Effect of Water Quality on Benthic Macro Invertebrates in Dikhu River, Nagaland, India

Background: Water quality monitoring is vital for the sustainable conservation of water resources. Benthic macro invertebrates have been documented as the best indicators of water quality serving as a vital link in the aquatic food web. The present work was carried out at Dikhu River, Mokokchung, Nagaland. Methods: The analysis of water samples was done by adopting standard methods and compared against the W.H.O. 1995 drinking quality standards of water. The macroinvertebrates were collected with the help of the Surber and preserved in 5% formalin and examined using inverted microscope. Pearson’s correlation analysis were performed by IBM SPSS package 16.0. Result: All water parameters were within the permissible limit of drinking quality standards except for alkalinity. A total of 646 individual benthic macro invertebrates belonging to three taxa (Annelida, Arthropoda and Mollusca) were recorded during the study. The study depicts the present status of macrobenthic structure of Dikhu River of Nagaland and aims to lay the foundation for further effective work as future prospect.

Investigating polystyrene nanoplastics-induced reproductive toxicity in vitro: Focus on Nrf2-PKM2-autophagy signaling pathway

As an issue of widespread concern, microplastics pollution has emerged as a harmful environmental pollutant. Nanoplastics (NaPs) has reported to accumulate in the testes and cause degeneration in the seminiferous tubules. However, the current research involving NaPs-induced reproductive toxicity remains poorly understood. The current work aimed to investigate the mechanisms of NaPs-induced reproductive injury in vitro. At first, we found that 80 nm fluorescent NaPs could enter into GC-2spd(ts) cells by fluorescent inverted microscope. Our results also demonstrated that suppression of reactive oxygen species (ROS) inhibited NaPs-triggered mitochondrial apoptosis and autophagy in GC-2spd(ts) cells. We also found that NaPs treatment did not change the interaction between nuclear factor erythroid-derived 2-related factor (Nrf2) and Kelch-like ECH associated protein 1 (Keap1), while inhibiting nuclear accumulation of Nrf2 protein. Further in vitro experiments showed that NaPs-induced reproductive toxicity associated with reducing dimerize pyruvate kinase M2 (PKM2), which are ascribed to the loss of Nrf2. Meanwhile, improving nuclear accumulation of Nrf2 might interact with PKM2 to rescue mitochondrial apoptosis caused by NaPs. Together, this study highlight that disturbing Nrf2-PKM2 signaling is essential process of NaPs-induced reproductive toxicity and provide valuable insights into the mechanism of microplastics-induced reproductive toxicity.

MАR-test and Spermiological Research Indices

Antisperm antibodies are detected in 3% to 25% of cases in men and women diagnosed with infertility. They can also be diagnosed in 1–10% of healthy fertile men. The presence of a high titer of AST is one of the factors of male infertility, which can be «hidden», i. e. not cause symptoms and deterioration of the overall spermogram. The objective: to analyze the results of sperm testing in men with antisperm antibodies, which were detected by MAR-test. Material and methods. A retrospective analysis of the examination results of 555 men was conducted on the basis of the Medical Center for Infertility Treatment in Chernivtsi. Spermograms were examined according to WHO recommendations in 2000, using an inverted microscope Olympus CKX41 in a Broker chamber. Determination of the percentage of sperm coated with antisperm antibodies was performed using the MAR-test (MAR-test, MAR-mixed antiglobulin reaction). Results. If we calculate the percentage of patients in whom we assume the connection between the presence of antisperm antibodies and undertaken surgery, genital infections and allergy history, we get only 48.3% of cases. It has been found that most changes in sperm counts correlate with the percentage of sperm that are coated with IgG. These data indicate that increased IgG levels play an important role in the development of pathospermia. A negative medium-strength correlation was found between the percentage of Category A sperm and the percentage of IgG-coated sperm. Persistence of IgG in semen is accompanied by a probable decrease in the morphological quality of sperm. Conclusion. In 52% of patients it was not possible to establish the etiological factor for the presence of Ig in the ejaculate. It has been found that most changes in sperm counts correlate with the percentage of sperm that are coated with IgG. It has been determined that an increase in the level of IgA and IgG leads to a probable increase in the percentage of pathological sperm forms and decrease in sperm motility. In the presence of 30% of sperm that are coated with IgG, with a probability of 95%, the ejaculate of patients will have 100% of pathologicoalr mf s of sperm.

