A Simple Protocol for Periodic Live Cell Observation of Flagellate Stages in the Lichen Alga Trebouxia

This article presents the concept and development of a prototype diagnostic decision support system for real-time control and monitoring of dynamical processes. This decision support system, known as Diagnostic Evaluation and Corrective Action (DECA), employs qualitative reasoning, in conjunction with quantitative models, for monitoring and diagnosis of malfunctions in dynamical processes under routine operations and emergency situations. DECA is especially suited for application to time-constrained environments where an immediate action is needed to avoid catastrophic failure(s). DECA is written in common Lisp and has been implemented on a Symbolics 3670 machine; its efficacy has been verified using the data from the Three Mile Island No. 2 Nuclear Reactor Accident.

Trebouxia is one of the most frequent genera of green microalgae that occur as photobionts in lichens. However, its life cycle is still poorly understood. The contradictory information about the flagellate cells impairs our knowledge of possible sexual reproduction. The aim of this study was to investigate the behaviour and fate of flagellate cells in four Trebouxia species, to better understand their role in the life cycle.Axenic cultures of Trebouxia were grown under controlled conditions. The cell cycle of flagellate cells was observed using a novel technique for real‐time monitoring with light microscopy, validated in more detail by scanning electron microscopy (SEM). Additionally, a molecular approach was used to investigate genomic evidence of sexual reproduction.The zoospores had two significantly different morphotypes, elongated and subspherical, suggesting that this dimorphism should be recognised in future species descriptions. Fusions of elongated gametes were observed in three species, with formation and development of a zygote documented in one case. SEM images provided further evidence of plasmogamic events in unprecedented detail. Molecular analyses confirmed the presence of key meiotic genes in eight genomes and one transcriptome of Trebouxia, providing further strong evidence of sexual reproduction in this genus.This study provides a new method to monitor the fate of flagellate cells over time which allowed demonstration of the presence of two types of flagellate cell: zoospores with two well‐defined morphologies which are involved in asexual reproduction, and gametes involved in sexual reproduction.

Gas chromatography (GC) is frequently used in qualitative and quantitative analysis of volatile organic molecules. In its most common embodiment, samples are prepared in the laboratory, and injected into the capillary column using an autosampler. Analytes are separated, and detected by one of several available detection systems, including mass spectrometry (MS). However, this mode of operation is not convenient when monitoring dynamic chemical systems – for example, reaction mixtures. Here we present a facile approach for automated sampling, and subsequent introduction of samples to a GC apparatus in order to enable monitoring of dynamic chemical processes in real time. The proposed system incorporates two pinch valves, a peristaltic pump, a thermoshaker, and an electronic control unit (Raspberry Pi microcomputer) with several relays. The device facilitates dosing small volumes of liquid samples to the injection port of a commercial GC-MS apparatus. A program in C language controls the operation of the pump (on/off, direction of flow) and actuation of the pinch valves, and it initiates GC-MS runs. The system has been characterised with artificial samples, and successfully used in the monitoring of transesterification reactions catalysed by single microbeads containing immobilised lipase, as well as in the monitoring of the extraction of natural products from plant tissue samples. We believe that – following further improvements – this automated approach may be adapted for monitoring industrial processes and environmental analysis.

The paper presents the concept and development of a prototype diagnostic decision support system for real-time control and monitoring of dynamical processes. This decision support system, known as Diagnostic Evaluation and Corrective Action (DECA), employs qualitative reasoning, in conjunction with quantitative models, for monitoring and diagnosis of malfunctions and disruptions in dynamical processes under routine operations and emergency situations. DECA is especially suited for application to time-constrained environments where an immediate action is needed to avoid catastrophic failure(s). DECA is written in common Lisp and has been implemented on a Symbolics 3670 machine; its efficacy has been verified using the data from the Three Mile Island No. 2 Nuclear Reactor Accident.

