Conventional Light Microscopy Research Articles

Global nuclear reorganization during heterochromatin replication in the giant-genome plant Nigella damascena L.

Among flowering plants, genome size varies remarkably, by >2200-fold, and this variation depends on the loss and gain of noncoding DNA sequences that form distinct heterochromatin complexes during interphase. In plants with giant genomes, most chromatin remains condensed during interphase, forming a dense network of heterochromatin threads called interphase chromonemata. Using super-resolution light and electron microscopy, we studied the ultrastructure of chromonemata during and after replication in root meristem nuclei of Nigella damascena L. During S-phase, heterochromatin undergoes transient decondensation locally at DNA replication sites. Due to the abundance of heterochromatin, the replication leads to a robust disassembly of the chromonema meshwork and a general reorganization of the nuclear morphology visible even by conventional light microscopy. After replication, heterochromatin recondenses, restoring the chromonema structure. Thus, we show that heterochromatin replication in interphase nuclei of giant-genome plants induces a global nuclear reorganization.

One-step nanoscale expansion microscopy reveals individual protein shapes.

The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.

Mosquito Tissue Ultrastructure-Expansion Microscopy (MoTissU-ExM) enables ultrastructural and anatomical analysis of malaria parasites and their mosquito

BackgroundStudy of malaria parasite cell biology is challenged by their small size, which can make visualisation of individual organelles difficult or impossible using conventional light microscopy. In recent years, the field has attempted to overcome this challenge through the application of ultrastructure expansion microscopy (U-ExM), which physically expands a biological sample approximately 4.5-fold. To date, U-ExM has mostly been used to visualise blood-stage parasites and used exclusively on parasites in vitro.MethodsHere we develop Mosquito Tissue U-ExM (MoTissU-ExM), a method for preparing dissected mosquito salivary glands and midguts by U-ExM. MoTissU-ExM preserves both host and parasite ultrastructure, enabling visualisation of oocysts and sporozoites in situ. We also provide a point-by-point protocol for how to perform MoTissU-ExM.ResultsWe validate that MoTissU-ExM samples expand as expected, provide a direct comparison of the same dissected tissues before and after MoTissU-ExM, and highlight some of the key host and parasite structures that can be visualised following MoTissU-ExM.DiscussionWe discuss potential use cases for MoTissU-ExM for study of malaria parasite biology, and more broadly. We detail drawbacks or challenges MoTissU-ExM and imaging these expanded tissues, along with information troubleshooting this technique. Finally, we discuss how MoTissU-ExM could be applied and adapted in future to increase its utility.

Quantitative real-time PCR assays for species-specific detection and quantification of Baltic Sea spring bloom dinoflagellates.

In the Baltic Sea, the dinoflagellates Apocalathium malmogiense, Biecheleria baltica, and Gymnodinium corollarium are important contributors to the spring bloom. However, their relative contribution to the bloom community cannot be unambiguously determined by conventional light microscopy due to a lack of resolution of distinctive morphological features of the three species. Here, we describe a molecular approach based on a quantitative real-time polymerase chain reaction (qPCR) primer and probe system, targeting the ITS1 and ITS2 regions of the rRNA gene for all three species and enabling their quantification. The specificity of the method was demonstrated using monocultures of A. malmogiense, B. baltica, G. corollarium as well as three other dinoflagellate species co-occurring in the Baltic Sea during spring and validated using field-collected phytoplankton samples.

Super-resolved highly multiplexed immunofluorescence imaging for precise protein localization and podocyte ultrastructure.

Deep insights into the complex cellular and molecular changes occurring during (patho-)physiological conditions are essential for understanding the interactions and regulation of proteins. This understanding is crucial for research and diagnostics. However, the effectiveness of conventional immunofluorescence and light microscope, tools for visualizing the spatial distribution of cells or proteins, are limited both in resolution and multiplexity in complex tissues. This is mainly due to challenges such as the spectral overlap of fluorophore wavelengths, a limited range of antibody types, the inherent variability of samples and the optical resolution limit. The herein demonstrated combination of multiplex immunofluorescence imaging and super resolution microscopy offers a solution to these limitations by enabling the identification of different cell types and precise subcellular localization of proteins in tissue sections. In this study, we demonstrate the cyclic staining and de-staining of paraffin kidney sections, making it suitable for routine use and compatible with super-resolution microscopy for podocyte ultrastructural studies. We have further developed a computerized workflow for data processing which is accessible through available reagents and open-access code. As a proof of principle, we identified CDH2 as a marker for cellular lesions of sclerotic glomeruli in the nephrotoxic serum nephritis mouse model and cross-validated this finding with a human Nephroseq dataset indicating its translatability. In summary, our work represents an advance in multiplex imaging, which is crucial for understanding the localization of numerous proteins in a single FFPE kidney section and the compatibility with super-resolution microscopy to study ultrastructural changes of podocytes.

