Vital Dye Research Articles

Apoptotic cell death of stomach cancer lines (AGS) induced by Co-NTB complex through cellular organelles and DNA damage.

Given that stomach cancer is the fourth leading cause of cancer-related death, there is a need to develop new drugs. Among various methods, metal-based coordination compounds are considered as an efficient strategy against this type of cancer. Similarly, the benzimidazole moiety plays a crucial role in biology; thus, various benzimidazole-based compounds have been found to be active as potential anticancer drugs and are currently used in clinical trials. In this study, we explored the benzimidazole-based cobalt(ii) complex as an anticancer agent against AGS stomach cancer cell lines. Interestingly, the MTT assay of the Co-NTB complex shows a lower IC50 value of 4.25 μg mL-1 compared to cisplatin, which has an IC50 of 7.5 μg mL-1 against AGS cell lines. Light microscopy and Hoechst/propidium iodide dye staining clearly indicate that the complex damages DNA, leading to cell death through an apoptotic pathway. The apoptotic cell death pathway was further complemented by Lysotracker and Mitotracker staining, as well as transmission electron microscopy (TEM) imaging. Overall, the Co-NTB complex acts as an effective anticancer agent against AGS stomach cancer cell lines, with apoptotic cell death induced by targeting cellular organelles and DNA.

Flavuside B exhibits antioxidant and anti-inflammatory properties in Staphylococcus aureus infected skin wound and affect the expression of genes controlling bacterial quorum sensing.

The aim of this study was to evaluate the antioxidant and anti-inflammatory effects of marine fungal cerebroside flavuside B (FlaB) on Staphylococcus aureus-infected keratinocytes in in vitro skin wounds and to identify FlaB targets in bacterial and human cells. A combination of enzyme-linked immunosorbent assay (ELISA), plate spectrofluorimetry, and flow cytometry with fluorescence dye staining, scratch assay, and real-time cell imaging techniques was used to investigate the effects of FlaB on S. aureus-infected HaCaT keratinocytes. FlaB decreased reactive oxygen species levels, nitrite oxide levels, and TNF-α and IL-18 release in S. aureus-infected HaCaT cells. FlaB reversed the inhibition of HaCaT cell proliferation caused by S. aureus infection. FlaB significantly increased keratinocyte migration and wound healing in an in vitro S. aureus-infected wound skin model. Using real-time qPCR, we found that FlaB caused a 1.7-fold reduction in agrA expression, which controls quorum sensing system in S. aureus. Bioinformatics analysis and molecular docking, together with experimental data, suggest that FlaB targets the pro/antioxidant defense system in human cells. Thus, FlaB can play a dual role as an antibacterial and pro/antioxidant machinery modulator, providing an observable positive effect in S. aureus-infected in vitro skin wounds. Staphylococcal sortase A enzyme and Arg systems are the targets of FlaB in bacterial cells. Nrf2/Bach1 dependent pro/antioxidant defense system is a target of FlaB in human cells. Some suggestions have also been made regarding the biological role of this marine fungal metabolite and its therapeutic possibilities.

Isolation of viable follicles from cryopreserved human ovarian tissue

Introduction: Ovarian tissue freezing is the most effective method to maintain fertility for immature girls and women diagnosed with cancer. Nonetheless, because of the chance that malignant cells might reappearing following tissue transplantation, it is crucial to isolate the follicles from the frozen-thawed ovarian tissue of these individuals and employ them in the process of in vitro maturation process or artificial ovarian framework. This study aimed to assess the application of neutral red (NR) vital dye alongside collagenase IA for effectively isolating viable follicles from the vitrified human ovarian tissue samples. Methods: Two categories existed: the category with NR and the group without NR. Chopped (0.5×0.5 mm) strips of vitrified-warmed ovarian tissue from 10 transsexual individuals were placed into two falcon tubes with HTCM and Collagenase IA (1mg/ml). Neutral red (NR) was introduced to one of the falcons. Follicles were then isolated mechanically. The morphology, size, and viability of the follicles were assessed. The condition of the follicles was evaluated using fluorescent staining methods involving Calcein-AM and Ethidium homodimer-I. The t-test method was used to evaluate the data. Results: The number of isolated follicles with Neutral Red (46.50±25/02) exceeded those without NR (6.6±5.58; P < 0/0001). Additionally, according to the morphological studies, a majority of the isolated follicles from the transsexual ovarian cortex were primordial (77.4%), and primary (21.12%) follicles, with only a small number of secondary follicles (1.4%) identified in these tissues. Live/dead staining verified the viability of isolated follicles by displaying a green hue. Conclusion: The finding indicates that combining collagenase I with the vital dye Neutral red significantly facilitates the of follicles from dense human ovarian tissue.