The antibiotic oxytetracycline (OTC) is a fluorochrome marker, and fluorescence microscopy is used to view OTC marks in fishes' calcified structures. However, OTC marks have been observed in calcified structures using standard light microscopy for multiple species. Therefore, we conducted an experiment to investigate potential factors (i.e., season, total length of fish, growth rate, and sex) influencing the observation of OTC in calcified structures (otoliths and fin rays or spines) from channel catfish Ictalurus punctatus, gray redhorse Moxostoma congestum, Guadalupe bass Mircopterus treculii, and redbreast sunfish Lepomis auritus viewed using standard light and fluorescence microscopy. OTC stains were not observed in any otoliths under standard light; however, OTC marks were commonly observed in I. punctatus spines using standard light microscopy (56.2%). Ninety-nine percent of otoliths and 88.9% of spines and fin rays had a visible fluorescent OTC mark when viewed using fluorescence microscopy. There was a negative relationship between the observed OTC mark and total length of fish for each season, but fish injected in the summer had the most structures with an observed OTC mark under either light condition. Understanding how OTC marking is affected by biological processes and environmental conditions will assist in future studies that rely on chemical marking of calcified structures by increasing efficacy of OTC marking and interpretation of marks.

Nerve morphometry is a quantitative diagnostic laboratory technique used to analyze nerve structures, allowing clinicians to detect morphological differences in myelination and assess the progression of peripheral neuropathies. To quantify myelin and axon pathology, a nerve biopsy is prepared for observation using both light microscopy (LM) and transmission electron microscopy (TEM). Standardized morphometry enables the assessment of metrics, including myelinated fiber profile equivalent circle diameter and axon density. This process remains laborious and complex. This quality improvement study aimed to investigate a consolidated approach to conducting nerve morphometry using only TEM, or high-throughput whole-slide imaging (WSI) to replace standard LM imaging, reducing the complexity and time to conduct the procedure. Cases previously biopsied and analyzed for diagnosis by light microscopy were reimaged and assessed utilizing WSI and TEM. Data were then compared to those previously obtained for diagnosis by the standard LM procedure. While the WSI procedure resulted in good sensitivity and specificity (positive and negative predictive values, respectively) compared to the morphometric analyses originally obtained by the standard diagnostic LM, the results from the TEM analysis failed these statistical tests because of the inability to correctly characterize any of the normal cases. Therefore, we conclude that WSI may be an effective alternative to conducting standard LM myelinated fiber morphometry, reducing the time needed to complete the procedure, whereas the TEM morphometry protocol cannot be recommended at this time.

Virtual microscopy (VM) holds promise to reduce subjectivity as well as intra- and inter-observer variability for the histopathological evaluation of prostate cancer. We evaluated (i) the repeatability (intra-observer agreement) and reproducibility (inter-observer agreement) of the 2014 Gleason grading system and other selected features using standard light microscopy (LM) and an internally developed VM system, and (ii) the interchangeability of LM and VM. Two uro-pathologists reviewed 413 cores from 60 Swedish men diagnosed with non-metastatic prostate cancer 1998–2014. Reviewer 1 performed two reviews using both LM and VM. Reviewer 2 performed one review using both methods. The intra- and inter-observer agreement within and between LM and VM were assessed using Cohen’s kappa and Bland and Altman’s limits of agreement. We found good repeatability and reproducibility for both LM and VM, as well as interchangeability between LM and VM, for primary and secondary Gleason pattern, Gleason Grade Groups, poorly formed glands, cribriform pattern and comedonecrosis but not for the percentage of Gleason pattern 4. Our findings confirm the non-inferiority of VM compared to LM. The repeatability and reproducibility of percentage of Gleason pattern 4 was poor regardless of method used warranting further investigation and improvement before it is used in clinical practice.

