Differential Interference Contrast Research Articles

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

An exploratory study on the effect of kynurenine metabolites on sEnd-2 endothelioma cells.

Cancer is the second leading cause of mortality worldwide. The development of anticancer therapy plays a crucial role in mitigating tumour progression and metastasis. Epithelioid hemangioendothelioma is a very rare cancer, however, with a high systemic involvement. Kynurenine metabolites which includel-kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and quinolinic acid have been shown to inhibit T-cell proliferation resulting in a decrease in cell growth of natural killercells and T cells. Furthermore, metabolites such as l-kynurenine have been shown to inhibit proliferation of melanoma cells in vitro. Considering these metabolite properties, the present study aimed to explore the in vitro effects of l-kynurenine, quinolinic acid and kynurenic acid on endothelioma sEnd-2 cells and on endothelial (EA. hy926 cells) (control cell line). The in vitro effect at 24, 48, and 72 h exposure to a range of 1-4 mM of the respective kynurenine metabolites on the two cell lines in terms of cell morphology, cell cycle progression and induction of apoptosis was assessed. The half inhibitory concentration (IC50), as determined using nonlinear regression, for l-kynurenine, quinolinic acid and kynurenic acid was 9.17, 15.56, and 535.40 mM, respectively. Optical transmitted light differential interference contrast and hematoxylin and eosin staining revealed cells blocked in metaphase, formation of apoptotic bodies and compromised cell density in l-kynurenine-treated cells. A statistically significant increase in the number of cells present in the sub-G1 phase was observed in l-kynurenine-treated sample. To our knowledge, this was the first in vitro study conducted to investigate the mechanism of action of kynurenine metabolites on endothelioma sEnd-2 cells. It can be concluded that l-kynurenine exerts an antiproliferative effect on the endothelioma sEnd-2 cell line by decreasing cell growth and proliferation as well as a metaphase block. These hallmarks suggest cell death via apoptosis. Further research will be conducted on l-kynurenine to assess the effect on cell adhesion in vitro and in vivo as cell-cell adhesion has been shown to increase metastasis to distant organs therefore, the inhibition of adhesion may lead to a decrease in metastasis.

The In Vitro Effect of Kynurenine Metabolites on Cell morphology, Cell cycle progression and the Induction of Apoptosis in Tumour Endothelioma Cells

Background: Cancer is one of the leading causes of death worldwide. The development of anticancer therapies plays a crucial role in mitigating tumour progression and metastasis. Kynurenine metabolites which include L-kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and quinolinic acid have been shown to inhibit T-cell proliferation resulting in a decrease in cell growth of natural killer (NK) cells and T cells. Research showed that L-kynurenine inhibits proliferation of melanoma cells in vitro. Methods: The present study aimed to explore the in vitro influence of L-kynurenine, quinolinic acid and kynurenic acid on endothelioma sEnd-2 cells at concentration ranges of 1 mM – 4 mM for 24 h, 48 h and 72 h on cell morphology, cell cycle progression and induction of apoptosis. Results and Discussion: The half inhibitory concentration (IC50), as determined using GraphPad Prism, for L-kynurenine, quinolinic acid and kynurenic acid was 10.77 mM, 14.78 mM and 535.40 mM respectively. Optical transmitted light differential interference contrast and hematoxylin and eosin staining revealed the presence of cells blocked in metaphase, formation of apoptotic bodies and compromised cell density in L-kynurenine- and quinolinic acid- treated cells. A statistically significant increase in the number of cells present in the sub-G1 phase was observed in L-kynurenine-treated samples. Conclusion: It can be concluded that L-kynurenine exerts an antiproliferative effect on the sEnd-2 cell line by a decrease in cell growth and proliferation and metaphase block. These hallmarks suggest cell death via apoptosis. Further research will be conducted on L-kynurenine to assess the effect on cell adhesion in vitro and in vivo. This research was supported by the following grants: The Research Development Programme at the University of Pretoria awarded to Dr YN Hlophe; National Research Foundation awarded to Dr YN Hlophe (A1F4685 & N1F580); The University Capacity Development Programme (UCDP) awarded to Ms Tatchum (A1D783 & N1F120). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparing the laser-induced damage distribution in POFs with raytracing simulations

