RhoU forms homo-oligomers to regulate cellular responses.

RhoU is an atypical member of the Rho family of small G-proteins, which has N- and C-terminal extensions compared to the classic Rho GTPases RhoA, Rac1 and Cdc42, and associates with membranes through C-terminal palmitoylation rather than prenylation. RhoU mRNA expression is upregulated in prostate cancer and is considered a marker for disease progression. Here, we show that RhoU overexpression in prostate cancer cells increases cell migration and invasion. To identify RhoU targets that contribute to its function, we found that RhoU homodimerizes in cells. We map the region involved in this interaction to the C-terminal extension and show that C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension reduces RhoU-induced activation of p21-activated kinases (PAKs), which are known downstream targets for RhoU, and induces cell morphological changes consistent with inhibiting RhoU function. Our results show for the first time that the activity of a Rho family member is stimulated by self-association, and this is important for its activity.

cellPLATO - an unsupervised method for identifying cell behaviour in heterogeneous cell trajectory data.

Advances in imaging, segmentation and tracking have led to the routine generation of large and complex microscopy datasets. New tools are required to process this 'phenomics' type data. Here, we present 'Cell PLasticity Analysis Tool' (cellPLATO), a Python-based analysis software designed for measurement and classification of cell behaviours based on clustering features of cell morphology and motility. Used after segmentation and tracking, the tool extracts features from each cell per timepoint, using them to segregate cells into dimensionally reduced behavioural subtypes. Resultant cell tracks describe a 'behavioural ID' at each timepoint, and similarity analysis allows the grouping of behavioural sequences into discrete trajectories with assigned IDs. Here, we use cellPLATO to investigate the role of IL-15 in modulating human natural killer (NK) cell migration on ICAM-1 or VCAM-1. We find eight behavioural subsets of NK cells based on their shape and migration dynamics between single timepoints, and four trajectories based on sequences of these behaviours over time. Therefore, by using cellPLATO, we show that IL-15 increases plasticity between cell migration behaviours and that different integrin ligands induce different forms of NK cell migration.

TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii.

Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt-loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme-coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum, TrmB-dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii. The ∆trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name trmB suppressor, or TbsP (a.k.a. "tablespoon"). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild-type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose-dependent transcription of gapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii.

Cross-modal sensitivities to auditory and visual stimulations in the first-order somatosensory thalamic nucleus.

The ventral posterolateral nucleus (VPL), being categorized as the first-order thalamic nucleus, is considered to be dedicated to uni-modal somatosensory processing. Cross-modal sensory interactions on thalamic reticular nucleus cells projecting to the VPL, on the other hand, suggest that VPL cells are subject to cross-modal sensory influences. To test this possibility, the effects of auditory or visual stimulation on VPL cell activities were examined in anaesthetized rats, using juxta-cellular recording and labelling techniques. Recordings were obtained from 70 VPL cells, including 65 cells responsive to cutaneous electrical stimulation of the hindpaw. Auditory or visual alone stimulation did not elicit cell activity except in three bi-modal cells and one auditory cell. Cross-modal alterations of somatosensory response by auditory and/or visual stimulation were recognized in 61 cells with regard to the response magnitude, latency (time and jitter) and/or burst spiking properties. Both early (onset) and late responses were either suppressed or facilitated, and de novo cell activity was also induced. Cross-modal alterations took place depending on the temporal interval between the preceding counterpart and somatosensory stimulations, the intensity and frequency of sound. Alterations were observed mostly at short intervals (< 200 ms) and up to 800 ms intervals. Sounds of higher intensities and lower frequencies were more effective for modulation. The susceptibility to cross-modal influences was related to cell location and/or morphology. These and previously reported similar findings in the auditory and visual thalamic nuclei suggest that cross-modal sensory interactions pervasively take place in the first-order sensory thalamic nuclei.

Investigation of stratum corneum cell morphology and content using novel machine-learning image analysis.