Objective To evaluate the effect of ethanol on cell migration and motility in tongue squamous cell carcinoma (TSCC) cell lines and investigate its potential mechanisms. Methods Two TSCC cell lines SCC9 and SCC15 were cultured with DMEM/F12 medium and treated with or without ethanol. Cell morphology and growth patterns were observed under the light microscope. Wound healing and transwell assay were performed to determine the ability of migration and invasion. Western blot and immunofluorescence were used to detect the expressions of E-cadherin and Vimentin. Data were analyzed by Chi square test using SPSS 19.0 statistical software, and the protein level of E-cadherin and Vimentin were evaluated by One-Way ANOVA. Results SCC9 and SCC15 cells presented with cobblestone-like pattern and an apical-basal polarity, whereas, both of them demonstrated a dispersed, spindle-shaped morphology with migratory protrusions after treated with ethanol. In wounding healing assay, the cell motility of SCC9 and SCC15 cells was enhanced significantly after alcohol treatment (χ2SCC9, 24 h=15.668, PSCC9, 24 h= 0.001; χ2SCC15, 24 h=4.703, PSCC15, 24 h=0.028; χ2SCC9, 48 h=3.391, PSCC9, 48 h=0.042; χ2SCC15, 48 h = 2.386, PSCC15, 48 h= 0.136) , while in transwell assay, the relative invasion rate was 384.24% (χ2= 26.917, P<0.001) in SCC9 compared to 428.49% (χ2= 32.880, P<0.001) in SCC15, which confirmed that invasion ability was significantly increased in SCC9 and SCC15 cells treated with ethanol. Moreover, E-cadherin expression was decreased (F= 131.533, PSCC9<0.001, PSCC15= 0.001) , but Vimentin expression was increased (F= 355.388, PSCC9<0.001, PSCC15<0.001) after administration of ethanol. Conclusion Ethanol could induce epithelial mesenchymal transition and promote migration and invasion in tongue squamous cell carcinoma. Key words: Tongue; Carcinoma, squamous cell; Ethanol; Epithelial-mesenchymal transition; E-cadherin

A Decision Support System for Real-Time Control and Monitoring of Dynamical Processes

SH3BP1, an Exocyst-Associated RhoGAP, Inactivates Rac1 at the Front to Drive Cell Motility

The Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associated carcinogenesis. CagA is thought to be necessary and sufficient to induce the motogenic response observed in response to CagA+ strains, as CagA interacts with proteins involved in adhesion and motility. We report that H. pylori strain 60190 stimulated AGS cell motility through a CagA- and TFSS-dependent mechanism, because strains 60190DeltacagA or 60190DeltacagE (TFSS-defective) did not increase motility. The JNK pathway is critical for H. pylori-dependent cell motility, as inhibition using SP600125 (JNK1/2/3 inhibitor) or a JNK2/3-specific inhibitor blocked motility. JNK mediates H. pylori-induced cell motility by activating paxillin, because JNK inhibition blocked paxillinTyr-118 phosphorylation, and paxillin expression knockdown completely abrogated bacteria-induced motility. Furthermore, JNK and paxillinTyr-118 were activated by 60190DeltacagA but not 60190DeltacagE, demonstrating CagA-independent signaling critical for cell motility. A beta1 integrin-blocking antibody significantly inhibited JNK and paxillinTyr-118 phosphorylation and cell scattering, demonstrating that CagA-independent signaling required for cell motility occurs through beta1. The requirement of both Src and focal adhesion kinase for signaling and motility further suggests the importance of integrin signaling in H. pylori-induced cell motility. Finally, we show that JNK activation occurs independent of known upstream kinases and signaling molecules, including Nod1, Cdc42, Rac1, MKK4, and MKK7, which demonstrates novel signaling leading to JNK activation. We report for the first time that H. pylori mediates CagA-independent signaling that promotes cell motility through the beta1 integrin pathway.