Species of the Genus Paroniella Fuhrmann, 1920 (Cestoda: Davaineidae) from Bulbuls (Passeriformes: Pycnonotidae): Redescription of P. perreti (Mahon, 1954) and Description of P. neolestes n. sp. from Africa.

The purpose of the present study is to contribute to the knowledge on the diversity of cestodes of the genus Paroniella Fuhrmann, 1920 parasitising passerine birds of the family Pycnonotidae (bulbuls) in the Afrotropical Region. The only known species of this groups, Paroniella perreti (Mahon, 1954) from Pycnonotus tricolor (Hartlaub) in the Democratic Republic of the Congo, is poorly described. Therefore, it needed a detailed redescription in order to make a reliable comparison and provide differentiation among this species and newly-collected davaineid specimens. Based on new materials collected from Neolestes torquatus Cabanis in Gabon, Paroniella neolestes n. sp. is described. The type series of P. perreti from the collection of the Natural History Museum of Geneva is redescribed and figured using conventional light microscopy and interference-contrast microscopy. The specimens of the new species were stained by iron acetocarmine and mounted in Canada balsam. The present study provided more detailed and accurate data on P. perreti (compared to its original description) in relation mostly to the armament of suckers (not mentioned in the original description), the structure of the copulative apparatus, the number of rostellar hooks and the number of testes. The morphological comparison of the specimens from Neolestes torquatus from Gabon with the known 53 species of the genus Paroniella (presented in a table format) characterised them as belonging to a new species. The new combination Paroniella oitaensis (Kugi, 1990) n. comb. was proposed. Based on the present state of knowledge, P. perreti and P. neolestes seem to be specific each to a single host species, i.e. Pycnonotus tricolor and Neolestes torquatus, respectively. Further studies of davaineid cestodes from pycnonotids from Africa as well as from South and Southeast Asia may result in discovering much greater diversity of this group than currently known.

Pathology students' perceptions of virtual learning: A case study of students in Saudi Arabia.

Pathology laboratory classes are traditionally conducted using a conventional light microscope. The Coronavirus Disease 2019 (COVID-19) pandemic and recent technological advances necessitated remote learning through online classes using virtual slides (VS) instead of glass slides (GS). The purpose of this study was to gauge the perception of learning pathology using virtual slides (VS) as opposed to glass slides (GS) for medical students in Saudi Arabia. This study would help modify teaching methods with the advancement of the application of newer methods in online teaching. This two-phased study evaluated learning outcomes and perceptions in pathology online education for medical students. Using a questionnaire, Phase one analyzed second and third-year students' perceptions of the teaching methods after an online pathology course. Phase Two assessed the learning outcomes of third-year students during online practical sessions using a pretest and post-test design. Statistical data were collected using a simple additive approach. Statistical tools were used to determine the factors affecting students' perceptions. The accessibility of VS at any possible time, location, or device was the most advantageous trait of virtual learning (mean = 2.94±0.9). Students agreed the least with virtual slides as the only optimal method of learning pathology (mean = 2.25±0.9). Most enjoyed the virtual lab experience (51.7%) but still prefer both laboratory-GS and virtual-VS classes (83.5%). VS had the benefit of accessibility and efficiency. The acceptance of VS was significantly affected by the orientation prior to the online class. Findings showed that VS cannot completely replace GS and more aspects such as technical difficulties and prior VS experience should be explored.

Rapid and Comprehensive Screening for Urogenital and Gastrointestinal Schistosomiasis with Handheld Digital Microscopy Combined with Circulating Cathodic Antigen Testing.