BACKGROUND: The morphology and content of stratum corneum (SC) cells provide information on the physiological condition of the skin. Although the morphological and biochemical properties of the SC are known, no method is available to fully access and interpret this information. This study aimed to develop a method to comprehensively decode the physiological information of the skin, based on the SC. Therefore, we established a novel image analysis technique based on artificial intelligence (AI) and multivariate analysis to predict skin conditions. MATERIALS AND METHODS: SC samples were collected from participants, imaged, and annotated. Nine biomarkers were measured in the samples using enzyme-linked immunosorbent assay. The data were then used to teach machine-learning models to recognize individual SC cell regions and estimate the levels of the nine biomarkers from the images. Skin physiological indicators (e.g., skin barrier function, facial analysis, and questionnaires) were measured or obtained from the participants. Multivariate analysis, including biomarker levels ​​and structural parameters of the SC as variables, was used to estimate these physiological indicators. RESULTS: We established two machine-learning models. The accuracy of recognition was assessed according to the average intersection over union (0.613), precision (0.953), recall (0.640), and F-value (0.766). The predicted biomarker levels significantly correlated with the measured levels. Skin physiological indicators and questionnaire answers were predicted with strong correlations and correct answer rates. CONCLUSION: Various physiological skin conditions can be predicted from images of the SC using AI models and multivariate analysis. Our method is expected to be useful for dermatological treatment optimization.

Serum Procalcitonin, Hematology Parameters, and Cell Morphology in Multiple Clinical Conditions and Sepsis.

BACKGROUND: The clinical value of procalcitonin (PCT) in infection diagnosis and antibiotic stewardship is still unclear. This study aimed to investigate the association between serum PCT and different clinical conditions as well as other infectious/inflammatory parameters in different septic patients in order to elucidate the value of PCT detection in infection management. METHODS: Chemiluminescence immunoassay was used for serum PCT analysis. Hematology analysis was used for complete blood cell count. Digital automated cell morphology analysis was used for blood cell morphology examination. Blood, urine, and stool cultures were performed according to routine clinical laboratory standard operating procedures. C-reactive protein (CRP) was analyzed by immunoturbidimetry. Erythrocyte sedimentation rate test was performed using natural sedimentation methods. RESULTS: Outpatients, ICU patients, and patients under 2 years of age with respiratory infections had higher serum PCT levels. Septic patients had the highest-serum PCT levels and other infection indexes. PCT levels in the blood, urine, and stool culture-positive patients were significantly higher than in culture-negative patients. The neutrophil granulation and reactive lymphocytes were observed together with the PCT-level increments in different septic patients, and these alterations were lessened after treatment. There was no significant change in monocyte morphology between pre- and posttreatment septic patients. CONCLUSIONS: Serum PCT is associated with neutrophil cytotoxicity and lymphocyte morphology changes in sepsis; thus, the combination of neutrophil and lymphocyte digital cell morphology evaluations with PCT detection may be a useful examination for guiding the clinical management of sepsis.

Elucidating the effect of TiO2 nanoparticles on mung bean rhizobia via in vitro assay: Influence on growth, morphology, and plant growth promoting traits.

Titanium dioxide nanoparticles (TiO2 NPs) are among the most commonly used nanomaterials and are most likely to end up in soil. Therefore, it is pertinent to study the interaction of TiO2 NPs with soil microorganisms. The present in vitro broth study evaluates the impacts of low-dose treatments (0, 1.0, 5.0, 10.0, 20.0, and 40.0 mg L-1 ) of TiO2 NPs on cell viability, morphology, and plant growth promoting (PGP) traits of rhizobia isolated from mung bean root nodule. Two types of TiO2 NPs, that is, mixture of anatase and rutile, and anatase alone were used in the study. These TiO2 NPs were supplemented in broth along with a multifunctional isolate (Bradyrhizobium sp.) and two reference cultures. The exposure of TiO2 (anatase+rutile) NPs at low concentrations (less than 20.0 mg L-1 ) enhanced the cell growth, and total soluble protein content, besides improving the phosphate solubilization, Indole-3-acetic acid (IAA) production, siderophore, and gibberellic acid production. The TiO2 (anatase) NPs enhanced exopolysaccharide (EPS) production by the test rhizobial cultures. The radical scavenging assay was performed to reveal the mode of action of the nano-TiO2 particles. The study revealed higher reactive oxygen species (ROS) generation by the TiO2 (anatase) NPs as compared with TiO2 (anatase+rutile) NPs. Exposure to TiO2 NPs also altered the morphology of rhizobial cells. The findings suggest that TiO2 NPs could act as promoters of PGP traits of PGP bacteria when applied at appropriate lower doses.

