High-quality sperm cells are crucial to reproductive success for both males and post-mating females in animals. Sperm viability, defined as the proportion of viable sperm cells, is used as a sperm quality index and this method has provided new insights into research on reproductive strategies. Sperm viability has been assessed by fluorescent staining of sperm cells. However, current staining protocols could potentially underestimate viability due to cell damage caused by cell treatments such as high dye concentration and long time for post-mounting. In this study, we established a method that enables rapid sperm viability assessment, has low sperm cell toxicity, and provides precise results regardless of operator expertise, and cost-effective using sperm cells from an ant, Crematogaster osakensis (Hymenoptera). First, to shorten the time for observation of a sufficient number of sperm cells, the volume per field of view was increased by height elevation between the glass slide and the coverslip, thereby we increased the number of sperm cells in a field of view. Second, to reduce sperm cell toxicity, we optimized the minimum dye concentration and incubation time using acridine orange (AO) and Hoechst in addition to SYBR 14 and propidium iodide (PI), which has been used in most previous studies. We determined the optimal protocol to be 1 µg/mL AO and 150 µM PI without incubation. Besides, we automated counting sperm cells with ImageJ software and combined with manual correction for more accurate results. We employed the improved method for sperm samples from mealworm beetles (Tenebrio molitor) and silkmoths (Bombyx mori). This method, established through our study, will advance research on reproductive strategies, including sperm competition and sperm quality maintenance in females.

The effect of non-ionizing millimeter range electromagnetic waves (MM EMW) (30-300 GHz) on the bovine serum albumin (BSA) interaction peculiarities with acridine orange (AO) has been studied in vitro. The frequencies 41.8 and 50.3 GHz were chosen, since the first one is nonresonant frequency for the water, while the second one is resonant for water. The binding constant and number of binding sites were calculated at both irradiation presence and absence. AO was revealed to bind to BSA, while after the protein irradiation the interaction force strengthens. However, it was also shown that there are differences of the interaction parameters while irradiating by 41.8 or 50.3 GHz. AO binds to BSA, irradiated by MM EMW with the frequency 41.8 GHz much more weaker, than to that, irradiated by MM EMW with the frequency 50.3 GHz.
The manuscript is devoted to the study of the effect of millimeter range electromagnetic waves with the frequencies 41.8 and 50.3 GHz on the model biological system, being on molecular level of organization. Nowadays millimeter range electromagnetic waves compose a significant part of electromagnetic pollution in the environment and affect biological material, besides these waves are used in extremely high frequencies-therapy ((30–300 GHz), which are millimeter range electromagnetic waves (1–10 mm)). On the other hand, the problem of their effect mechanism is mainly connected to water, since the resonant frequencies for water molecules are in the interval of millimeter waves. In the present study, as such biological molecule, the bovine serum albumin has been chosen, which interacts with acridine orange. Serum albumins are known to carry and transport various endogenous and exogenous agents, including drugs throughout circulatory system. In turn, acridine orange has been extensively used for biological staining to differentiate DNA from RNA by fluorescence emission for years, Nowadays, it is considered as a promising agent for antitumorous treatment and diagnosis.The data obtained show that the interaction between bovine serum albumin and acridine orange changes, when the solution of albumin is irradiated by the millimeter waves with the frequencies 41.8 and 50.3 GHz. However, the interaction alteration depends on the frequency as well. Thus, the irradiation with the frequency 41.8 GHz makes insignificant changes, while that with the frequency 50.3 GHz induces significant changes of measured parameters. The studies were conducted by absorption, fluorescence and CD spectroscopy methods.

Mg2+, the most abundant divalent cation in living cells, plays a pivotal role in numerous enzymatic reactions and is of particular importance for organisms performing oxygenic photosynthesis. Its significance extends beyond serving as the central ion of the chlorophyll molecule, as it also acts as a counterion during the light reaction to balance the proton gradient across the thylakoid membranes. In this study, we investigated the effects of Mg2+ limitation on the physiology of the well-known model microorganism Synechocystis sp. PCC6803. Our findings reveal that Mg2+ deficiency triggers both morphological and functional changes. As seen in other oxygenic photosynthetic organisms, Mg2+ deficiency led to a decrease in cellular chlorophyll concentration. Moreover, the PSI-to-PSII ratio decreased, impacting the photosynthetic efficiency of the cell. In line with this, Mg2+ deficiency led to a change in the proton gradient built up across the thylakoid membrane upon illumination.

