to their toxic effect. At present, synthetic drugs are replaced by several herbal drugs, the natural source of medicine which has many therapeutic values. C. amada has strong antioxidant activity due to the presence of bio‐active compound(s) that can able to manage streptozotocin‐induced diabetes linked to oxidative damage of male gonadal organs. Therefore, these bio‐active compound(s)‐containing said medicinal plant may use as a good source of antioxidative food in the food industry as nutraceuticals and in pharmaceutical industries for the development of the herbal drug to manage diabetes‐linked male gonadal hypofunctions. At present, WHO also gives emphasis for developing one drug‐multi‐disease therapy. From such a viewpoint, this active fraction‐containing phytomolecules may have corrective efficacy against diabetes as well as oxidative stress‐linked testicular complications.
Schematic form of experimental work.
The potentiality of nanomedicine in the cancer treatment being widely recognized in the recent years. In the present investigation, the synergistic effects of chitosan-modified selenium nanoparticles ...loaded with paclitaxel (PTX-chit-SeNPs) were studied. These selenium nanoparticles were tested for drug release analysis at a pH of 7.4 and 5.5, and further characterized using FTIR, DLS, zeta potential, and TEM to confirm their morphology, and the encapsulation of the drug was carried out using UPLC analysis. Quantitative evaluation of anti-cancer properties was performed via MTT analysis, apoptosis, gene expression analysis, cell cycle arrest, and over-production of ROS. The unique combination of phytochemicals from the seed extract, chitosan, paclitaxel, and selenium nanoparticles can be effectively utilized to combat cancerous cells. The production of the nanosystem has been demonstrated to be cost-effective and have unique characteristics, and can be utilized for improving future diagnostic approaches.
Cytosine methylation is the leading epigenetic modification on DNA playing a role in gene regulation. Methylation can occur in cytosines of any nucleic acids in cytosol (as mitochondrial DNA, mtDNA) ...and in nuclear DNA (ncDNA). mtDNA exists as multiple copies within numerous mitochondria. This suggests that the number of mitochondria and mtDNA copy number can indicate the presence of a significant amount of DNA methylation within total DNA methylation detected. However, immunofluorescence method does not have a step to discriminate the staining between ncDNA and mtDNA. Antibodies used in immunological methods are methylation-specific but not selective for DNA type and they can bind to methylated cytosines in any DNA within the specimen. Current study aimed to understand whether mtDNA methylation interferes with the detection of nuclear DNA methylation by immunofluorescence and affinity enrichment (ELISA) in different mammalian cells. Experiments were performed to distinguish methylation between mtDNA and ncDNA. Immunofluorescence showed that there was no significant difference in the detected amount of methylation between mitochondrial and nuclear DNA. But ELISA revealed that up to 25% of cellular methylation was derived from mitochondria. This suggests that significant contamination of mtDNA methylation with ncDNA methylation can result in overestimation of the quantitative level of nuclear methylation.
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•Mitochondrial DNA methylation interferes with the detection of global DNA methylation in cells.•ELISA-based detection of nuclear DNA methylation needs attention for significant contribution of organelle DNA methylation.•Mitochondrial DNA copy number is not related to the level of mtDNA methylation.
Magnesium ion (Mg+2) plays an important role in various biological processes. All the commercial indicators available share a common drawback, i.e., they have a higher affinity towards calcium ions ...(Ca+2) than Mg+2. In this study, we reported a new robust green fluorescent indicator, Mag-520, for detection of Mg+2 in live cells. Our results showed that Mag-520 has 10 fold higher affinity towards Mg+2 than Ca+2, while mag-fluo-4 has less than 0.5 fold affinity to Mg+2 than Ca+2 under the same conditions using flow cytometry and fluorescence microscopy. The results demonstrated that Mag-520 provides a better tool to measure Mg+2 with less interference from Ca+2.
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•Mag-520 provides more specificity and selectivity towards magnesium than calcium, unlike other magnesium detection dyes.•Mag-520 offers higher sensitivity for physiological changes that occur in cells.•The acetoxymethyl ester form of Mag-520 is useful to detect changes in magnesium for in vitro analysis.•Mag-520 is compatible with widely available 488 nm laser excitation and 530/30 nm emission filter in flow cytometers.
Hepatocellular carcinoma (HCC) is asymptomatic at an early stage which delays its timely diagnosis and treatment. Circulating tumor cells (CTCs), derived from a primary or secondary tumor, may help ...in the management of HCC. Here, we evaluate and characterize CTCs in liver disease patients.
