Cellular & Molecular Imaging

Background Microsporidia sp. MB, a microsporidian symbiont found naturally in Anopheles mosquitoes, has potential as a novel malaria control tool since it can inhibit Plasmodium development and transmission. The most feasible MB-based Plasmodium control strategy would involve dissemination through live mosquito releases, or release of spores infective to mosquito larvae. To implement either strategy, establishment of stable mosquito colonies carrying MB at a high frequency is likely to be essential. The progeny of field caught An. gambiae s.l from Burkina Faso were isolated for individual egg laying and tested for MB. The progeny of the MB positive females were pooled and this process was repeated for multiple generations. The relative density of MB in different life stages and tissues of the An. coluzzii host was examined using a novel duplex qPCR assay. We also examined the impact of MB on fecundity through individualization for egg laying and counting of eggs. Finally, we examined laid eggs for presence of MB spores. Results Three An. coluzzii colonies and one An. gambiae s.l hybrid colony were established with high prevalence and density of MB and were maintained for more than two years with minimal intervention. MB prevalence and density was highest in eggs and adult females and lowest in L4 larvae; in adults density was highest in the gonads. Additionally, MB density increased in ovary samples following blood feeding which was likely due to the activation of sporogony. The production of spores is the reason why MB-carrying females lay more white non-hatching eggs and show a small reduction in fecundity. Conclusions Establishment of several stable MB carrying An. gambiae s.l colonies and understanding the impact of spores on fecundity are significant steps forward in developing MB as a malaria control tool.

Aberrant biomolecular condensates are implicated in multiple incurable neurological disorders, including Amyotrophic Lateral Sclerosis, Frontotemporal Dementia, and DYT1 dystonia. However, the role of condensates in driving disease etiology remains incompletely understood. Here, we establish myeloid leukemia factor 2 (MLF2) as a disease-associated phase transition biomarker and develop a scalable high-content platform that identifies condensate modulators across broad chemical and genetic space. We uncover FDA-approved drugs that remodel aberrant condensate composition, validating the approach for drug discovery. A genome-wide CRISPR/Cas9 screen identifies genes linked to microcephaly and related neurodevelopmental disorders whose loss drives nuclear condensate accumulation. Machine learning resolves two phenotypic clusters: RNF26 deletion induces nuclear envelope condensates reminiscent of nuclear pore defects, whereas loss of microcephaly-associated ZNF335 drives accumulation of distinct nucleoplasmic condensates. Our study provides a scalable resource for identifying corrective modulators of aberrant condensates and establishes a link between dysregulated phase transitions and neurodevelopmental disorders.

OME-NGFF converted study from idr0090. Drug resistance threatens the effective prevention and treatment of an ever-increasing range of human infections. This highlights an urgent need for new and improved drugs with novel mechanisms of action to avoid cross-resistance. Current cell-based drug screens are, however, restricted to binary live/dead readouts with no provision for mechanism of action prediction. Machine learning methods are increasingly being used to improve information extraction from imaging data. Such methods, however, work poorly with heterogeneous cellular phenotypes and generally require time-consuming human-led training. We have developed a semi-supervised machine learning approach, combining human- and machine-labelled training data from mixed human malaria parasite cultures. Designed for high-throughput and high-resolution screening, our semi-supervised approach is robust to natural parasite morphological heterogeneity and correctly orders parasite developmental stages. Our approach also reproducibly detects and clusters drug-induced morphological outliers by mechanism of action, demonstrating the potential power of machine learning for accelerating cell-based drug discovery.