The Frequency of Trichomonas vaginalis Infection in Married Women in Al-Khums City, Libya

Trichomonas vaginalis is a parasitic protozoan that infects the urogenital tract of individuals globally, affecting both women and men. There are various methods to detect T. vaginalis, including clinical diagnosis and laboratory examination. "The purpose of this study was to determine the prevalence of T. vaginalis among married women in Al-Khums, Libya. The comparison between four different diagnostic methods to detect T. vaginalis, vaginal swab samples from women who visited Souq Al-Kamlesh Hospital during the period from February 2022 to July 2022 from different regions of the city (Al-Khums, Lebdah, Seline, Souq AlKhamees, Kaam and Caucasus) and collected 350 samples and analyzed them. The highest infection rate is recorded at pH < 7 in 68.75% of cases. The Whiff test resulted in 13 positive samples (3.71%), the culture medium method Cystine-Peptone-Liver infusion- Maltose (CPLM) recorded 16 positive samples (4.57%), and the Giemsa dye staining method recorded 16 positive samples (4.57%). The age groups were more affected by women aged 45 years (28.5%), followed by the age groups between 35 and 45 years at a rate of 4.59%. The lowest infection rate was in the 15-25 (1.90%) age group, with significant differences (P ? 0.05). Furthermore, the most common injuries among women were from Al-Khums region (7.76%), followed by Souq Al-Khamees region (3.94%) and finally Kaam region (2.94%). Cases of miscarriage (10.15%) are found more frequently in this study. The culture and staining methods were used to detect the highest infection rate of Trichomonas vaginal and achieve better results than other methods.

Natural Molecule-Derived Nanogels from Hematoxylin and l-lysine for Biomedical Use with Antimicrobial Properties.

Hematoxylin (HT) is a natural staining dye used in histopathology, often combined with Eosin for H&E staining. A poly(hematoxylin-co-l-lysine) (p(HT-co-l)) nanonetwork was synthesized through a one-step Mannich condensation reaction using formaldehyde as a linking agent. The resulting p(HT-co-l) nanogels had an average size of about 200 nm and exhibited a smooth surface and desirable functional groups such as -OH, -NH2, and -COOH, as recognized by FT-IR analysis. The isoelectric point (IEP) of the p(HT-co-l) nanogel was determined as pH 7.9, close to physiological environments, despite HT being acidic IEP at pH 1.7 and l-lysine being basic IPE at pH 8.7. The time-dependent swelling studies of p(HT-co-l) nanogels were carried out using dynamic light scattering (DLS) in different salt solutions, e.g., MgCl2, KNO3, KCl, PBS, and DI water environments revealed that nanogels have high swelling ability depending on the medium, e.g., >10-fold in a saline solution compared to distilled water within 1.5 h. Hydrolytic degradation studies in PBS demonstrated a linear release profile up to 125 h at 37.5 °C. The p(HT-co-l) nanogels also demonstrated significant antimicrobial and antifungal activities against E. coli (ATCC 8739), S. aureus (ATCC 6538), and C. albicans (ATCC 10231). Furthermore, biocompatibility tests indicated that p(HT-co-l) nanogels are more biocompatible than HT alone, as tested with human Nuli-1 bronchial epithelial cells.