The role of oxygen tension in cell fate and regenerative medicine: implications of hypoxia/hyperoxia and free radicals.

Oxygen pressure plays an integral role in regulating various aspects of cellular biology. Cell metabolism, proliferation, morphology, senescence, metastasis, and angiogenesis are some instances that are affected by different tensions of oxygen. Hyperoxia or high oxygen concentration, enforces the production of reactive oxygen species (ROS) that disturbs physiological homeostasis, and consequently, in the absence of antioxidants, cells and tissues are directed to an undesired fate. On the other side, hypoxia or low oxygen concentration, impacts cell metabolism and fate strongly through inducing changes in the expression level of specific genes. Thus, understanding the precise mechanism and the extent of the implication of oxygen tension and ROS in biological events is crucial to maintaining the desired cell and tissue function for application in regenerative medicine strategies. Herein, a comprehensive literature review has been performed to find out the impacts of oxygen tensions on the various behaviors of cells or tissues.

The Unique N-Terminal Domain of Chlamydial Bactofilin Mediates Its Membrane Localization and Ring-Forming Properties.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen. In evolving to the intracellular niche, Chlamydia has reduced its genome size compared to other bacteria and, as a consequence, has a number of unique features. For example, Chlamydia engages the actin-like protein MreB, rather than the tubulin-like protein FtsZ, to direct peptidoglycan (PG) synthesis exclusively at the septum of cells undergoing polarized cell division. Interestingly, Chlamydia possesses another cytoskeletal element-a bactofilin ortholog, BacA. Recently, we reported BacA is a cell size-determining protein that forms dynamic membrane-associated ring structures in Chlamydia that have not been observed in other bacteria with bactofilins. Chlamydial BacA possesses a unique N-terminal domain, and we hypothesized this domain imparts the membrane-binding and ring-forming properties of BacA. We show that different truncations of the N terminus result in distinct phenotypes: removal of the first 50 amino acids (ΔN50) results in large ring structures at the membrane whereas removal of the first 81 amino acids (ΔN81) results in an inability to form filaments and rings and a loss of membrane association. Overexpression of the ΔN50 isoform altered cell size, similar to loss of BacA, suggesting that the dynamic properties of BacA are essential for the regulation of cell size. We further show that the region from amino acid 51 to 81 imparts membrane association as appending it to green fluorescent protein (GFP) resulted in the relocalization of GFP from the cytosol to the membrane. Overall, our findings suggest two important functions for the unique N-terminal domain of BacA and help explain its role as a cell size determinant. IMPORTANCE Bacteria use a variety of filament-forming cytoskeletal proteins to regulate and control various aspects of their physiology. For example, the tubulin-like FtsZ recruits division proteins to the septum whereas the actin-like MreB recruits peptidoglycan (PG) synthases to generate the cell wall in rod-shaped bacteria. Recently, a third class of cytoskeletal protein has been identified in bacteria-bactofilins. These proteins have been primarily linked to spatially localized PG synthesis. Interestingly, Chlamydia, an obligate intracellular bacterium, does not have PG in its cell wall and yet possesses a bactofilin ortholog. In this study, we characterize a unique N-terminal domain of chlamydial bactofilin and show that this domain controls two important functions that affect cell size: its ring-forming and membrane-associating properties.

Microglia contribute to cognitive decline in hypercholesterolemic LDLr-/- mice.