OBJECTIVE: The increasing incidence of renal cell carcinoma (RCC) and its associated bone metastasis pose challenges in surgical interventions, warranting the exploration of novel therapeutic approaches. Therefore, this study aimed to assess the impact of hematogenously administering acridine orange (AO) alone and in combination with zoledronic acid (ZA) on bone metastasis in RCC.
METHODS: RENCA cells (1.0×106 cells/10 μl) were directly injected into the right femur of male BALB/c mice. The mice were categorized into four groups based on the applied therapeutic intervention and were euthanized after five weeks. Micro-computed tomography was performed to quantify the extent of periosteal reaction, indicative of bone metastasis, along the entire length of the femur. Tumor weight and volume were measured at euthanization. Hematoxylin and eosin staining was used to examine the extent of tumor development in the bone. Apoptotic cell, osteoclast, and vascular endothelial growth factor (VEGF)-positive cell counts were assessed using TdT-mediated dUTP-biotin nick end labeling, tartrate-resistant acid phosphatase staining, and VEGF staining, respectively.
RESULTS: The periosteal reaction was significantly reduced in the intervention groups compared to the control group (p<0.05). The apoptotic cell numbers in the intervention groups surpassed that in the control group (p<0.05), whereas those of osteoclasts and VEGF-positive cells in the intervention groups were lower than those in the control group (p<0.05).
CONCLUSIONS: AO hinders bone metastasis progression in RCC, and combination therapy with ZA may be more effective than AO administration alone.

Microbial dysbiosis is an important trigger in the development of oral diseases. Oral keratinocytes or gingival epithelial cells (GECs) offer protection against various microbial insults. Recent studies suggest that GECs expressed higher level of bitter taste receptor 14 (T2R14) compared to other taste receptors and toll-like receptors and act as innate immune sentinels. Macroautophagy or autophagy is a cellular conserved process involved in the regulation of host innate immune responses against microbial infection. Here, we describe a robust method for evaluation of T2R14-dependent autophagy flux in GECs. Autophagy flux was detected using Western blot analysis in GECs and further was confirmed using Acridine Orange-dependent flow cytometry analysis.

Macroautophagy (hereafter autophagy) is a lysosomal degradation pathway that functions in nutrient recycling and as a mechanism of innate immunity. Previously, we reported a novel host-bacteria interaction between cariogenic S. mutans and bitter taste receptor (T2R14) in gingival epithelial cells (GECs), leading to an innate immune response. Further, S. mutans might be using the host immune system to inhibit other Gram-positive bacteria, such as S. aureus. To determine whether these bacteria exploit the autophagic machinery of GEC, it is first necessary to evaluate the role of T2R14 in modulating autophagic flux. So far, the role of T2R14 in the regulation of autophagy is not well characterized. Therefore, in this study, for the first time, we report that T2R14 downregulates autophagy flux in GECs, and T2R14 knockout increases acidic vacuoles. However, the treatments of GEC WT with a T2R14 agonist and antagonist did not lead to a significant change in acidic vacuole formation. Transmission electron microscopy morphometric results also suggested an increased number of autophagic vesicles in T2R14-knockout GEC. Further, our results suggest that S. mutans competence stimulating peptide CSP-1 showed robust intracellular calcium release and this effect is both T2R14- and autophagy protein 7-dependent. In this study, we provide the first evidence that T2R14 modulates autophagy flux in GEC. The results of the current study could help in identifying the impact of T2R in regulation of the immuno-microenvironment of GEC and subsequently oral health.

Normoxic inactivation of prolyl hydroxylase-2 (PHD-2) in tumour microenvironment paves the way for cancer cells to thrive under the influence of HIF-1α and NF-κB. Henceforth, the present study is aimed to identify small molecule activators of PHD-2. A virtual screening was conducted on a library consisting of 265,242 chemical compounds, with the objective of identifying molecules that exhibit structural similarities to the furan chalcone scaffold. Further, PHD-2 activation potential of screened compound was determined using in vitro 2-oxoglutarate assay. The cytotoxic activity and apoptotic potential of screened compound was determined using various staining techniques, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 4\',6-diamidino-2-phenylindole (DAPI), 1,1\',3,3\'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), and acridine orange/ethidium bromide (AO/EB), against MCF-7 cells. 7,12-Dimethylbenz[a]anthracene (DMBA) model of mammary gland cancer was used to study the in vivo antineoplastic efficacy of screened compound. [(E)-1-(4-fluorophenyl)-3-(furan-2-yl) prop-2-en-1-one] (BBAP-7) was screened and validated as a PHD-2 activator by an in vitro 2-oxo-glutarate assay. The IC50 of BBAP-7 on MCF-7 cells is 18.84 µM. AO/EB and DAPI staining showed nuclear fragmentation, blebbing and condensation in MCF-7 cells following BBAP-7 treatment. The red-to-green intensity ratio of JC-1 stained MCF-7 cells decreased after BBAP-7 treatment, indicating mitochondrial-mediated apoptosis. DMBA caused mammary gland dysplasia, duct hyperplasia and ductal carcinoma in situ. Carmine staining, histopathology, and scanning electron microscopy demonstrated that BBAP-7, alone or with tirapazamine, restored mammary gland surface morphology and structural integrity. Additionally, BBAP-7 therapy significantly reduced oxidative stress and glycolysis. The findings reveal that BBAP-7 activates PHD-2, making it a promising anticancer drug.