Polyphasic studies on cyanobacterial biodiversity are scarce in the Caribbean and have never been conducted in the Dominican Republic. In this study we analysed sixteen cyanobacterial isolates sampled from biofilms growing on rocks and related to freshwater, saline and thermal environments across the country. A polyphasic approach was employed, incorporating 16S rRNA gene phylogenetic analysis, molecular identity assessments (p-distance), 16S–23S ITS sequence comparison and secondary structure analysis, alongside morphological characterization and habitat comparison. The strains were distributed across the orders Nodosilineales, Oculatellales, Oscillatoriales and Nostocales. Eleven strains were identified as belonging to Almyronema, Euryhalinema, Salileptolyngbya, Nodosilinea, Reticulonema, Vacuolonema, Capilliphycus, Purpureonostoc, Violetonostoc, Desmonostoc and Hapalosiphon. Additionally, four strains formed three distinct clades and presented low 16S rRNA gene identities with their phylogenetically closest genera, suggesting the existence of three undescribed lineages pending comprehensive polyphasic study. At the species level, four strains were considered undescribed lineages pending comprehensive polyphasic study and one strain (LEGE 171504) was described as a new species of Salileptolyngbya. This study represents the first investigation into the cyanobacterial diversity of Dominican Republic expanding the distribution of the identified taxa. Moreover, this is the first report of Salileptolyngbya in the Caribbean region and in brackish waters, as well as the first newly described cyanobacterial species from the Dominican Republic.

Tara Expeditions are global scientific voyages that probe morphological and molecular diversity, evolution and ecology of marine plankton to explore how they are impacted by changes in the Earth's climate. The first expeditions collected samples of marine plankton containing viruses, bacteria, archaea, protists and planktonic metazoans living in the photic layer of the world's oceans. These expeditions, the first taking place between 2009 and 2013, include Tara Oceans: a global view, and Tara Oceans Polar Circle, both of which followed the same sampling protocol. This dataset includes 1074 pictures of 284 planktonic taxa (mainly microalgae and other Ciliate and Radiolarian protists) collected from the vicinity of the Marquesas Islands in the Southern Pacific Ocean during the Tara Oceans expedition. Multiple samples particularly of the size fractions 5-20 and 20 180 um from four sites and two depths were processed with different methods and studied in detail using scanning electron microscopy.

Manuscript abstract: Malaria transmission relies on sporozoite formation in the mosquito midgut and subsequent salivary gland invasion. Despite their importance, the cell biology of these processes remains poorly understood. We apply Mosquito Tissue Ultrastructure Expansion Microscopy (MoTissU-ExM), which physically expands infected mosquito tissues while preserving host and parasite ultrastructure. MoTissU-ExM reveals parasite structures and organelles, including features previously seen only by electron microscopy and novel structures not observed before. We use MoTissU-ExM to investigate sporozoite formation and salivary gland invasion, focusing on rhoptries - secretory organelles critical for host cell invasion. We establish a timeline for rhoptry biogenesis, show that two rhoptries are consumed during salivary gland invasion, and provide the first evidence that rhoptry pairs are specialized for different invasion events. We further characterize RON11 as the first protein involved in sporozoite rhoptry biogenesis; its disruption produces sporozoites that specifically fail to invade salivary gland epithelial cells, blocking parasite transmission. Dataset description: This dataset contains all microscopy data associated with the linked publication "Unlocking new understanding of Plasmodium sporozoite biology with expansion microscopy". All samples were prepared by ultrastructure-expansion microscopy (U-ExM). All samples were imaged on either a Zeiss LSM900 or LSM980 microscope, using either Airyscan-SR or Airyscan-MPLX modes. File names will include the magnification of the objective lens used as follows: 5x = EC Plan-Neofluar 5x/0.16NA Air 10x = Ziess Plan-Apochromat 10x/0.45NA air 20x = Ziess Plan-Apochromat 20x/0.8NA air 40x = Zeiss C-Apochromat 40x/1.2NA water-immersion autocorr M27 63x = Zeiss Plan-Apochromat 63x/1.4NA oil-immersion M27 Images are of mosquito tissues, or isolated parasites, from three Plasmodium species - berghei (Pb), falciparum (Pf), and yoelii (Py). Images are sorted and named as follows (folder name, file name) Plasmodium species > Tissue type/site of isolation > Parasite strain > Species abbreviation, MG/SG, Harvest day(dpi), Dye/Fluorophores (405nm -> 647nm), Objective, Image number (1->X), as (airyscan) For example, the third image taken of a P. berghei oocyst with the RON11iKD parasite line, that was harvested on Day 14 post infection, stained with NHS Ester AF405, BODIPY-FL, anti-Tubulin AF555, and Sytox Red, and imaged on the 40x-objective would be listed as follows: Plasmodium berghei > Infected midguts > RON11iKD > RON11KD MG 14dpi NHSBFlTub-SytR 40x 1 as The majority of images in this dataset are z-stacked images, but for many oocysts a single-slice image of the whole oocyst was taken. When this is the case, the single-slice image will be indicated with "SNAP". A list of the acronyms and abbreviations used in file names are as follows MG = Midgut SG = Salivary gland Spz = Sporozoite HC = Haemocoel dpi = Days post infection NHS = NHS Ester Alexa Fluor 405 BFl = Bodipy-FL-Ceramide BTRc = Bodipy-TR-Ceramide SytR = Sytox Deep Red Tub = anti-tubulin antibody CSP = anti-circumsporozoite protein antibody RAP1 = anti-rhoptry associated protein 1 antibody iKD = Inducible knockdown Ctrl = Control KD = Knockdown RON4 = anti-rhoptry neck protein 4 antibody GFP = anti-green fluorescent protein antibody WGA = Wheat germ aglutinnin BIP = anti-BiP antibody ERD2 = anti-ERD2 antibody