Organized Thrombus Masquerading as Spontaneous Coronary Artery Dissection: A Set of Two Cases

Background Spontaneous coronary artery dissection (SCAD) often impacts young women who lack traditional risk factors. Diagnosing SCAD can be challenging due to its diverse angiographic presentations and resemblance to other conditions, such as organized thrombus. Case Summary We present two cases involving middle-aged male smokers, each experiencing chest pain lasting over 12 hours. Both patients exhibited ST-segment elevation in the anterior leads on ECG, anterior wall hypokinesia, and an LVEF of 40% on 2D echocardiography. Immediate coronary angiography revealed dye staining and multiple radiolucent lumens in the LAD, indicative of SCAD. Optical coherence tomography (OCT) identified an organized thrombus with a distinctive “Swiss cheese” pattern, differing from the usual radiolucent false lumen associated with SCAD. Both patients were successfully treated with PCI, including thrombus aspiration and stent placement. Take-home message Relying solely on angiography for SCAD diagnosis may overlook organized thrombus. Redefining SCAD criteria and utilizing multimodal imaging like OCT in all cases is essential.

Laparoscopic varicocelectomy: Does intraoperative lymphography with vital dye influence the outcome?

Vital dyes lymphography during laparoscopic varicocelectomy (LV) has been suggested to allow for lymphatic mapping thereby allowing for lymphatic sparing (LS) and subsequent reduction of hydrocele formation. Intratesticular injection of the dye seems to achieve the best lymphatic visualization, however, the potential toxicity of some agents, is a cause of concern for the risk of testicular pathological changes. We report our experience with LV, to test the hypothesis that Intraoperative lymphography with methylene blue (BL), is safe and reduces the rate of post-operative hydrocelectomy. Secondary aim was to evaluate the effect of intratesticular injection of the dye. Retrospective review of all consecutive patients undergoing LV<18 years of age, between 2018 and 2022, and with at least 12-month follow-up. We compared hydrocele rate after LS-LV vs. non-LS-LV and between patients undergoing LS with vs. without BL and according to injection site. Post-operative ultrasounds were reviewed to rule out testicular anomalies in patients undergoing BL. Reoperations for varicocele were also noted. 116 patients undergoing LV at a median (range) age of 14 (10-17) years were included. Post-operative hydrocelectomy rate was significantly more common (p=0.033) in patients undergoing non-LS (3/38, 8%) vs. LS (0/78) LV. No hydrocelectomy was performed in patients undergoing LS-LV with (0/47) or without (0/31) BL. Vital dye injection site was intra-testicular in 21/47 (45%) cases. Avascular intra-testicular lesions were identified in 4 (19%) patients and persisted after a median (range) follow-up time of 21 (14-28) months. Two varicoceles were reoperated for recurrence, one in each group. Vital dyes lymphography improves lymphatics visualization during LV. Although, in present study, overall hydrocele rate was not significantly different in patients undergoing LS vs. non-LS procedures, hydrocelectomy was required only after non-LS procedures. In keeping with other 2 recent studies, avascular intratesticular lesions were observed in one fifth of cases undergoing Intratesticular dye injection. These lesions did not change during follow-up, but their clinical significance remains to clarify. Limitations of study include the retrospective design and the small number of patients undergoing intratesticular dye injection, but still this is one of the largest series with long-term follow-up of intratesticular lesions after BL. LS-LV significantly reduced the risk of hydrocelectomy, irrespective to the use of BL. Intratesticular methylene blue injection was associated with persistent testicular lesions on follow-up ultrasounds in 19% of cases. More studies are necessary to prove the safety of dye injections into the testis.

Annexin A1 Mitigates Blood-Brain Barrier Disruption in a Sepsis-Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens-1 (ZO-1).

