Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

NLRP3 inflammasome activity in RPE : role in AMD pathogenesis

Purpose: Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), an important cell type afflicted in AMD, undergoes cell death in the late stages of the disease. Salient factors underlying AMD pathogenesis are aging, drusen components and NLRP3 inflammasome activity. The purpose of this dissertation is to elucidate the molecular interactions among these factors and how they contribute to RPE damage. Methods: The effects of aging on drusen components, in particular the membrane attack complex (MAC) and amyloid beta (Aβ) were examined in rats at different age. To determine the role of MAC in inflammasome activation in RPE, aurin tricarboxylic acid complex (ATAC), was administrated to naïve rats. To understand Aβ’s role in inflammasome activation, Aβ intravitreal injections were made into rat eyes in vivo and Vinpocetine was used to ameliorate the inflammatory responses. An in vitro RPE cell culture model was established to further investigate the relationship between inflammasome and X-chromosome linked inhibitor of apoptosis (XIAP). Statistical significance was set at p ≤ 0.05. Results: An age-dependent increase in MAC, Aβ, and NF-κB activation was observed in the RPE-choroid in vivo. Blocking MAC formation with ATAC led to a prominent reduction in inflammasome activity (caspase-1 cleavage and cytokine secretion), but not in NF-κB activity. Aβ intravitreal injections triggered inflammasome activation evidenced by enhanced caspase-1 cleavage and IL-1β/IL-18 release, which was suppressed by Vinpocetine mediated NF-κB inhibition. The robust inflammasome activity further led to gasdermin D-mediated activation of the pyroptotic pathway and a significant reduction in XIAP, which in turn enhanced IL-18 secretion. Conclusion: Aging is a strong risk factor for AMD, which increases the deposition of MAC and Aβ in the outer retina. The elevated levels of MAC and Aβ are triggers for inflammasome activation. By demonstrating a causal relationship between inflammasome activation and XIAP reduction, this dissertation suggests the precise regulation of XIAP, together with the suppression of MAC and NF-κB, may be crucial for controlling inflammasome activity and hence provides new avenues to prevent AMD.Medicine, Faculty ofGraduat

A novel active contour model for unsupervised low-key image segmentation

Unsupervised image segmentation is greatly useful in many vision-based applications. In this paper, we aim at the unsupervised low-key image segmentation. In low-key images, dark tone dominates the background, and gray level distribution of the foreground is heterogeneous. They widely exist in the areas of space exploration, machine vision, medical imaging, etc. In our algorithm, a novel active contour model with the probability density function of gamma distribution is proposed. The flexible gamma distribution gives a better description for both of the foreground and background in low-key images. Besides, an unsupervised curve initialization method is designed, which helps to accelerate the convergence speed of curve evolution. The experimental results demonstrate the effectiveness of the proposed algorithm through comparison with the CV model. Also, one real-world application based on our approach is described in this paper

Application Progress of the Single Domain Antibody in Medicine

The camelid-derived single chain antibody (sdAb), also termed VHH or nanobody, is a unique, functional heavy (H)-chain antibody (HCAb). In contrast to conventional antibodies, sdAb is a unique antibody fragment consisting of a heavy-chain variable domain. It lacks light chains and a first constant domain (CH1). With a small molecular weight of only 12~15 kDa, sdAb has a similar antigen-binding affinity to conventional Abs but a higher solubility, which exerts unique advantages for the recognition and binding of functional, versatile, target-specific antigen fragments. In recent decades, with their unique structural and functional features, nanobodies have been considered promising agents and alternatives to traditional monoclonal antibodies. As a new generation of nano-biological tools, natural and synthetic nanobodies have been used in many fields of biomedicine, including biomolecular materials, biological research, medical diagnosis and immune therapies. This article briefly overviews the biomolecular structure, biochemical properties, immune acquisition and phage library construction of nanobodies and comprehensively reviews their applications in medical research. It is expected that this review will provide a reference for the further exploration and unveiling of nanobody properties and function, as well as a bright future for the development of drugs and therapeutic methods based on nanobodies

LogDet divergence-based metric learning with triplet constraints and its applications

How to select and weigh features has always been a difficult problem in many image processing and pattern recognition applications. A data-dependent distance measure can address this problem to a certain extent, and therefore an accurate and efficient metric learning becomes necessary. In this paper, we propose a LogDet divergence-based metric learning with triplet constraints (LDMLT) approach, which can learn Mahalanobis distance metric accurately and efficiently. First of all, we demonstrate the good properties of triplet constraints and apply it in LogDet divergence-based metric learning model. Then, to deal with high-dimensional data, we apply a compressed representation method to learn, store, and evaluate Mahalanobis matrix efficiently. Besides, a dynamic triplets building strategy is proposed to build a feedback from the obtained Mahalanobis matrix to the triplet constraints, which can further improve the LDMLT algorithm. Furthermore, the proposed method is applied to various applications, including pattern recognition, facial expression recognition, and image retrieval. The results demonstrate the improved performance of the proposed approach

Evidence for the activation of pyroptotic and apoptotic pathways in RPE cells associated with NLRP3 inflammasome in the rodent eye