The understanding of the laser-induced damage behavior in polymer optical materials is of high interest to prevent their damage and to increase the laser damage resistance of optical components. Moreover, compared with optical components made from high-quality glass materials, nanosecond laser-induced damage for wavelengths in the visible and near-infrared (NIR) occurs inside the bulk material of PMMA and not at its surface. This phenomenon complicates the determination of the laser-induced damage threshold (LIDT) in PMMA fibers. Since the bulk material itself determines the LIDT, knowledge of the intensity distribution in the multimode fiber is of utmost importance. Our fibers were irradiated at a wavelength of 532 nm with an ns-pulsed laser system with a 10 Hz repetition rate. To investigate the damage behavior in polymer optical fibers, we applied different imaging and analysis techniques. To our knowledge, those techniques are used here for the first time in order to study damaged polymer materials. With the help of a Nomarski microscope, axial and radial damage distributions within the multimode PMMA fibers were determined and compared with ray-tracing simulations of the intensity distribution within the optical fiber. Moreover, extruded PMMA plates were irradiated with the aim of comparing the damage behavior of materials with different manufacturing. In addition, the planar geometry of the plates allows for a more reliable application of the different measurement methods. Overall, investigations with a thermal imaging camera and EDX analysis indicate that the damage behavior of polymer optical material is thermally driven during the ns-pulsed irradiation. Furthermore, voids are formed during the damaging process within the polymer optical fibers and plates, as indicated by both SEM images and X-ray computed microtomography (µ-CT) investigations. Finally, we investigated the damages in fiber preforms and PMMA plates using two photon-microscopy. By doing this, we detected fluorescence signals from the damaged material, indicating that the damage process leads to a major modification of the polymer.

Quantitative phase imaging by gradient retardance optical microscopy

Quantitative phase imaging (QPI) has become a vital tool in bioimaging, offering precise measurements of wavefront distortion and, thus, of key cellular metabolism metrics, such as dry mass and density. However, only a few QPI applications have been demonstrated in optically thick specimens, where scattering increases background and reduces contrast. Building upon the concept of structured illumination interferometry, we introduce Gradient Retardance Optical Microscopy (GROM) for QPI of both thin and thick samples. GROM transforms any standard Differential Interference Contrast (DIC) microscope into a QPI platform by incorporating a liquid crystal retarder into the illumination path, enabling independent phase-shifting of the DIC microscope's sheared beams. GROM greatly simplifies related configurations, reduces costs, and eradicates energy losses in parallel imaging modalities, such as fluorescence. We successfully tested GROM on a diverse range of specimens, from microbes and red blood cells to optically thick (~ 300 μm) plant roots without fixation or clearing.

Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Interfaces-engineered M-structure for infrared detectors

In this paper, the authors report strain-balanced M-structures InAs/GaSb/AlSb/GaSb superlattice growth on GaSb substrates using two kinds of interfaces (IFs): GaAs-like IFs and InSb-like IFs. The in-plane compressive strain of 60-period and 100-period InAs��/GaSb/AlSb��/GaSb with different InAs (��) and AlSb (��) monolayers are investigated. The M-structures InAs/GaSb/AlSb/GaSb represent type II superlattices (T2SL) and at present are under intensive investigation. Many authors show theoretical and experimental results that such structures can be used as a barrier material for a T2SL InAs/GaSb absorber tuned for long-wave infrared detectors (8 μm–14 μm). Beside that, M-structure can also be used as an active material for short-wave infrared detectors to replace InAs/GaSb which, for this region of infrared, are a big challenge from the point of view of balancing compression stress. The study of InAs/GaSb/AlSb/GaSb superlattice with the minimal strain for GaSb substrate can be obtained by a special procedure of molecular beam epitaxy growth through special shutters sequence to form both IFs. The authors were able to achieve smaller minimal mismatches of the lattice constants compared to literature. The high-resolution X-ray diffraction measurements prove that two types of IFs are proper for balancing the strain in such structures. Additionally, the results of Raman spectroscopy, surface analyses of atomic force microscopy, and differential interference contrast microscopy are also presented. The numerical calculations presented in this paper prove that the presence of IFs significantly changes the energy gap in the case of the investigated M-structures. The theoretical results obtained for one of the investigated structures, for a specially designed structure reveal an extra energy level inside the energy gap. Moreover, photoluminescence results obtained for this structure prove the good quality of the synthesized M-structures, as well as are in a good agreement with theoretical calculations.