Novel methods are required to aid the monitoring of schistosomiasis control and elimination initiatives through mass drug administration. Portable digital and mobile phone microscopy is a promising tool for this purpose. This cross-sectional study evaluated the diagnostic operating characteristics of a converted mobile phone microscope (the SchistoScope) for the detection of Schistosoma haematobium eggs, as determined by community-based field workers and expert microscopists, compared with a field gold standard of light microscopy. Three hundred sixty-five urine samples were evaluated by conventional light microscopy, with 49 (13.4%) positive for S. haematobium. Compared with light microscopy, the sensitivity and specificity of S. haematobium detection by field microscopists trained to use the SchistoScope were 26.5% (95% CI: 14.9-41.1%) and 98.4% (95% CI: 96.3-99.5%), respectively. The sensitivity and specificity of S. haematobium detection by expert microscopists using the SchistoScope was 74% (95% CI: 59.7-85.4%) and 98.1% (95% CI: 95.9-99.3%), respectively, compared with light microscopy. The sensitivity rose to 96.1% and 100% when evaluating for egg counts greater than five and 10 eggs per 10 mL, respectively. A point-of-care circulating cathodic anion (POC CCA) test was used to evaluate Schistosoma mansoni; however, there were too few positive samples to reliably comment on diagnostic characteristics. This study demonstrated that a "urine-only" approach to rapidly screen for schistosomiasis at the point of sample collection can be conducted with mobile phone microscopy (S. haematobium) coupled with POC CCA (S. mansoni). Such an approach may aid in streamlined schistosomiasis control and elimination initiatives.

The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part I: Super-Resolution’s STED, SIM, SMI, and SMLM

Abstract As technology advances, the field of microscopy offers unique opportunities for scientific exploration. However, the complex terminology used to describe these cutting-edge techniques often appears as a jumble of letters, akin to alphabet soup. Among the plethora of acronyms, some of the most prevalent are associated with super-resolution microscopy, including Stimulated Emission Depletion Microscopy (STED), Structured Illumination Microscopy (SIM), Spatially Modulated Illumination (SMI), and Single-Molecule Localization Microscopy (SMLM). These techniques push past the limitations of conventional light microscopy, achieving resolutions down to the tens of nanometers. This article aims to decode these common super-resolution techniques, fostering a deeper understanding of microscopy and igniting interest in their application.

Assessing Structural Traits in Triticum aestivum and Zea mays for C3 and C4 Photosynthetic Differentiation Using Free-hand and Semi-thin Sections.

The enhanced efficiency of C4 photosynthesis, compared to the C3 mechanism, arises from its ability to concentrate CO2 in bundle sheath cells. The effectiveness of C4 photosynthesis and intrinsic water use efficiency are directly linked to the share of mesophyll and bundle leaf cells,size and density of bundle sheaths, and size, density, and cell wall thickness of bundle sheath cells. Rapid microscopical analysis of these traits can be performed on free-hand and semi-thin sections using conventional light microscopy, providing valuable information about photosynthetic efficiency in C4 crops by means of identifying and examining specific cell types. Additionally, errors in freehand and semi-thin section preparation that affect anatomical measurements and cell type diagnoses are shown, as well as how to avoid these errors. This microscopical approach offers an efficient means of gathering insights into photosynthetic acclimation to environmental variation and aids in the rapid screening of crops for future climates.

Reproducibility of c-Met Immunohistochemical Scoring (Clone SP44) for Non-Small Cell Lung Cancer Using Conventional Light Microscopy and Whole Slide Imaging.

Emerging therapies for non-small cell lung cancer targeting c-Met overexpression have recently demonstrated promising results. However, the evaluation of c-Met expression can be challenging. We aimed to study the inter and intraobserver reproducibility of c-Met expression evaluation. One hundred ten cases with non-small cell lung cancer (40 biopsies and 70 surgical specimens) were retrospectively selected in a single laboratory (LPCE) and evaluated for c-Met expression. Six pathologists (4 seniors and 2 juniors) evaluated the H-score and made a 3-tier classification of c-Met expression for all cases, using conventional light microscopy (CLM) and whole slide imaging (WSI). The interobserver reproducibility with CLM gave global Cohen Kappa coefficients (ƙ) ranging from 0.581 (95% CI: 0.364-0.771) to 0.763 (95% CI: 0.58-0.92) using the c-Met 3-tier classification and H-score, respectively. ƙ was higher for senior pathologists and biopsy samples. The interobserver reproducibility with WSI gave a global ƙ ranging from 0.543 (95% CI: 0.33-0.724) to 0.905 (95% CI: 0.618-1) using the c-Met H-score and 2-tier classification (≥25% 3+), respectively. ƙ for intraobserver reproducibility between CLM and WSI ranged from 0.713 to 0.898 for the c-Met H-score and from 0.600 to 0.779 for the c-Met 3-tier classification. We demonstrated a moderate to excellent interobserver agreement for c-Met expression with a substantial to excellent intraobserver agreement between CLM and WSI, thereby supporting the development of digital pathology. However, some factors (scoring method, type of tissue samples, and expertise level) affect reproducibility. Our findings highlight the importance of establishing a consensus definition and providing further training, particularly for inexperienced pathologists, for c-Met immunohistochemistry assessment in clinical practice.