Familial hypercholesterolemia (FH) is caused by mutations in the gene that encodes the low-density lipoprotein (LDL) receptor, which leads to an excessive increase in plasma LDL cholesterol levels. Previous studies have shown that FH is associated with gliosis, blood-brain barrier dysfunction, and memory impairment, but the mechanisms associated with these events are still not fully understood. Therefore, we aimed to investigate the role of microgliosis in the neurochemical and behavioral changes associated with FH using LDL receptor knockout (LDLr-/- ) mice. We noticed that microgliosis was more severe in the hippocampus of middle-aged LDLr-/- mice, which was accompanied by microglial morphological changes and alterations in the immunocontent of synaptic protein markers. At three months of age, the LDLr-/- mice already showed increased microgliosis and decreased immunocontent of claudin-5 in the prefrontal cortex (PFC). Subsequently, 6-month-old male C57BL/6 wild-type and LDLr-/- mice were treated once daily for 30 days with minocycline (a pharmacological inhibitor of microglial cell reactivity) or vehicle (saline). Adult LDLr-/- mice displayed significant hippocampal memory impairment, which was ameliorated by minocycline treatment. Non-treated LDLr-/- mice showed increased microglial density in all hippocampal regions analyzed, a process that was not altered by minocycline treatment. Region-specific microglial morphological analysis revealed different effects of genotype or minocycline treatment on microglial morphology, depending on the hippocampal subregion analyzed. Moreover, 6-month-old LDLr-/- mice exhibited a slight but not significant increase in IBA-1 immunoreactivity in the PFC, which was reduced by minocycline treatment without altering microglial morphology. Minocycline treatment also reduced the presence of microglia within the perivascular area in both the PFC and hippocampus of LDLr-/- mice. However, no significant effects of either genotype or minocycline treatment were observed regarding the phagocytic activity of microglia in the PFC and hippocampus. Our results demonstrate that hippocampal microgliosis, microglial morphological changes, and the presence of these glial cells in the perivascular area, but not increased microglial phagocytic activity, are associated with cognitive deficits in a mouse model of FH.

Identification of a morphogene required for tapered filament termini in filamentous cyanobacteria.

Although the photosynthetic cyanobacteria are monophyletic, they exhibit substantial morphological diversity across species, and even within an individual species due to phenotypic plasticity in response to life cycles and environmental signals. This is particularly prominent among the multicellular filamentous cyanobacteria. One example of this is the appearance of tapering at the filament termini. However, the morphogenes controlling this phenotype and the adaptive function of this morphology are not well defined. Here, using the model filamentous cyanobacterium Nostoc punctiforme ATCC29133 (PCC73102), we identify tftA, a morphogene required for the development of tapered filament termini. The tftA gene is specifically expressed in developing hormogonia, motile trichomes where the tapered filament morphology is observed, and encodes a protein containing putative amidase_3 and glucosaminidase domains, implying a function in peptidoglycan hydrolysis. Deletion of tftA abolished filament tapering inidcating that TftA plays a role in remodelling the cell wall to produce tapered filaments. Genomic conservation of tftA specifically in filamentous cyanobacteria indicates this is likely to be a conserved mechanism among these organisms. Finally, motility assays indicate that filaments with tapered termini migrate more efficiently through dense substratum, providing a plausible biological role for this morphology.

Geometrical analysis identified morphological features of hydrogel-induced cancer stem cells in synovial sarcoma model cells.

Cancer stem cells (CSCs) has been a key target to cure cancer patients completely. Although many CSC markers have been identified, they are frequently cancer type-specific and those expressions are occasionally variable, which becomes an obstacle to elucidate the characteristics of the CSCs. Here we scrutinized the relationship between stemness elevation and geometrical features of single cells. The PAMPS hydrogel was utilized to create the CSCs from mouse myoblast C2C12 and its synovial sarcoma model cells. qRT-PCR analysis confirmed the significant increase in expression levels of Sox2, Nanog, and Oct3/4 on the PAMPS gel, which was higher in the synovial sarcoma model cells. Of note, the morphological heterogeneity was appeared on the PAMPS gel, mainly including flat spreading, elongated spindle, and small round cells, and the Sox2 expression was highest in the small round cells. To examine the role of morphological differences in the elevation of stemness, over 6,400 cells were segmented along with the Sox2 intensity, and 12 geometrical features were extracted at single cell level. A nonlinear mapping of the geometrical features by using uniform manifold approximation and projection (UMAP) clearly revealed the existence of relationship between morphological differences and the stemness elevation, especially for C2C12 and its synovial sarcoma model on the PAMPS gel in which the small round cells possess relatively high Sox2 expression on the PAMPS gel, which supports the strong relationship between morphological changes and the stemness elevation. Taken together, these geometrical features can be useful for morphological profiling of CSCs to classify and distinguish them for understanding of their role in disease progression and drug discovery.

Reducing Cancer Cell Adhesion using Microtextured Surfaces.