This study investigated the protective role of Annexin A1 (ANXA1) in sepsis-associated encephalopathy (SAE) by examining its effects on brain vascular endothelium and blood-brain barrier (BBB) integrity. Mice were divided into four groups: wild type (WT), cecal ligation and puncture (CLP), ANXA1 knockout (ANXA1[-/-]), and ANXA1(-/-) with CLP. Neurobehavioral changes were assessed using the Y-maze test, while BBB integrity was evaluated through Evans blue dye (EBD) staining and permeability tests with fluorescein isothiocyanate (FITC)-dextran. Results showed that ANXA1 levels were reduced in septic mice, and its deficiency exacerbated cognitive impairment and survival rate reduction. ANXA1 deficiency also upregulated proinflammatory cytokines and adhesion molecules, worsened BBB impairment, and altered expression of tight junction proteins and VEGF-A/VEGF-R2. Invitro, ANXA1 Ac2-26 prevented LPS-induced increased permeability in bEnd.3 cells by restoring tight junction proteins and reducing VEGF-A/VEGF-R2 expression. Notably, VEGF-A negated the protective effects of ANXA1 Ac2-26. The study concludes that ANXA1 reduces BBB permeability to protect against sepsis-induced brain dysfunction via VEGF-A/VEGF-R2 regulation of tight junction proteins, suggesting ANXA1 as a potential therapeutic for SAE.

Phospholipid Nanoemulsion-Based Ocular Lubricant for the Treatment of Dry Eye Subtypes: A Multicenter and Prospective Study.

Dry eye disease (DED) is a multifactorial condition of the ocular surface (OS) characterized by loss of tear film homeostasis, ocular discomfort, and vision disturbances. Most available ocular lubricants target the aqueous deficiency of the tear, restoring only this layer, leaving the tear lipid stratum deficient, as occurs in most patients with evaporative DED. An innovative propylene glycol-hydroxypropyl guar enriched with a phospholipid nanoemulsion (PG-HPG-PH-N) is indicated to restore deficiencies in both the lipid and aqueous layers of the tear film, and its composition was designed to increase lubricant retention on the OS. The purpose of this study was to evaluate, through the Ocular Surface Disease Index (OSDI) and clinical assessment, the treatment of patients who had DED due to aqueous deficiency arising from mixed or evaporative DED subtypes with a PG-HPG-PH-N ocular lubricant at a reduced frequency of twice a day, in a prospective, multicenter, and single-arm study. Patients were screened from days - 7 to 0, and from day 1 (baseline and first day of treatment) to day 28 of treatment with this lubricant. After visit 1 (screening visit, days - 7 to 0), designed as pre-treatment OS assessment, patients returned to their research center on days 14 and 28 of treatment for a complete assessment, including anamnesis, the OSDI, corrected visual acuity, tear breakup time (TFBUT), OS staining evaluation, tolerability index, and environmental exposure questionnaire. Seventy patients were enrolled in this study (60 women, 10 men), with a mean age of 45 (range 27-64) years. TFBUT results showed an improvement in tear film stability as vital dyes sodium fluorescein and lysamine green showed a decrease in corneal staining after 14 and 28days of treatment. No significant adverse events were reported, demonstrating the good tolerability of the lubricant. The PG-HPG-PH-N nanoemulsion can be considered to be a safe and effective ocular lubricant for treating DED due to aqueous deficiency, both mixed and evaporative subtypes. Brazilian National Research Ethics Commission (ReBEC registration number 16055).

Luminescent ZnSe/ZnO Heterostructures for Photocatalytic Degradation of Azophloxine Dye and Bioimaging Applications.

Herein, we demonstrate a simple hydrothermal route to synthesizing ZnSe/ZnO type II heterostructure using L-cysteine as a capping agent. The use of nanomaterials in bioimaging and photocatalysis towards the degradation of Azophloxine dye is of potential interest. The synthesized ZnSe/ZnO nanomaterials were characterized by UV-Vis absorption spectroscopy, fluorescence spectroscopy, IR spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. It exhibits a strong sharp emission at 465 nm in fluorescence spectroscopy. The synthesized nanomaterials were used as an excellent fluorescence probe for imaging live-cell bacteria such as E. coli and S. aureus compared with the commercial staining dye PI (propidium iodide). The synthesized ZnSe/ZnO possess more photo-stability and showed excellent photocatalytic activity for the degradation of azophloxine dye under sunlight.