Identification of z-axis filopodia in growth cones using super-resolution microscopy.

A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three-dimensional-structured illumination microscopy, a type of super-resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z-axis ("z-filopodia") within the growth cone. Z-filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z-filopodia formation and dynamics were regulated by actin-regulatory proteins, such as vasodilator-stimulated phosphoprotein, fascin, and cofilin. Chromophore-assisted laser inactivation of cofilin induced the rapid turnover of z-filopodia. An axon guidance receptor, neuropilin-1, was concentrated in z-filopodia and was transported together with them, whereas its ligand, semaphorin-3A, was selectively bound to them. Membrane domains associated with z-filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin-1. The results suggest that z-filopodia have unique turnover properties, and unlike xy-filopodia, do not function as force-generating structures for axon extension.

Machine Learning-Assisted Classification of Paraffin-Embedded Brain Tumors with Raman Spectroscopy.

Raman spectroscopy (RS) has demonstrated its utility in neurooncological diagnostics, spanning from intraoperative tumor detection to the analysis of tissue samples peri- and postoperatively. In this study, we employed Raman spectroscopy (RS) to monitor alterations in the molecular vibrational characteristics of a broad range of formalin-fixed, paraffin-embedded (FFPE) intracranial neoplasms (including primary brain tumors and meningiomas, as well as brain metastases) and considered specific challenges when employing RS on FFPE tissue during the routine neuropathological workflow. We spectroscopically measured 82 intracranial neoplasms on CaF2 slides (in total, 679 individual measurements) and set up a machine learning framework to classify spectral characteristics by splitting our data into training cohorts and external validation cohorts. The effectiveness of our machine learning algorithms was assessed by using common performance metrics such as AUROC and AUPR values. With our trained random forest algorithms, we distinguished among various types of gliomas and identified the primary origin in cases of brain metastases. Moreover, we spectroscopically diagnosed tumor types by using biopsy fragments of pure necrotic tissue, a task unattainable through conventional light microscopy. In order to address misclassifications and enhance the assessment of our models, we sought out significant Raman bands suitable for tumor identification. Through the validation phase, we affirmed a considerable complexity within the spectroscopic data, potentially arising not only from the biological tissue subjected to a rigorous chemical procedure but also from residual components of the fixation and paraffin-embedding process. The present study demonstrates not only the potential applications but also the constraints of RS as a diagnostic tool in neuropathology, considering the challenges associated with conducting vibrational spectroscopic analysis on formalin-fixed, paraffin-embedded (FFPE) tissue.

Digital Validation in Breast Cancer Needle Biopsies: Comparison of Histological Grade and Biomarker Expression Assessment Using Conventional Light Microscopy, Whole Slide Imaging, and Digital Image Analysis.

Given the widespread use of whole slide imaging (WSI) for primary pathological diagnosis, we evaluated its utility in assessing histological grade and biomarker expression (ER, PR, HER2, and Ki67) compared to conventional light microscopy (CLM). In addition, we explored the utility of digital image analysis (DIA) for assessing biomarker expression. Three breast pathologists assessed the Nottingham combined histological grade, its components, and biomarker expression through the immunohistochemistry of core needle biopsy samples obtained from 101 patients with breast cancer using CLM, WSI, and DIA. There was no significant difference in variance between the WSI and CLM agreement rates for the Nottingham grade and its components and biomarker expression. Nuclear pleomorphism emerged as the most variable histologic component in intra- and inter-observer agreement (kappa ≤ 0.577 and kappa ≤ 0.394, respectively). The assessment of biomarker expression using DIA achieved an enhanced kappa compared to the inter-observer agreement. Compared to each observer's assessment, DIA exhibited an improved kappa coefficient for the expression of most biomarkers with CLM and WSI. Using WSI to assess prognostic and predictive factors, including histological grade and biomarker expression in breast cancer, is acceptable. Furthermore, incorporating DIA to assess biomarker expression shows promise for substantially enhancing scoring reproducibility.