For the past century, trypsin has been the primary method of cell dissociation, largely without any major changes to the process. Enzymatic cell detachment strategies for large-scale cell culturing processes are popular but can be labor-intensive, potentially lead to the accumulation of genetic mutations, and produce large quantities of liquid waste. Therefore, engineering surfaces to lower cell adhesion strength could enable the next generation of cell culture surfaces for delicate primary cells and automated, high-throughput workflows. In this study, a process for creating microtextured polystyrene (PS) surfaces to measure the impact of microposts on the adhesion strength of cells is developed. Cell viability and proliferation assays show comparable results in two cancer cell lines between micropost surfaces and standard cell culture vessels. However, cell image analysis on microposts reveals that cell area decreases by half, and leads to an average twofold increase in cell length per area. Using a microfluidic-based method up to a seven times greater percentage of cells are removed from micropost surfaces than the flat control surfaces. These results show that micropost surfaces enable decreased cell adhesion strength while maintaining similar cell viabilities and proliferation as compared to flat PS surfaces.

Spatial molecular profiling: platforms, applications and analysis tools.

Molecular profiling technologies, such as genome sequencing and proteomics, have transformed biomedical research, but most such technologies require tissue dissociation, which leads to loss of tissue morphology and spatial information. Recent developments in spatial molecular profiling technologies have enabled the comprehensive molecular characterization of cells while keeping their spatial and morphological contexts intact. Molecular profiling data generate deep characterizations of the genetic, transcriptional and proteomic events of cells, while tissue images capture the spatial locations, organizations and interactions of the cells together with their morphology features. These data, together with cell and tissue imaging data, provide unprecedented opportunities to study tissue heterogeneity and cell spatial organization. This review aims to provide an overview of these recent developments in spatial molecular profiling technologies and the corresponding computational methods developed for analyzing such data.

Clinical and biochemical footprints of inherited metabolic disease. XVI. Hematological abnormalities.

Many classical inherited metabolic diseases (IMDs) are associated with significant hematological complications such as anemia or thrombosis. While these may not be the prominent presenting feature of these conditions, management of these issues is important for optimal outcomes in people with IMDs. Some disorders that are included in the nosology of inherited metabolic disorders, such as inherited disorders of red cell energy metabolism, have purely hematological features, and have typically been cared for by a hematologist. In the 16th issue of the Footprints series, we identified 265 IMDs associated with hematological abnormalities. We review the major hematological manifestations of IMDs, suggest further investigation of hematological findings, and discuss treatment options available for specific hematological complications of IMDs.

The fate of adipose tissue and adipose-derived stem cells in allograft.

Utilizing adipose tissue and adipose-derived stem cells (ADSCs) turned into a promising field of allograft in recent years. The therapeutic potential of adipose tissue and ADSCs is governed by their molecular secretions, ability to sustain multi-differentiation and self-renewal which are pivotal in reconstructive, genetic diseases, and cosmetic goals. However, revisiting the existing functional capacity of adipose tissue and ADSCs and their intricate relationship with allograft is crucial to figure out the remarkable question of safety to use in allograft due to the growing evidence of interactions between tumor microenvironment and ADSCs. For instance, the molecular secretions of adipose tissue and ADSCs induce angiogenesis, create growth factors, and control the inflammatory response; it has now been well determined. Though the existing preclinical allograft studies gave positive feedback, ADSCs and adipose tissue are attracted by some factors of tumor stroma. Moreover, allorecognition is pivotal to allograft rejection which is carried out by costimulation in a complement-dependent way and leads to the destruction of the donor cells. However, extensive preclinical trials of adipose tissue and ADSCs in allograft at molecular level are still limited. Hence, comprehensive immunomodulatory analysis could ensure the successful allograft of adipose tissue and ADSCs avoiding the oncological risk.

Functional assessment of donated human embryos for the generation of pluripotent embryonic stem cell lines.