Addition of hydrogen peroxide to natural ferruginous water improves the efficacy of SODIS method against the waterborne pathogen Cryptosporidium

The enhancement of SODIS method against the waterborne pathogen Cryptosporidium by incorporating H2O2 in natural ferruginous water (NFW) was evaluated. Polyethylene terephthalate bottles containing distilled water or different NFW with added different concentrations of H2O2 (0–100 mg L−1) were spiked with Cryptosporidium parvum oocysts and exposed to simulated and natural solar radiation. Oocyst viability was evaluated by induced excystation and inclusion/exclusion of the vital dye propidium iodide (PI). The experiments performed under sunlight showed better C. parvum inactivation rates than those under simulated solar radiation. Oocyst viability rates of 2.26±1.91% and 16.52±3.83% were determined by induced excystation and inclusion/exclusion of PI, respectively, after exposure to natural sunlight during 6 h in NFW with 100 mg L−1 of H2O2. This study proves the enhancement in the effectiveness of conventional SODIS by the addition of H2O2 to natural ferruginous waters, speeding up the C. parvum oocyst inactivation. Since surface waters in countries where the SODIS method can be used often contain dissolved iron salts, the addition of small amounts of H2O2 will increase the microbiological quality of drinking waters and consequently would decrease the risk of diarrhoeal diseases in the population, having a positive impact in terms of development and poverty reduction.

An AIE-based fluorescent dye for selective staining of polyamide microplastics without pretreatment: Applications to environmental samples and zebrafish

A novel staining dye, BEM ((1E,1'E)-1,1'-([2,2'-bithiophene]-5,5'-diyl)bis(N-(9-ethyl-9H-carbazol-3-yl)methanimine)) was synthesized for selective identification of polyamide (PA) micrplastics. BEM showed unique photophysical properties such as solvatochromism, intramolecular charge transfer (ICT), and aggregation induced emission (AIE) which were demonstrated through spectroscopic analysis and density functional theory (DFT) calculations. The optimal staining conditions for selective staining of PA by BEM were established by evaluating the staining efficiency according to the variation of the solvent compositions, concentrations of BEM, and staining durations. BEM demonstrated outstanding selective staining of PA among 11 types of microplastics (MPs) and 5 types of non-plastics through the emission of green fluorescence. BEM successfully identified PA without any noticeable influence on the size change of PA, aging of PA, and pH alteration of the solvent. In addition, BEM was practically applied to environmental samples like river water, seawater, and soil for selective identification of PA without pretreatment. In particular, the cost-effective technique of BEM-labeled PA allowed to monitor the location and accumulation of PA in living zebrafish. The interaction between PA and BEM was investigated through scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS), which suggested that BEM might be adsorbed onto the surface of PA. Moreover, non-covalent interaction (NCI) analysis demonstrated that the intermolecular hydrogen bonds and van der Waals interactions would play a significant role in the adsorption process between PA and BEM.

Can preoperative percutaneous injection of ultrasound contrast agent locate sentinel lymph nodes of breast cancer?

We evaluated the ability and accuracy of preoperative identification and localization of sentinel lymph nodes (SLNs) using intradermal injection of ultrasound contrast agent. Prospectively recruited 191 early breast cancer patients with clinically negative axillary lymph nodes (ALNs). All participants received intradermal injection of microbubble contrast agent. Following the identification and localization of SLNs using contrast-enhanced ultrasound (CEUS), Markers were deployed in the SLNs US-guided. Subsequently, the SLNs with Markers were stained and marked with a suspension of nano-carbon US-guided to assist in intraoperative localization of SLNs. Standard SLNB with methylene blue tracing was performed intraoperatively to assess the consistency between the two methods of SLNs localization, thereby determining the ability and accuracy of CEUS in identifying and localizing SLNs. A total of 179 patients were included in the final evaluation analysis, in which a microbubble contrast agent was injected subcutaneously in the areolar region. A total of 201 SLNs were identified, with a median of 1 SLN per patient. Each SLN was identified in 157 patients, and two SLNs were identified in 22 patients. Among the 201 SLNs from the 179 patients, the proportion that could be individually matched between CEUS and the blue dye method was 95.5% (192/201), and the consistency evaluation in SLNs identification between CEUS and blue dye staining was excellent (Kappa value = 0.62, P < 0.001). The consistency of identification and localization of SLNs in early breast cancer patients between CEUS and the blue dye method was strong.