Construction and preservation of a stable and highly expressed recombinant Helicobacter pylori vacuolating cytotoxin A with apoptotic activity

Background H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer. Results A 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA34–854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay. Conclusions A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

Morphological evaluation of MDA-MB-231 human breast cancer cells treated with DMEM extract of Turkish propolis

Purpose: To evaluate the influence of DMEM extract of Turkish propolis (TP) on the morphology of metastatic MDA-MB-231 cells. Methods: The cells were incubated with DMEM extract of TP (collected from Trabzon in Turkey) at a dose of 2.5 mg/mL for 72 h. The effect of DMEM extract on proliferation and cytotoxicity of the cells was determined using 3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assay. MDA-MB-231 cells incubated with or without extracts were randomly photographed with a camera-coupled inverted microscope. Treated and control MDA-MB-231 cells were classified as monopolar, bipolar or multipolar, and their dimensions measured with an electronic caliper. Results: Although the extract reduced the proliferation of the cells, the effect was not statistically significant (p < 0.05). Moreover, no cytotoxic effect was observed. Field diameters, process length and cell body diameters of the treated cells were increased by DMEM extract treatment in bipolar and multipolar cell types, but these parameters were decreased in monopolar cell type, although insignificantly (p < 0.05). In addition, the process thickness of treated MDA-MB-231 cells increased insignificantly in all cell types (p < 0.05). Conclusion: These findings indicate that DMEM extract of TP at a dose of 2.5 mg/mL morphologically suppresses monopolar MDA-MB-231 cells. Future studies would examine the morphological effects of different concentrations of the propolis extract in anti-proliferation, cytotoxicity and morphological investigations in MDA-MB-231 cells.

High content 3D imaging method for quantitative characterization of organoid development and phenotype.

Quantitative analysis on a large number of organoids can provide meaningful information from the morphological variability observed in 3D organotypic cultures, called organoids. Yet, gathering statistics of growing organoids is currently limited by existing imaging methods and subsequent image analysis workflows that are either restricted to 2D, limited in resolution, or with a low throughput. Here, we present an automated high content imaging platform synergizing high density organoid cultures with 3D live light-sheet imaging. The platform is an add-on to a standard inverted microscope. We demonstrate our capacity to collect libraries of 3D images at a rate of 300 organoids per hour, enabling training of artificial intelligence-based algorithms to quantify the organoid morphogenetic organization at multiple scales with subcellular resolution. We validate our approach on different organotypic cell cultures (stem, primary, and cancer), and quantify the development of hundreds of neuroectoderm organoids (from human Embryonic Stem Cells ) at cellular, multicellular and whole organoid scales.

Observation of Cells on a Simulated Titanium Surface with Transparency

The main driving force of osseointegration on titanium implants is believed to be the calcification caused by cellular activity. However, owing to the opacity of bulk titanium, live cells on titanium surfaces cannot be observed using an inverted microscope. To overcome this limitation, this study proposes a transparent titanium thin layer as a simulated titanium surface that allows live-cell observation from below. The titanium layer was fabricated on a polystyrene culture dish by magnetron DC sputtering using a pure Ti(JIS1) target. The titanium layer was characterized by transparency, composition, structure, and wettability. Osteoblast-like cells were cultured in the titanium-coated dishes. The cell culture was observed periodically using an inverted microscope, and the images were compiled into time-lapse videos. Cells on the titanium layer were characterized by movement speeds and doubling times. The titanium-coated dish was transparent gray, and its transmittance profile was consistent with that of the polystyrene dish. The titanium layer showed similarities to bulk titanium surfaces in terms of composition and structure; that is, it showed an oxidized titanium outermost layer and titanium metal basal layer. The wettability of the titanium layer was hydrophilic with mean contact angles of 67.52°. Osteoblast-like cells successfully adhered to the titanium layer and proliferated to confluence. The time-lapse videos demonstrated active movement of the cells on the titanium layer, which suggested the involvement of the titanium surface in cellular motility. The cell culture on the titanium layer can be considered cell culture on a titanium surface. In short, the titanium layer enabled the acquisition of information for living cells on titanium that has either been unknown or analogically understood based on cell culture on polystyrene dishes.

Long-Term Imaging of Living Adult Zebrafish

ABSTRACTThe zebrafish has become a widely used animal model due in large part to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior, and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels.

Systematic Quantification of Cell Confluence in Human Normal Oral Fibroblasts

Background: The accurate determination of cell confluence is a critical step for generating reasonable results of designed experiments in cell biological studies. However, the cell confluence of the same culture may be diversely predicted by individual researchers. Herein, we designed a systematic quantification scheme implemented on the Matlab platform, the so-called “Confluence-Viewer” program, to assist cell biologists to better determine the cell confluence. Methods: Human normal oral fibroblasts (hOFs) seeded in 10 cm culture dishes were visualized under an inverted microscope for the acquisition of cell images. The images were subjected to the cell segmentation algorithm with top-hat transformation and the Otsu thresholding technique. A regression model was built using a quadratic model and shape-preserving piecewise cubic model. Results: The cell segmentation algorithm generated a regression curve that was highly correlated with the cell confluence determined by experienced researchers. However, the correlation was low when compared to the cell confluence determined by novice students. Interestingly, the cell confluence determined by experienced researchers became more diverse when they checked the same images without a time limitation (up to 1 min). Conclusion: This tool could prevent unnecessary human-made mistakes and meaningless repeats for novice researchers working on cell-based studies in health care or cancer research.