Quantification of cell shape, intracellular flows and transport based on DIC object detection and tracking.

Computational image analysis combined with label-free imaging has helped maintain its relevance for cell biology, despite the rapid technical improvements in fluorescence microscopy with the molecular specificity of tags. Here, we discuss some computational tools developed in our lab and their application to quantify cell shape, intracellular organelle movement and bead transport in vitro, using differential interference contrast (DIC) microscopy data as inputs. The focus of these methods is image filtering to enhance image gradients, and combining them with segmentation and single particle tracking (SPT). We demonstrate the application of these methods to Escherichia coli cell length estimation and tracking of densely packed lipid granules in Caenorhabditis elegans one-celled embryos, diffusing beads in solutions of different viscosities and kinesin-driven transport on microtubules. These approaches demonstrate how improvements to low-level image analysis methods can help obtain insights through quantitative cellular and subcellularmicroscopy.

A new species of Agauopsis (Acari, Halacaridae) from South Korea belonging to A. brevipalpus group

A new marine halacarid mite of the genus Agauopsis Viets, 1927 is recorded from mussels in the intertidal bedrock of the southern coast of South Korea. Agauopsis bisetosa n. sp. belonging to the A. brevipalpus group, is characterized by an ‘H’-shaped areola on the anterior dorsal plate, two pairs of costae on the posterior dorsal plate, a tectum with a truncate anterior margin, posterior epimeral plate with two ventral setae, telofemur I with one ventral and two ventromedial dentate spines, and tibia II with one ventral and one ventromedial dentate spines. Notably, the presence of two ventral setae on the posterior epimeral plate is a unique characteristic of the new species. This study deals with a description of A. bisetosa n. sp., accompanied by detailed illustrations drawn under a differential interference contrast microscope and scanning electron microscope photomicrographs. Additionally, it includes a character comparison table of the A. brevipalpus group and a key for known Agauopsis species in the northwest Pacific, both including the new species.

The Mechanism of Ovule Abortion in Self-Pollinated 'Hanfu' Apple Fruits and Related Gene Screening.

Apples exhibit S-RNase-mediated self-incompatibility and typically require cross-pollination in nature. 'Hanfu' is a cultivar that produces abundant fruit after self-pollination, although it also shows a high rate of seed abortion afterwards, which greatly reduces fruit quality. In this study, we investigated the ovule development process and the mechanism of ovule abortion in apples after self-pollination. Using a DIC microscope and biomicroscope, we found that the abortion of apple ovules occurs before embryo formation and results from the failure of sperm-egg fusion. Further, we used laser-assisted microdissection (LAM) cutting and sperm and egg cell sequencing at different periods after pollination to obtain the genes related to ovule abortion. The top 40 differentially expressed genes (DEGs) were further verified, and the results were consistent with switching the mechanism at the 5' end of the RNA transcript (SMART-seq). Through this study, we can preliminarily clarify the mechanism of ovule abortion in self-pollinated apple fruits and provide a gene reserve for further study and improvement of 'Hanfu' apple fruit quality.