Homoplasy in the embryonic development of terrestrial and epiphytic orchids from the subtribe Malaxidinae (Orchidaceae).

During evolution, similar vectors of adaptive radiation may have evolved in the subtribe Malaxidinae. This was manifested in homologous series of variability in suspensor shape and seed coat ultrasculpture in clades of terrestrial and epiphytic orchids. The present study examines the variability of embryonic traits across clades and subclades of subtribe Malaxidinae (Orchidaceae), previously identified by molecular genetic data. Ovules and seeds from fruits of orchids of the genera Crepidium, Liparis (sections Cestichis and Blepharoglossum), Dienia, and Oberonia were examined by confocal laser microscopy with fluorescent dye staining. The branched or rounded suspensor in the studied species was unicellular and originated from the nondividing basal cell cb. The elongated or flattened transmission structure of one or two cells was located at the junction of the suspensor and the embryo proper. Two species (Oberonia gammiei and Liparis elliptica) were found to have unitegmal ovules. Three morphological groups of seeds were identified based on the shape and sculpture of the periclinal cell wall. A comparative analysis of the embryological characters in Malaxidinae species reveals that the lobed suspensor is a homoplasy present in different subclades of terrestrial and epiphytic orchids. The flat transmission cell is an apomorphy in the Cestichis subclade. Similarly, the independent formation of the unitegmal ovule occurred in two subclades of epiphytic orchids. The results of our study suggest that similar adaptive radiation vectors may have evolved in the subtribe Malaxidinae in the orchids we studied.

Efficacy and Safety of Keratosept Eye Drops in Patients with Punctate Keratitis: Clinical and Microbiological Evaluation on 50 Eyes.

We evaluated the efficacy and safety of eye drops with antiseptic and re-epithelizing properties (Keratosept®, Bruschettini, Genova, Italy) on 50 eyes affected by punctate keratitis of suspected microbial origin. A biomicroscopic examination, fluorescein dye staining test (Fluotest), tear break-up time test (TBUT), and the ocular surface disease Index (OSDI) questionnaire were used to assess treatment efficacy. Treatment success was defined as a negative Fluotest and an OSDI score <12 on the 15th day of treatment. According to this definition, Keratosept®, either alone or in combination with antibiotics, was effective in over 80% of microbial and nonmicrobial forms. Conjunctival swabs were taken from all patients for culture examination with an antibiogram and sensitivity test for Keratosept®; 32/43 (74.4%) forms with positive culture for the bacteria were sensitive to Keratosept®. A total of 35/38 (92%) eyes treated with this product alone achieved full treatment success without any apparent adverse effects. These results suggest the use of Keratosept® eye drops in punctate keratitis as an alternative or in combination with established antibiotic therapies. Further studies are needed to evaluate its efficacy in different infectious forms and identify other indications for using this product.

Dissociation Constant (Kd) Measurement for Small-Molecule Binding Aptamers: Homogeneous Assay Methods and Critical Evaluations.