Background: Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), the primary cell type that is afflicted in AMD, undergoes programmed cell death in the late stages of the disease. However, the exact mechanisms for RPE degeneration in AMD are still unresolved. The prevailing theories consider that each cell death pathway works independently and without regulation of each other. Building upon our previous work in which we induced a short burst of inflammasome activity in vivo, we now investigate the effects of prolonged inflammasome activity on RPE cell death mechanisms in rats. Methods: Long-Evans rats received three intravitreal injections of amyloid beta (Aβ), once every 4 days, and were sacrificed at day 14. The vitreous samples were collected to assess the levels of secreted cytokines. The inflammasome activity was evaluated by both immunohistochemistry and western blot. The types of RPE cell death mechanisms were determined using specific cell death markers and morphological characterizations. Results: We found robust inflammasome activation evident by enhanced caspase-1 immunoreactivity, augmented NF-κB nuclear translocalization, increased IL-1β vitreal secretion, and IL-18 protein levels. Moreover, we observed elevated proteolytic cleavage of caspase-3 and gasdermin D, markers for apoptosis and pyroptosis, respectively, in RPE-choroid tissues. There was also a significant reduction in the anti-apoptotic factor, X-linked inhibitor of apoptosis protein, consistent with the overall changes of RPE cells. Morphological analysis showed phenotypic characteristics of pyroptosis including RPE cell swelling. Conclusions: Our data suggest that two cell death pathways, pyroptosis and apoptosis, were activated in RPE cells after exposure to prolonged inflammasome activation, induced by a drusen component, Aβ. The involvement of two distinct cell death pathways in RPE sheds light on the potential interplay between these pathways and provides insights on the future development of therapeutic strategies for AMD.Medicine, Faculty ofOphthalmology and Visual Sciences, Department ofReviewedFacult

The reduction of XIAP is associated with inflammasome activation in RPE: implications for AMD pathogenesis

Background: Age-related macular degeneration (AMD) is a multifactorial chronic disease of the eye. Several candidate pathways have been hypothesized to play a role in AMD pathogenesis. Our work and those of others suggests inflammasome activity as a mechanism associated with retinal pigment epithelial (RPE) cell demise. X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptosis factor, has recently been shown to regulate inflammasome activity in non-ocular cells. The purpose of this study is to characterize XIAP’s regulatory role in RPE. Methods: Protein lysates of eye tissues from rats (vinpocetine- or aurin tricarboxylic acid complex-treated, ATAC, vs naïve) and mice (wild type vs Caspase-4−/−) were utilized to analyze XIAP protein levels. Immunohistochemistry was used to detect NLRP3 levels in the RPE layer. In vitro inflammasome activation on RPE cells was achieved with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) stimulation. Levels of XIAP mRNA and 18S RNA were quantified by RT-PCR. Cell culture supernatants were tested directly for secreted IL-1β by ELISA or concentrated for the detection of secreted IL-18 by western blot. Protein lysates from RPE in cell culture were collected for the measurement of cleaved caspase-1 p20, XIAP, and GAPDH. Data are presented as Mean ± SD. p < 0.05 is considered statistically significant. Results: The XIAP protein level was significantly increased when the inflammasome was inhibited at the “activation” step by ATAC, but not the “priming” step, in vivo. Concomitantly, NLRP3 immunoreactivity was lower in the RPE layer of animals fed with ATAC. In mice where caspase-1 cleavage was impaired by the genetic deficiency in caspase-4, the XIAP protein level increased in eye tissues. In RPE cell culture, Leu-Leu-OMe stimulation led to caspase-1 cleavage, cytokine secretion, and XIAP reduction, which can be abolished by Z-YVAD-FMK. When XIAP siRNA was given as a pre-treatment to RPE in vitro, Leu-Leu-OMe induced IL-1β/IL-18 secretion was enhanced, whereas overexpressing XIAP reduced IL-1β secretion under inflammasome activation, both compared to controls cells. Conclusions: Together, these data suggest XIAP-mediated inhibition of inflammasome activity in RPE may provide insights into the biological consequences of inflammasome activation in RPE and reveals the caspase-1/XIAP/IL-1β/IL-18 axis as a target for broader applications in AMD biology and treatment design.Medicine, Faculty ofOphthalmology and Visual Sciences, Department ofReviewedFacult

NLRP3 Inflammasome: Activation and Regulation in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly in industrialized countries. AMD is a multifactorial disease influenced by both genetic and environmental risk factors. Progression of AMD is characterized by an increase in the number and size of drusen, extracellular deposits, which accumulate between the retinal pigment epithelium (RPE) and Bruch’s membrane (BM) in outer retina. The major pathways associated with its pathogenesis include oxidative stress and inflammation in the early stages of AMD. Little is known about the interactions among these mechanisms that drive the transition from early to late stages of AMD, such as geographic atrophy (GA) or choroidal neovascularization (CNV). As part of the innate immune system, inflammasome activation has been identified in RPE cells and proposed to be a causal factor for RPE dysfunction and degeneration. Here, we will first review the classic model of inflammasome activation, then discuss the potentials of AMD-related factors to activate the inflammasome in both nonocular immune cells and RPE cells, and finally introduce several novel mechanisms for regulating the inflammasome activity