Single-exposure quantitative differential interference contrast microscopy using bandlimited image and its Fourier transform constraints

Phase microscopy that records the bandlimited image and its Fourier image simultaneously (BIFT) is a phase retrieval method with unique and rapid convergence. In this paper, we present a single-exposure quantitative differential interference contrast (DIC) microscopy based on BIFT method. The contrasts of the recorded DIC image and its Fourier image, analyzed by simulation and experiment, can be largely improved by the initial phase difference between two sheared lights (bias), however their trends with biases are opposite. By adding the optimized bias with the compromise of the contrasts in image and Fourier space, the phase sensitivity can be improved than BIFT method only. We have experimentally demonstrated that a sample of 25 nm height can be successfully recovered from a single exposure. The presented single-exposure quantitative DIC microscopy provides a promising technique for real-time phase imaging.

Abstract 2306: A deep learning framework for automated segmentation and analysis of 3D breast cancer spheroid invasion dynamics

Abstract Invasion of neighboring tissues is a cardinal feature of malignancy, notably observed in aggressive cancers like breast cancer, where it can lead to significant morbidity. To explore cancer invasion dynamics, the implementation of a three-dimensional (3D) tumor spheroid invasion assay offers a swift approach to mimicking a tumor micro-region or micro-metastasis. Differential Interference Contrast (DIC) time-lapse imaging was chosen for its fluorescence-free and non-destructive advantages in capturing live spheroid movement. However, the subsequent analysis posed challenges, including out-of-focus cells and a voluminous dataset with diverse microscope planes and time points. Our study introduces a segmentation framework featuring in-focus spatial stacking algorithm and novel training architecture that enabling in-depth automated analysis of 3D spheroid invasion behaviors within a microenvironment. Firstly, the MDA-MB-231 breast cancer spheroids were cultured in a microwell dish and then incorporated into a collagen type I matrix for the 3D spheroid invasion assay. Capturing spheroid invasion dynamics involved acquiring 15 z-plane images (spaced at 20 µm intervals) per timepoint for each spheroid using DIC microscopy at 15-minute intervals over a 28-hour period, employing a 20X objective lens. We refined the Focus Stacking framework into Multiple Slices Blurry Stacking (MSB-Stack) by pre-filtering blurred areas before stacking images. This process produced 6390 slices consolidated into 426 labeled stacked images, highlighting Invasive area, Spheroid core, and Invasive cells post-invasion. We then enhanced Mask R-CNN (Region based-Convolutional Neural Networks) with a weak-to-strong augmentation mechanism, leveraging well-segmented samples from MSB-Stack for training on both fully stacked and blurred images. Blurry Consistency Mask R-CNN (BCMask R-CNN) demonstrates overall improvement across feature extractors, outperforming Mask R-CNN. Using Mean average precision (mAP) as the main metric for instance segmentation and detection, our proposed model achieves 73.0% and 65.2% on mAP @[0.5, 0.95], showcasing robustness. Notably, our best results for mAP @0.5 reach 96.6% and 96.7% on both tasks, underscoring the effectiveness of consistency training in mitigating unclear boundary issues during preprocessing. In conclusion, our proposed method, MSB-Stack, effectively addresses the challenge of unclear boundaries in 3D breast cancer cell DIC microscopy datasets. The integration of weak-to-strong consistency training in our model, BCMask R-CNN, not only mitigates biases but also enhances performance across different backbones, setting the stage for future advancements in cell-tracking tasks and broader migratory analyses. Citation Format: Thi Kim Ngan Ngo, Thanh-Huy Nguyen, Fang-Yu Lin, Ting-Yuan Tu. A deep learning framework for automated segmentation and analysis of 3D breast cancer spheroid invasion dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2306.