Anatomy of vegetative organs of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. (Araliaceae)

Eleutherococcus senticosus (Araliaceae) is widely used as adaptogen in herbal medicine. Since comprehensive anatomical analysis of its vegetative organs was not available, the present study aimed at providing the reference data on the structure of leaves and roots, under- and aboveground stems of 1–4 year-old plants by means of conventional light microscopy. In the primary structure, roots were di- or triarch, with secretory canals facing protoxylem. Concurrently with cambium initiation, additional pericyclic secretory canals differentiated close to the existing ones. In the root secondary structure, secretory canals were formed in the conductive secondary phloem and maintained in the nonconductive one. Stem primary structure encompassed uniseriate epidermis with scant prickles, primary cortex and stele. Primary cortex was composed of collenchyma, chlorenchyma, ground parenchyma with secretory canals and ca. triseriate starch sheath. In outer stele (pericycle) strands of sclerenchyma fibers (stereids) differentiated, alternated with parenchyma. In the ring of open collateral bundles, cambium became continuous concurrently with (sub)epidermal initiation of phellogen. In the wide pith, ground parenchyma occurred, with a few secretory canals close to protoxylem. Secondary structure stems retained the cortical tissues in the 4th year; new secretory canals appeared in the conductive secondary phloem. The epithelial cells/sheath cells complexes were maintained even in the oldest nonconductive phloem (and in the cortex) in stems of 4 year-old plants, while the successively formed sieve tube-companion cell complexes functioned till the end of the vegetation periods only. The bifacial leaves exhibited shade adaptation in chlorenchyma structure; secretory canals were formed mainly in veins’ phloem.

Quantification of C. neoformans Capsule Diameter.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.

In Situ Imaging of Proteins Using DNA-PAINT Super-Resolution Microscopy.

The spatial resolution of conventional light microscopy is restricted by the diffraction limit to hundreds of nanometers. Super-resolution microscopy enables single digit nanometer resolution by circumventing the diffraction limit of conventional light microscopy. DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) belongs to the family of single-molecule localization super-resolution approaches. Unique features of DNA-PAINT are that it allows for sub-nanometer resolution, spectrally unlimited multiplexing, proximity detection, and quantitative counting of target molecules. Here, we describe prerequisites for efficient DNA-PAINT microscopy.

Investigation of the Effect of Virtual Microscopy on Pathology Education in a Medical Faculty