Droplet microfluidics is becoming an enabling technology for synthesizing microscale particles and an effective real-time method is essential to monitor the variations in a dynamic droplet generation process. Here, a novel real-time cosine similarity algorithm (RT-CSA) method was developed to investigate the droplet generation process by measuring the droplet generation frequency continuously. The RT-CSA method uses a first-in-first-out (FIFO) similarity vector buffer to store calculated cosine similarities, so that these cosine similarities are reused to update the calculation results once a new frame is captured and stored. For the first time, the RT-CSA method achieved real-time monitoring of dynamic droplet generation processes by updating calculation results over 2,000 times per second, and two pre-microgel droplet generation processes with or without artificial disturbances were monitored closely and continuously. With the RT-CSA method, the disturbances in dynamic droplet generation processes were precisely determined, and following changes were monitored and recorded in real time. This highly effective RT-CSA method could be a powerful tool for further promoting research of droplet microfluidics.

Background: Plant reproduction ensures the survival and diversity of plant species. Plants use both sexual and asexual methods of reproduction, each offering distinct advantages. Sexual reproduction involves the combination of genetic material from two parent plants, leading to genetic diversity. Asexual reproduction, on the other hand, allows plants to reproduce without the fusion of gametes, producing genetically identical offspring. Methods: This study explored sexual and asexual reproduction methods across different plant species, analyzing their mechanisms through a review of literature and observational data from selected plant species. Data was collected from field and greenhouse experiments involving species like Arabidopsis thaliana for sexual reproduction and Bryophyllum for asexual reproduction. Pollination, fertilization, seed germination, and vegetative propagation were observed and analyzed using microscopic, genetic, and statistical tools. Results: Sexual reproduction was found to increase genetic variation, contributing to greater resilience in changing environments. Asexual reproduction provided faster population growth in stable conditions. Sexual reproduction was more common in flowering plants, while asexual reproduction was predominant in species with poor pollination opportunities or those in extreme environments. Conclusion: Sexual and asexual reproduction methods provide evolutionary advantages based on environmental factors and species-specific needs. The choice between the two strategies is influenced by the availability of resources, environmental conditions, and genetic diversity requirements. A combination of both methods is seen in some species, enabling adaptation and survival across diverse ecosystems.

Cell cycle studies of the dinoflagellates Gonyaulax polyedra Stein and Gyrodinium uncatenum Hulburt during asexual and sexual reproduction

Glycosphingolipids, due to their tendency to form laterally separated liquid-ordered phases, possess a high potential for the creation of order in biological membranes. The formation of glycosphingolipid-rich membrane domains within the membrane has profound consequences on the membrane organization at different levels, and on the conformational and biological properties of membrane-associated proteins and multimolecular protein complexes. Alterations in the structures of carbohydrate epitopes associated with glycosphingolipids are a common feature of tumors and tumor cells (“aberrant glycosylation”). In particular, tumors are characterized by the peculiar ability to manipulate sialylation processes (1). This abnormal sialylation process generates peculiar antigenic determinants, which are normally absent in healthy cells, and affects cell homeostasis, altering the normal signaling pathways. Indeed, glycosphingolipids in tumor cells have been implicated in the regulation of cell adhesion, motility, recognition, survival and proliferation (2). Thus, an ever-increasing interest to this regard is being devoted to gangliosides, sialic acid-containing glycolipids, and to the enzymes affecting sialylation. Both sialyltransferases and sialidases seem to be involved in the phenomenon of aberrant sialylation in tumor cells. The genetic (stable overexpression sialyltransferase I SAT-I or GM3 synthase) or pharmacological (selective pressure by N-(4-hydroxyphenyl)retinamide)) manipulation of A2780 human ovarian carcinoma cells allowed us to obtain monoclonal cells characterized by higher GM3 synthase activity respect to wild type cells (3-5). High GM3 synthase expression resulted in 1) elevated ganglioside levels, 2) reduced in vitro cell motility and increased adhesion to fibronectin, 3) enhanced expression of the membrane adaptor protein caveolin-1, an integral membrane protein playing multiple roles as negative regulator in the progression of several types of human tumors (6,7). Administration of exogenous gangliosides was able to strongly reduce in vitro cell motility and to increase cell adhesive ability to fibronectin in wild type cells, which