Abstract 1505: Unraveling diepafitaxis: Investigating ECM stiffness and microarchitecture in tumor spheroid migration

Abstract Background: Metastasis, accounting for up to 90% of cancer-related deaths, remains one of the least understood aspects of cancer progression. Recent research has shifted focus from solely biological hallmarks to the physical characteristics of cancer, such as the stiffness and microarchitecture of the extracellular matrix (ECM), which play pivotal roles in metastatic advancement. We previously introduced the concept of 'diepafitaxis' to describe a unique mechanism driving the interface-mediated tropism of tumor spheroids at matrix-surface boundaries. This phenomenon suggests that cancer cells preferentially migrate along a 2D 'interfacial' track formed by heterogeneous 3D ECM structures, facilitating invasion and dispersion. However, the exact mechanisms by which interfacial components, stiffness, and structural attributes influence cell movement remain debated. To investigate this, we have developed a microfluidic chip with a precisely controlled stiffness of the hydrogel matrix-matrix interface (MMI), aiming to shed light on specific diepafitatic behaviors. Method: We developed a polydimethylsiloxane (PDMS)-based microfluidic chip using a traditional photolithographic process. This chip features three channels, with the center channel designed to precisely control various MMI stiffness combinations: stiff vs soft (t-o), stiff vs stiff (t-t) and soft vs soft (o-o). MDA-MB-231 breast cancer spheroids (BCS), representing an in vitro tumor model, were embedded in the top gel. We observed the dynamic behavior of these cancer spheroids over a 24-h period using differential interference contrast (DIC) microscopy. Result: The study revealed that BCS exhibited more pronounced dispersion in a soft homogeneous ECM compared to a stiff ECM in 3D environments. Interestingly, when BCS were near an MMI, a stiffer MMI (t-t) significantly enhanced BCS invasiveness and single-cell migration, more so than t-o, and notably more than o-o. We previously described this phenomenon as '2.5D migration,' where cells migrate in a ventral 2D manner within a sandwiched 3D gel structure. This diepafitatic behavior was further analyzed for its response to gravitational effects and the influence of chemotaxis, induced by introducing VEGF on one side of the microfluidic channel. Conclusion: Conclusively, our study demonstrates that the stiffness and structure of the ECM, particularly at MMI, significantly influence the invasive behavior and migration patterns of BCS, underscoring the critical role of mechanical microenvironments in cancer metastasis. Citation Format: Hsiang-Pei Chen, Ya-Chu Tsai, Ting-Yuan Tu. Unraveling diepafitaxis: Investigating ECM stiffness and microarchitecture in tumor spheroid migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1505.

Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Larval Neuromuscular Junction.

Focal recording is an extracellular method for studying synaptic transmission at the Drosophila larval neuromuscular junction (NMJ) designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all the boutons as occurs with intracellular recording or two-electrode voltage-clamp. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons. A compound microscope with epifluorescent imaging capability is very helpful but not essential; any GFP Drosophila strain that labels the nerve terminal or synaptic boutons can be used to locate the boutons. A particularly useful strain is Mhc-CD8-Sh-GFP, containing a GFP molecule that is expressed in muscle, localizes to the postsynaptic apparatus, and outlines boutons. Vital fluorescent dyes (such as 4-Di-2-Asp) may also be applied to the dissected preparation to help locate boutons. The microscope should be equipped for differential interference contrast (DIC or Nomarski) optics if fluorescence is not used.

Isolation of phase edges using off-axis q-plate filters.

Edge-enhanced microscopes with a q-plate have attracted more attention to enhance the edges of phase-amplitude objects in biological samples due to their capacity for all-directional edge enhancement, while differential interference-contrast microscopy enhances edges in only one-direction. However, the edge-enhanced microscopes cannot distinguish the edges of phase and amplitude objects, as both edges are equally enhanced. This study introduces a novel method for isolating the edge of a phase object from an amplitude object using an off-axis q-plate filter in a 4f system. Herein, we combined off-axis q-plates with four different displacements to isolate the phase object edge from the amplitude object. To demonstrate the proposed method, we conducted experiments using two distinct samples. The first sample comprised a phase test target surrounded by an aperture, and the second sample involved an overlap between the phase test target and a white hair with non-zero transmittance. In the samples, the isolated phase object edge is in good agreement with the theoretical expectations, and the amplitude object edge was reduced by approximately 93%. The proposed method is a novel and effective approach for isolating the edge of a phase object from an amplitude object and can be useful in various biological imaging applications.