Light microscopy and scanning electron microscopy images of snow algae field samples containing Rosetta gen. nov., including type material.

Bacteria occupy heterogeneous environments, attaching and growing within pores in materials, living hosts, and matrices like soil. Systems that permit high-resolution visualization of dynamic bacterial processes within the physical confines of a realistic and tractable porous media environment are rare. Here we use microfluidics to replicate the grain shape and packing density of natural sands in a 2D platform to study the flow-induced spatial evolution of bacterial biofilms underground. We introduce a wildtype strain (Pantoea sp. YR343, n=3) or an EPS-defective strain (Pantoea sp. YR343 ΔUDP, n=3) to the porous media platform and then simulate a rainfall event using gravity-driven flow of bacterial growth media.

The cyanoHAB forming cyanobacteria Microcystis and Planktothrix frequently produce high intracellular amounts of microcystins (MCs) or anabaenopeptins (APs). In this study, chemically modified MCs and APs have been localized on a subcellular level in Microcystis and Planktothrix applying copper-catalyzed alkyne-azide cycloaddition (CuACC). For this purpose, three different non-natural amino acids carrying alkyne or azide moieties were fed to individual P. agardhii strains No371/1 and CYA126/8 as well as to M. aeruginosa strain Hofbauer showing promiscuous incorporation of various amino acid substrates during non-ribosomal peptide synthesis (NRPS). Moreover, CYA126/8 peptide knock-out mutants and non-toxic strain Synechocystis PCC6803 were processed under identical conditions. Simultaneous labelling of modified peptides with ALEXA405 and ALEXA488 and lipid staining with BODIPY 505/515 were performed to investigate the intracellular location of the modified peptides. Pearson correlation coefficients (PCC) obtained from confocal images were calculated between the different fluorophores and the natural autofluorescence (AF), and between labelled modified peptides and dyed lipids to investigate the spatial overlap between peptides and the photosynthetic complex, and between peptides and lipids. Overall, labelling of modified MCs (M. aeruginosa) and APs (P. agardhii) using both fluorophores revealed in-creased intensity in MC/AP producing strains. For Synechocystis lacking NRPS, no labelling using either ALEXA405 or ALEXA488 was observed. Lipid staining in M. aeruginosa and Synechocystis was intense while in Planktothrix it was more variable. When compared with AF, both modified peptides and lipids showed a heterologous distribution. In comparison, the correlation between stained lipids and labelled peptides was not increased suggesting a reduced spatial overlap.

ARDS was induced in pigs as described in Stenlo et al. AJP Lung 2020 via dual intratracheal and intravenous administration of lipolysaccharide until two consecutive blood gases confirmed the onset of ARDS according to the Berlin criteria. In Stenlo et al. Physiological Reports 2021, animals were also treated with extracorporeal membrane oxygenation in addition to mechanical ventilation. Samples were subsequently processed for routine histopathological examination. Biopsies of 5mm thickness were formalin fixed overnight at 4 degrees Celsius followed by a xylene free tissue processing routine and paraffin embedding. Paraffin embedded slices of 5um thickness were subjected to hematoxylin and eosin staining followed by whole slide scanning for subsequent blinded scoring.