In total, 65 patients, categorized into liver cirrhosis (LC) (n = 30) and HCC (n = 35), were enrolled. Using ImagestreamX MkII imaging flow cytometer, CTCs were detected and characterized using biomarker expression of EpCAM, CK, AFP, CD45, and DRAQ5 in LC and HCC patients.
CTCs were detected in 33/35 (94%) HCC patients and in 28/30 (93%) LC patients. In the HCC group, the number of biomarker-positive CTCs was higher in BCLC stage D when compared with others. EpCAM + CK was the most expressed biomarker on CTCs in LC versus HCC (83.3% vs. 77.14%), followed by AFP (80% vs. 65.71%), EpCAM (30% vs. 28.57%), and CK (16.6% vs. 14.28%). The EpCAM cell area was significantly associated (P value = 0.031) with the CTC-positive status. The combination biomarker expression of CTCs cell area (EpCAM, CK, and AFP) performed well with the area under the curve of 0.92, high sensitivity, and specificity in detecting early-stage and AFP-negative HCC as well as in AFP-negative LC cases.
Enumeration and cell area of CTCs may be used as a biomarker for early detection of HCC and guiding treatment.
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•CTCs were characterized using a biomarker panel (+EpCAM, +CK, +AFP, −CD45, +DRAQ) in LC and HCC patients using IFC.•Surface markers, EpCAM + CK, were the most expressed biomarkers on CTCs in LC vs. HCC followed by AFP, EpCAM and CK.•The cell area of AFP was significantly higher in the LC group when compared with the HCC group (P value = 0.035).•CTCs cell area (EpCAM+CK+ AFP) performed well with an AUC of 0.92, in detecting early-stage and AFP-negative HCC and LC cases.
This paper presents an innovative micro flow cytometer which is capable of counting and sorting cells or particles. This compact device employs electrokinetic forces rather than the more conventional ...hydrodynamic forces technique for flow focusing and sample switching, and incorporates buried optical fibers for the on-line detection of cells or particles. This design approach results in a compact microfluidic system and an easier integration process. The proposed cytometer integrates several critical modules, namely electrokinetic-focusing devices, built-in control electrodes, buried optical fibers for on-line detection, and electrokinetic flow switches for bio-particle collection. A linear relationship exists between the focused stream width (
d) and the focusing ratio (
F/φ), which is estimated to be
D≈134.5−53.8
F/
φ. The relationship between the particle velocity (
U) and the applied voltage (
V) is also investigated. Numerical and experimental data confirm the effectiveness of the device when applied to the counting and sorting of 10
μm diameter particles and red blood cells.
Quantitative detections of proteins for single cells have made great contributions to tumor heterogeneity and immune variance while effective tools of measuring single-cell intracellular proteins are ...under developments. This study reported a flow cytometer based on constriction microchannel and light modulation, which was capable of quantifying intracellular proteins for single cells with high resolution. Cells tagged with fluorescein labelled antibodies were squeezed into the constriction microchannel as either the calibration or detection structure where a light source with a carrier wave was used for fluorescent modulation, which was further demodulated into the low-frequency domain and translated into numbers of single-cell proteins. In platform characterization, the inclusion of light modulation was shown to effectively improve the detection resolution from ~1000 intracellular proteins per cell without modulation to ~100 intracellular proteins per cells with modulation. As demonstrations, expressions of mutant p53 or phosphorylated p53 from tens of thousands of single T47D and/or A431 cells were quantitatively measured by the microfluidic quantitative flow cytometer, which were 1.78 ± <inline-formula> <tex-math notation="LaTeX">0.96 \times10 </tex-math></inline-formula> 5 mutant p53 per cell for T47D cells (<inline-formula> <tex-math notation="LaTeX">{N}_{c} =10\,315 </tex-math></inline-formula>), 1.99 ± <inline-formula> <tex-math notation="LaTeX">1.49\times10 </tex-math></inline-formula> 5 mutant p53 per cell for A431 cells (<inline-formula> <tex-math notation="LaTeX">{N}_{c} =15\,665 </tex-math></inline-formula>), and 6.34 ± <inline-formula> <tex-math notation="LaTeX">7.50\times10 </tex-math></inline-formula> 3 phosphorylated p53 per cell for A431 cells treated with doxorubicin (<inline-formula> <tex-math notation="LaTeX">{N}_{c} =12\,812 </tex-math></inline-formula>). In conclusion, the inclusion of light modulation can effectively decrease electrical and optical noises and therefore improve detection resolution in microfluidic quantitative flow cytometry.