Modified diff-quick staining for canine sperm morphology

Sperm morphology assessment requires specialized microscopes or stains. Diff-quick (DQ) is considered a universal stain that is cost-effective; however, morphological evaluation of sperm using DQ staining is poor and not encouraging. Therefore, this study investigated modifications to the DQ protocol to improve identification of morphological defects of dog sperm and compared the modified DQ techniques with eosin-nigrosin, Karras, and differential interference contrast microscopy [DIC]). One ejaculate from each of 9 dogs was used. To perform the proposed modified DQ techniques (DQ1 and DQ2), dried semen smears were fixed by immersing for 10 seconds in solution 1 of DQ and 5 minutes each in solutions II and III of the kit. After the third stain solution, slides in DQ1 were rinsed in water whereas slides in DQ2 were not rinsed but were vertically supported to facilitate stain drainage. Results suggested that the standard DQ protocol overestimated normal sperm and detached heads whereas underestimated abnormal heads and total defects compared to DIC, Karras, eosin-nigrosin, and DQ2. Acrosome abnormalities were only detectable with Karras, DIC, and DQ2. In conclusion, prolonging exposure to DQ staining solutions enhanced sensitivity in sperm morphological evaluation, and avoiding rinse as a final step in the DQ protocol improved visualization of certain acrosome defects in dog sperm. Therefore, modified DQ techniques can serve as a viable alternative for dog sperm morphology evaluation in clinical practice.

Morphological and Molecular Characterization of Two New Species of Ceratomyxa Thélohan, 1892 (Cnidaria: Myxosporea) from the Marine Ornamental Fish Zanclus cornutus (Linnaeus, 1758) off Lakshadweep Islands, Arabian Sea.

The present study provides the complete morphological and molecular description of two new species of myxosporeans, Ceratomyxa zancli n. sp. and Ceratomyxa cornuti n. sp. infecting the gallbladder of Zanclus cornutus from the Lakshadweep Islands, Arabian Sea. Zanclus cornutus were screened for the presence of myxosporeans, and the recovered myxospores were morphologically characterized using Nomarski Differential Interference Contrast (DIC) optics. The sequences of SSU rDNA were employed for molecular and phylogenetic studies. Both the parasites exhibited a prevalence of 21% each. C. zancli n. sp. is characterized by broadly cresentic myxospores with convex anterior and slightly concave to straight posterior margins and rounded ends. Spore valves two, unequal, measured 9.6 ± 0.7μm × 25.2 ± 1.3μm. Polar capsules two, unequal, spherical, measured 4 ± 0.6μm × 3.5 ± 0.6μm. Polar filament exceptionally long and arranged irregularly. Myxospores of C. cornuti n. sp. are elongated with convex anterior and slightly concave to straight posterior margins. Spore valves two, unequal, measured 7.00 ± 0.4μm × 26.56 ± 1.8μm. Polar capsules spherical, unequal, measured 3.52 ± 0.2 × 3.36 ± 0.35. Molecular analysis of C. zancli n. sp. (ON818297) and C. cornuti n. sp. (ON818298) resulted in 1469 and 1491bp long SSU rDNA sequences, respectively. Molecularly C. zancli n. sp. is close to C. diplodae and C. barnesi with 91.39% similarity, while C. cornuti n. sp. appears closer to C. robertsthomsoni with 97.46% similarity. In phylogenetic analyses, C. zancli n. sp. branched separately within the Ceratomyxa clade while C. cornuti n. sp. clustered with C. robertsthomsoni and C. thalassomae. Based on the differences in morphological, morphometric, molecular, and phylogenetic characteristics, as well as differences in the host and geographic location, the above two species of myxosporeans are considered novel. The study forms the first report of a species of Ceratomyxa from Z. cornutus.