Leishmaniasis is a neglected tropical disease caused by numerous species of Leishmania parasites, including Leishmania major. The parasite is transmitted by several species of sandfly vectors and infects myeloid cells leading to a myriad of inflammatory responses, immune dysregulations, and disease manifestations. Every cell undergoes autophagy, a self-regulated degradative process that permits the cells to recycle damaged or worn-out organelles in order to maintain cellular health and homeostasis. Studies have shown that Leishmania modulates their host cell autophagic machinery and there are indications that the parasite-specific autophagic processes may be valuable for parasite virulence and survival. However, the role of autophagy in Leishmania is inconclusive because of the limited tools available to study the Leishmania-specific autophagic machinery. Here, we describe methods to study and definitively confirm autophagy in Leishmania major. Transmission electron microscopy (TEM) allowed us to visualize Leishmania autophagosomes, especially those containing damaged mitochondrial content, as well as dividing mitochondria with ongoing fusion/fission processes. Flow cytometry enabled us to identify the amount of acridine orange dye accumulating in the acidic vacuolar compartments in Leishmania major by detecting fluorescence in the red laser when autophagic inhibitors or enhancers were included. These methods will advance studies that aim to understand autophagic regulation in Leishmania parasites that could provide insights into developing improved therapeutic targets against leishmaniasis.

Sex determination in forensic medicine is considered one of the first and foremost steps in personal identification. The need for identifying the exact sex of the individual arises when deciding whether a person can exercise certain civil rights reserved for one particular sex, for competing in sex-specific athletic and sports events, legitimacy, divorce, paternity disputes and also to some criminal offenses. Nuclear sexing by Barr body examination can be done using buccal smears to establish the sex of the individual when routine methods fail to disclose the exact gender of the individual.
UNASSIGNED: To determine and compare the Barr bodies present in exfoliated buccal epithelial cells in males, females and transgender populations using light and fluorescence microscopy.
UNASSIGNED: A total of 90 patients were recruited for the study. Group I consisted of 30 female patients. Group II consisted of 30 male patients and group III consisted of 30 transgender patients. The buccal mucosa was then scraped using a wooden spatula and the cells obtained were fixed in 95% ethanol. Two smears per individual were made and stained. One smear was stained with papanicolaou (PAP) stain and the other with Acridine orange and viewed under light microscopy and fluorescent microscopy, respectively.
UNASSIGNED: When PAP stained slides were examined, the percentage of Barr-bodies in females ranged from 3% to 5% and in males it was 0% and in transgenders, it ranged from 0% to 5%. In Acridine orange stained smears, the percentage of Barr bodies in females ranged from 1% to 3% and in males it was 0% and in transgenders, it was 0%. Kruskal-Wallis test to study the relation of Barr body percentage in females, males and transgender subjects demonstrated significant differences between the groups (P < 0.001). Wilcoxon signed rank test was done for pairwise comparison, which showed that the distribution of percentage of positive cells in females are statistically significant from males and transgenders (P < 0. 001).
UNASSIGNED: Nuclear sexing using Barr bodies offers a simple yet effective method for determining the sex of transgender patients which could help them in understanding their gender identity better and diagnose any underlying chromosomal aberration.

Acridine orange is a nucleic acid binding dye that emits green fluorescence when bound to double-stranded DNA or RNA and red fluorescence when bound to single-stranded DNA or RNA under ultraviolet light. This unique characterization allows it to be used for distinguishing or visualization of dsRNA. Here, we present a convenient and efficient protocol for detecting dsRNA in polyacrylamide gels.