The objective of this study was to evaluate the effect of antifreeze protein type III (AFP III) on the freezing of epididymal spermatozoa of goats. A total of 16 pairs of testicles were collected in ...a slaughterhouse and transported at approximately 5 °C in a thermal box. Epididymal spermatozoa were recovered by retrograde lavage and evaluated using a phase contrast microscope. Then, they were cryopreserved in extender based on Tris-egg yolk, supplemented with AFP III (0, 1, 10, 100 μg/mL), using an automated system. After thawing (37 °C/30 s), the spermatozoa kinetics were evaluated using the CASA automated system; and plasma and acrosome membrane integrity, mitochondrial membrane potential, and intracellular ROS production, by flow cytometry. There was no difference (P ≥ 0.05) between the experimental groups for the parameters of spermatozoa kinetics, mitochondrial membrane potential, and ROS production. However, the integrity of plasma and acrosome membranes of frozen spermatozoa with 100 μg/mL of AFP III was lower (P < 0.05) than the control group. It was concluded that the addition of AFP III to the Tris-egg yolk extender, used in the freezing of sperm obtained from the epididymis of goats, did not improve the preservation of these cells.
•AFP III did not improve the preservation sperm from the epididymis of goats.•AFP III decreases intact spermatozoa plasma and acrosome membranes.•No effect on sperm kinetics, MMP, and intracellular ROS production.
Resumen OBJETIVO: Evaluar si la manipulación de gametos con sorter de citometría de flujo repercute negativamente en los indicadores clave de rendimiento de un laboratorio de reproducción asistida. ...MATERIALES Y MÉTODOS: Estudio descriptivo y retrospectivo, llevado a cabo en parejas a quienes se efectuó fecundación in vitro mediante inyección intracitoplasmática de espermatozoides (ICSI), con selección espermática, mediante un sorter de citometría de flujo, para selección de sexo. El estudio se efectuó en el New Hope Fertility Center de Guadalajara y Ciudad de México, de junio de 2014 a agosto de 2017. Los resultados se compararon con un grupo control seleccionado al azar. Se evaluaron los indicadores decisivos de rendimiento (KPI´s); tasa de fecundación normal, anormal (1PN, ≥ 3 PN) y fallida; tasa de degeneración posterior a ICSI; tasas de segmentación o división, blastocisto, implantación (segmentación y blastocisto) y recién nacido. Se utilizó la prueba t de Student para dos muestras y se consideró estadísticamente significativo el valor de p < 0.05. RESULTADOS: Se evaluaron 150 ciclos. Grupo 1: ICSI con selección espermática y sorter de citometría de flujo (n = 40); Grupo 2: ICSI sin sorter de citometría de flujo (n = 110). Los indicadores clave de rendimiento del grupo 1 disminuyeron; se reportaron tasas de fecundación fallida de 1.6%, blastocisto 17.4%, implantación en la segmentación 10%, implantación en blastocisto 14.2% y de recién nacido 14.5%. CONCLUSIONES: La manipulación de gametos con sorter de citometría de flujo reportó un efecto negativo en los indicadores clave de rendimiento del laboratorio de reproducción asistida, específicamente en las tasas de blastocisto, implantación de blastocisto y de recién nacido.
A stochastic model of the flow cytometer measurement process was developed to assess the nature of the observed coefficient of variation (CV%) of the mean fluorescence intensity (MFI) from a ...population of labeled microspheres (beads). Several sources of variability were considered: the total number of labels on a bead, the path through the laser beam, the optical absorption cross-section, the quantum yield, the numerical aperture of the collection optics, and the photoelectron conversion efficiency of the photomultiplier (PMT) cathode. The variation in the number of labels on a bead had the largest effect on the CV% of the MFI of the bead population. The variation in the path of the bead through the laser beam was minimized using flat-top lasers. The variability in the average optical properties of the labels was of minor importance for beads with sufficiently large number of labels. The application of the bead results to the measured CV% of labeled B cells indicated that the measured CV% was a reliable measure of the variability of antibodies bound per cell. With some modifications, the model can be extended to multicolor flow cytometers and to the study of CV% from cells with low fluorescence signal.
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