Phosphorylation-Regulated Dynamic Phase Separation of HIP-55 Protects Against Heart Failure.

Heart failure (HF), which is the terminal stage of many cardiovascular diseases, is associated with low survival rates and a severe financial burden. The mechanisms, especially the molecular mechanism combined with new theories, underlying the pathogenesis of HF remain elusive. We demonstrate that phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 (hematopoietic progenitor kinase 1-interacting protein of 55 kDa) protects against HF. Fluorescence recovery after photobleaching assay, differential interference contrast analysis, pull-down assay, immunofluorescence, and immunohistochemical analysis were used to investigate the liquid-liquid phase separation capacity of HIP-55 and its dynamic regulation in vivo and in vitro. Mice with genetic deletion of HIP-55 and mice with cardiac-specific overexpression of HIP-55 were used to examine the role of HIP-55 on β-adrenergic receptor hyperactivation-induced HF. Mutation analysis and mice with specific phospho-resistant site mutagenesis were used to identify the role of phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 in HF. Genetic deletion of HIP-55 aggravated HF, whereas cardiac-specific overexpression of HIP-55 significantly alleviated HF in vivo. HIP-55 possesses a strong capacity for phase separation. Phase separation of HIP-55 is dynamically regulated by AKT-mediated phosphorylation at S269 and T291 sites, failure of which leads to impairment of HIP-55 dynamic phase separation by formation of abnormal aggregation. Prolonged sympathetic hyperactivation stress induced decreased phosphorylation of HIP-55 S269 and T291, dysregulated phase separation, and subsequent aggregate formation of HIP55. Moreover, we demonstrated the important role of dynamic phase separation of HIP-55 in inhibiting hyperactivation of the β-adrenergic receptor-mediated P38/MAPK (mitogen-activated protein kinase) signaling pathway. A phosphorylation-deficient HIP-55 mutation, which undergoes massive phase separation and forms insoluble aggregates, loses the protective activity against HF. Our work reveals that the phosphorylation-regulated dynamic phase separation of HIP-55 protects against sympathetic/adrenergic system-mediated heart failure.

Analysis of the growth of sickle hemoglobin fibers observed by DIC microscopy

Analysis of the growth of sickle hemoglobin fibers observed by DIC microscopy

Efficiency and novelty of using environmental swabs for dry-surface biofilm recovery.

Studies on the epidemiology of dry-surface biofilms (DSBs) within healthcare settings have shown an almost universal distribution across frequently touched items. Despite a growing body of evidence for DSBs in hospitals, little attention has been paid to the recovery capacity of techniques used to detect these microbial communities. Biofilms are inherently difficult to remove from surfaces due to adhesive substances within their matrix and may act as sources of infection, but to what extent is largely unknown. In this study, we evaluate the recovery efficiencies of commonly used environmental swabs against DSBs containing 7.24 log10 Acinetobacter baumannii cm-2, using a drip flow reactor and desiccation cycle. Biofilm presence was visually confirmed using episcopic differential interference contrast microscopy combined with epifluorescence and quantified using sonicated viable plate counts. The swab materials used comprised foam, viscose and cotton, all of which were pre-moistened using a buffer solution. The surfaces were vigorously swabbed by each material type and the resultant microbe populations for both swabs and remaining DSBs were quantified. Our results found foam-tipped swabs to be superior, detecting on average 30 % of the original DSB contamination; followed by viscose (6 %) and cotton (3 %). However, no distinct difference was revealed in the concentration of microbes remaining on the surface after swabbing for each swab type, suggesting there is variation in the capacity for each swab to release biofilm-associated micro-organisms. We conclude whilst environmental swabs do possess the ability to detect biofilms on dry surfaces, the reduced efficiencies are likely to cause an underestimation of the microbes present and should be considered during clinical application.