Since 1990, numerous aptamers have been isolated and discovered for use in various analytical, biomedical, and environmental applications. This trend continues to date. A critical step in the characterization of aptamer binding is to measure its binding affinity toward both target and non-target molecules. Dissociation constant (Kd) is the most commonly used value in characterizing aptamer binding. In this article, homogenous assays are reviewed for aptamers that can bind small-molecule targets. The reviewed methods include label-free methods, such as isothermal titration calorimetry, intrinsic fluorescence of target molecules, DNA staining dyes, and nuclease digestion assays, and labeled methods, such as the strand displacement reaction. Some methods are not recommended, such as those based on the aggregation of gold nanoparticles and the desorption of fluorophore-labeled DNA from nanomaterials. The difference between the measured apparent Kd and the true Kd of aptamer binding is stressed. In addition, avoiding the titration regime and paying attention to the time required to reach equilibrium are discussed. Finally, it is important to include mutated non-binding sequences as controls.

Single-cell analysis of osmoregulation reveals heterogeneity of aquaporin 4 functionality in human astrocytes.

The water channel aquaporin 4 (AQP4) contributes to water flow and waste removal across the blood-brain barrier and its levels, organization and localization are perturbed in various neurological diseases, including Alzheimer's Disease. This renders AQP4 a potentially valuable therapeutic target. However, most functional assays aimed at identifying modulators of AQP4 function are performed with primary rodent cells and do not consider inter-cellular variations in AQP4 abundance and presentation. To address this, we have established and applied a robust live cell microscopy assay that captures the contribution of AQP4 in the osmotically driven (de-)quenching of the vital dye Calcein-AM with single-cell resolution. Using human astrocytoma cells, we found that performing measurements on cellular regions instead of whole fields of view yielded a more sensitive readout of the osmotic response, which correlated with AQP4 abundance. Stable co-expression of the two major AQP4 isoforms, but not of the individual isoforms, provoked a faster adaptation to osmotic changes, while siRNA-mediated knockdown of AQP4 had the opposite effect. Post-hoc correlation with the canonical membrane marker CD44 revealed that the speed of the osmotic response scaled with AQP4 membrane enrichment. Coating the substrate with laminin promoted AQP4 membrane enrichment, while cell confinement with fixed-size micropatterns further increased the speed of osmoregulation, underscoring the influence of extracellular factors. The osmotic response of primary fetal astrocytes and human iPSC-derived astrocyte models was comparable to AQP4-deficient astrocytoma cells, in line with their low AQP4 levels and indicative of their immature state. In conclusion, a correlative single-cell approach based on the quantification of Calcein-AM quenching capacity, AQP4 abundance and AQP4 membrane enrichment, allows resolving osmoregulation in a more sensitive manner and reveals heterogeneity between and within human astrocyte (-like) cultures, which could prove crucial for future screens aimed at identifying AQP4 modulators.

Fluorescent Staining and Quantification of Starch Granules in Chloroplasts of Live Plant Cells Using Fluorescein.

Plants use CO2, water, and light energy to generate carbohydrates through photosynthesis. During daytime, these carbohydrates are polymerized, leading to the accumulation of starch granules in chloroplasts. The catabolites produced by the degradation of these chloroplast starch granules are used for physiological responses and plant growth. Various staining methods, such as iodine staining, have previously been used to visualize the accumulation of chloroplast starch granules; however, these staining methods cannot be used to image live cells and/or provide confocal images with non-specific signals. In this study, we developed a new imaging method for the fluorescent observation of chloroplast starch granules in living plant cells by staining with fluorescein, a widely available fluorescent dye. This simple staining method, which involves soaking a leaf disk in staining solution, shows high specificity in confocal images. Fluorescent images of the stained tissue allow the cellular starch content of living cells to be quantified with the same level of accuracy as a conventional biochemical method (amyloglucosidase/α-amylase method). Fluorescein staining thus not only enables the easy and clear observation of chloroplast starch granules but also allows for precise quantification in living cells. Key features • Visualizes chloroplast starch granules stained with fluorescein in living cells. • Requires only simple specimen preparation with no reagents needed other than the staining solution. • Fluorescein is readily available worldwide. • Highly specific method for identifying chloroplast starch granules in confocal images. • Enables estimation of cellular starch content using fluorescent images.