RESEARCH QUESTION: Can discarded embryos at blastocyst stage, donated to research because of genetic abnormalities and poor morphological quality, become a reliable source of human embryonic stem cell (HESC) lines? DESIGN: This study was consecutively conducted with 23 discarded embryos that were donated to research between February 2020 and April 2021. All embryos, except one, were morphologically evaluated and underwent trophectoderm biopsy for preimplantation genetic testing using next-generation sequencing (NGS), and then vitrified. After warming, the embryos were placed in appropriate culture conditions for the generation of HESCs, which was functionally assessed with immunofluorescence and flow cytometry for pluripotency capacity and spontaneous in-vitro differentiation. Cytogenetic assessment of the HESC was conducted with multiplex ligation-dependent probe amplification, and micro array comparative genomic hybridization. RESULTS: From the 23 embryos initially included, 17 survived warming, and 16 of them presented viability. Overall, the embryos presented poor morphological quality after warming. Only the previously untested embryo was capable of generating a new HESC line. Further characterization of this line revealed fully functional, euploid HESCs with preserved pluripotency, becoming a useful resource for research into human development and therapeutic investigation. CONCLUSIONS: None of the donated blastocysts with poor morphological quality in association with genetic abnormalities detected by NGS had the capacity for further in-vitro expansion to originate pluripotent HESC lines. This finding seems to provide extra support to genetic counselling on the suitability of this type of embryo for clinical use.

Quantitative features to assist in the diagnostic assessment of chronic lymphocytic leukemia progression†.

The use of artificial intelligence methods in the image-based diagnostic assessment of hematological diseases is a growing trend in recent years. In these methods, the selection of quantitative features that describe cytological characteristics plays a key role. They are expected to add objectivity and consistency among observers to the geometric, color, or texture variables that pathologists usually interpret from visual inspection. In a recent paper in The Journal of Pathology, El Hussein, Chen et al proposed an algorithmic procedure to assist pathologists in the diagnostic evaluation of chronic lymphocytic leukemia (CLL) progression using whole-slide image analysis of tissue samples. The core of the procedure was a set of quantitative descriptors (biomarkers) calculated from the segmentation of cell nuclei, which was performed using a convolutional neural network. These biomarkers were based on clinical practice and easily calculated with reproducible tools. They were used as input to a machine learning algorithm that provided classification in one of the stages of CLL progression. Works like this can contribute to the integration into the workflow of clinical laboratories of automated diagnostic systems based on the morphological analysis of histological slides and blood smears. © 2021 The Pathological Society of Great Britain and Ireland.

Review on Cell Structure Regulation and Performances Improvement of Porous Poly(Lactic Acid).

Recent advances in the cell structure regulation and performances improvement of porous poly(lactic acid) materials (PPMs) are systematically reviewed in this feature article. First, the typical processing methods, including template method, non-solvent induced phase separation, freeze-drying, and supercritical CO2  foaming, of PPMs are introduced emphatically. Their various cell morphologies by different processing methods are summarized: finger-like, honeycomb-like, fiber-like, through cell, open cell, closed cell, ball-like, and flower-like. Meanwhile, the transformation among different cell morphologies as well as the changes in cell size and cell density, having impact on the performances, is described. Second, the influence of stereo-complex crystals on the cell structure of PPMs is emphatically reviewed. Furthermore, the relationships between cell structure and properties that includes mechanical properties, thermal stability, heat insulation, and hydrophobicity, are elaborated. Eventually, the issues of PPMs worthy of further study are discussed.

Comparative analysis of thioflavin T and other fluorescent dyes for fluorescent staining of Bacillus subtilis vegetative cell, sporulating cell, and mature spore.

Thioflavin T, a cationic benzothiazole dye, is typically used to detect amyloid fibrils. In this study, we analyzed the staining properties of Bacillus subtilis cells using several fluorescent dyes, including thioflavin T analogs, 2-(4'-methylaminophenyl) benzothiazole (BTA-1), and 2-(4-aminophenyl) benzothiazole (APBT). Thioflavin T stained vegetative cells in the early log phase and outer layer structures of forespores and mature spores. The inner parts of forespores and heat-killed mature spores were also stained with thioflavin T. Congo red, auramine O, and rhodamine B stained forespores and mature spores similar to thioflavin T. In contrast, APBT and BTA-1 fluorescence was detected in the outer layers of vegetative cells, mother cells, forespores, and mature spores, indicating that they bind to the cell membrane and/or cell wall. The combination of the fluorescent dyes used in this study will help analyze morphogenetic processes during the sporulation and the damage mechanisms of vegetative cells and spores.