Aim: Virtual microscopy technology has been widely used in histopathological diagnosis and pathology education, replacing the traditional light microscope, which is considered an important visual tool in pathology education. Slides scanned and stored online for use in virtual microscopy were first described in 1985 and have experienced significant developments since 2000. In this study. The aim of this study was to determine how virtual microscopy affected medical school students' pathology education. Methods: The study included the 3rd year students of Izmir Democracy University Faculty of Medicine. The study was conducted prior to the end-of-year pathology practice exam. As a result of the scanning performed by company received support, 102 slides were selected from the training archive and virtualized. Among them, forty slides representing lesions and organs from ten different systems, were chosen. The students were divided into two groups (group 1 and 2.) and the selected slides were used as educational material. Each student was given the opportunity to study both 20 virtual and 20 microscope slides. The students in the first group (G1) examined 20 slides (M1) of the ten systems with the light microscope and the other 20 slides on the virtual microscope (S1). On the other hand, the students in the second group (G2) examined with a light microscope (M2) the slides which shown to the first group in the virtual microscope (V1); and they examined with the virtual microscope (V2) the slides which shown to the first group in the light microscope (M1). This training course was completed in approximately eight hours. For the end-of-year pathology practice exam held the next day, the students were randomly divided into 4 groups and each group was asked questions about 10 slides from 10 different systems. These questions were about the descriptive characteristics of the organs and lesions that are examined. All slides were shown to the examinees one day later. Results: Eighty-nine students in total [n:52 (58.4%) men, n:37 (41.6%) women] took part in the survey. The study found no significant difference in overall exam scores between students using traditional light microscopy and those using virtual microscopy, with average scores being 17.2±7 on a scale of 5 to 40 points. Students who examined slides using light microscopy had an average score of 8.7±4.6, while those who used virtual microscopy had an average score of 8.6±4.1. It was found that the success rates were similar regardless of whether the students used traditional microscopy or virtual reality. Yet when the results were grouped by gender, female students had higher overall (p=0.05) and light microscopy scores (p=0.001). When the groups were investigated separately, the exam scores attained were comparable in terms of from the exam content related to major system disorders did not differ between the groups. based on learning method without any statistical significance between groups. However, while group 1 students who participated in studying with light microscopy training before the exam had higher exam scores in the exam content related to bone and soft tissue pathology compared to group 2 students studying with virtual microscopy (in bone and soft tissue pathology had a mean score of 3±1.5 vs 1.2±1.7) points, virtual learners had a mean score of 1.2±1.7 points, with a statistically significant intergroup difference according to the Mann- Whitney U test (p=0.027). Conclusions: Virtual microscopy is a crucial component of medical students' pathology training in undergraduate medical education in terms of providing opportunities for medical students to see the rare cases and standardization of the training in different institutions. Particularly in undergraduate education, it is crucial for the understanding of rare cases and standardization of the pathology education. But as our study's findings on the pathology of bone and soft tissues showed, conventional light microscopy is still essential for understanding the pathology of some systems. Moreover, one's gender may affect student's capacity for virtual learning. Understanding the distinction between genders in terms of learning capacity may aid in providing students a better education.

Discovery of molecular and genetic mechanisms of cell cycle regulation: 2001 Nobel laureates leland Hartwell, Timothy Hunt and Paul Nurse

Leland Hartwell, Paul Nurse and Timothy Hunt in 2001 were awarded the Nobel Prize for their discovery of molecular and genetic mechanisms of the cell cycle. The review analyzes the features of the objects chosen by scientists, a wide range of methods from conventional light microscopy to recombinant DNA technology and complementation test, used for research, and covers a history of the discoveries made. Thanks to the work of these scientists, a modern­ understanding of the cell cycle checkpoints, the complexes formed by cyclin and cyclin-dependent­ kinases at different phases of the cell cycle, as well as the mechanism of periodic cyclin degradation and the universality of the cyclin mechanism of cellular division in all living organisms was gained. Keywords: cdc mutants, cdc2/cdc28, cdk protein kinase, cdk/cyclin complex, cell cycle, cyclin, Saccharomyces cere­visiae, Schizosaccharomyces pombe

In situ diets of the bloom-forming dinoflagellate Noctiluca scintillans in Daya Bay

Red Noctiluca scintillans is a common heterotrophic dinoflagellate that forms blooms in temperate, subtropical, and tropical coastal ecosystems. The diet of this species plays an important role in its cell growth, development, and reproduction. Because limited gene diversity data are available regarding prey of this species, its diet in Daya Bay during a boreal winter bloom is reported using an integrated approach involving light microscopy, single cell isolation and plastid 16S rDNA cloning, and 18S rDNA V4 and V9 region amplification using isolated cells and environmental DNA as templates with high-throughput sequencing. While conventional light microscopy reveals the diet of this species to comprise Coscinodiscus sp. and Stephanopyxis turris (diatoms), copepod eggs, and detritus, plastid gene diversity identifies a diet comprising diatoms, cyanobacteria, and bacteria, and 18S rDNA high-throughput sequencing reveals a diet comprising 36 eukaryote families (primarily copepods, as well as diatoms, dinoflagellates, Ochrophyta, Haptophytes, Chordata, Cercozoans, Chlorophyta, Polychaeta, and ciliates). Dietary staples include copepods, diatoms, dinoflagellates, Ochrophyta, and Synechococcus. High copepod abundance in prey may reflect their relatively high abundance in environmental seawater. Thus, N. scintillans is generally omnivorous but prefers dominant phytoplankton taxa, including Rhizosoleniaceae, Leptocylindraceae, and Cymatosiraceae (diatoms), as well as Gonyaulacaceae (dinoflagellates). An integrated multi-disciplinary approach provides a more comprehensive picture of N. scintillans diet in Daya Bay, and an improved understanding of this species’ ecological niche and trophic role in marine ecosystems.