Exploring the potential use of Caenorhabditis elegans as an animal model for evaluating chemical-induced intestinal dysfunction

Evaluating intestinal toxicity is crucial for identifying and preventing the harmful effects of environmental chemicals. Owing to the limitations of existing models in evaluating intestinal toxicity, the development of alternative models is urgently needed. This study explored the potential use of the nematode Caenorhabditis elegans as a model animal for assessing chemical-induced intestinal dysfunction. Changes in intestinal permeability and nutrient absorption in C. elegans individuals exposed to four intestine-disrupting chemicals (sodium dodecyl sulfate (SDS), dextran sulfate sodium (DSS), lipopolysaccharide (LPS) and ethanol) were examined using dye stain assays, an enzymatic photometric assay, and fluorescent probe uptake assays. Additionally, epigallocatechin-3-gallate (EGCG), an intestine-protecting phytochemical, was chosen to prevent ethanol-induced intestinal damage. The results indicated that SDS, DSS, LPS, and ethanol compromised the intestinal barrier in C. elegans. SDS had no effect on glucose absorption, but LPS, DSS, and ethanol inhibited or tended to inhibit glucose absorption. SDS, DSS, LPS, and ethanol reduced fatty acid absorption. LPS increased peptide absorption at a low dose but decreased it at a high dose; SDS, DSS, and ethanol attenuated peptide absorption. EGCG protected against the disruption of the intestinal barrier that was induced by ethanol treatment. These results suggest that C. elegans is a suitable surrogate model animal for evaluating chemical-induced intestinal dysfunction. These findings also provide new insights into the effects of SDS, DSS, LPS, and ethanol on intestinal function and highlight the potential of EGCG as a natural dietary intervention to protect individuals who use excess alcohol from intestinal injury.

Trichosanthin elicits antitumor activity via MICU3 mediated mitochondria calcium influx

IntroductionTrichosanthin (TK) is a glycoprotein extracted from the Chinese medicinal herb Trichosanthes kirilowi, which has anti-virus and anti-tumor activity. However, the target and detailed mechanism of TK remains elusive. ObjectivesWe aimed to identify novel antitumor targets of TK in lung adenocarcinoma and study its anti-tumor mechanism. MethodsWe utilized a Lewis lung carcinoma mouse model to evaluate the inhibition of TK on tumor growth. CCK8 assay was utilized to calculate IC50 of trichosanthin on A549 and H1299. In-vitro cellular assays and in-vivo xenograft mice studies were used to investigate MICU3 overexpression and TK treatment on tumor growth. Fluo-4 dye and JC-1 staining was used to measure the mitochondrial calcium levels and membrane potential. H&E and immunohistochemistry staining were applied the asses the effect of TK on tumor and microenvironment. RNA sequencing was applied to analyze transcriptome changes in TK-treated and MICU3-overexpressed tumor cells. The influence of trichosanthin on DNMT3B expression and MICU3 methylation were detected by qPCR and Western blotting. Transcriptional activity of the MICU3 gene was measured by ChIP-PCR and luciferase assays. ResultsTrichosanthin ihibited the tumor growth in vivo, resulting cancer cell growth inhibition and cell death, with almost no effect on normal cells. IC50 of trichosanthin in A549 and H1299 cells were 62.8 μg/ml and 39.7 μg/ml, respectively. Mitochondrial Calcium Uptake Family complex MICU3 was shown to associated with favorable prognosis and was upregulated upon trichosanthin treatment, along with reduces tumor cell growth and migration, and increased cell death both in vitro and in vivo. Increased mitochondrial calcium level was observed in MICU3 overexpression cells. Pathway analysis of RNA-seq data revealed that cytokine and receptor pathways were enriched in MICU3-overexpressing cells. Trichosanthin decreased DNMT3B expression and altered MICU3 methylation while increased FOSL2 expression and reduced methylation that correlated with increased transcription of the MICU3 gene. ConclusionTrichosanthin elicits antitumor activity in lung adenocarcinoma via repressing DNMT3B and increasing FOSL2, which in turn induces MICU3-mediated mitochondrial calcium influx and tumor cell death.