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[
Sci Total Environ,
2020]
This study aims to investigate the neuroprotective effects of 6-feruloylspinosin (6-FS), one of the main active flavonoid components in Sour Jujube seeds, on beta-amyloid (A) protein transgenic Caenorhabditis elegans (GMC101) and PC12 cells, and determine the molecular mechanism of its action. We found that 6-FS could ameliorate the progression of the Alzheimer's disease (AD) phenotype by delaying the aging, decreasing the rate of paralysis, enhancing resistance to heat stress, and increasing the chemotaxis ability, and promotes autophagy activity though autophagy/lysosome pathway in GMC101. Furthermore, 6-FS reduced A-induced toxicity by inhibiting the deposition of A and the aggregated proteins, increasing the level of mitophagy in PC12 through promoting the expression of Pink1/Parkin in the mitophagy pathway. Our findings suggest that 6-FS may be used as a medicinal supplement for treating AD.
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[
Nat Methods,
2023]
Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single-neuron resolution in model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate neurons with specific regeneration phenotypes from C. elegans. We present femtosecond laser microdissection (fs-LM), a single-cell isolation method that dissects specific cells directly from living tissue by leveraging the micrometer-scale precision of fs-laser ablation. We show that fs-LM facilitates sensitive and specific gene expression profiling by single-cell RNA sequencing (scRNA-seq), while mitigating the stress-related transcriptional artifacts induced by tissue dissociation. scRNA-seq of fs-LM isolated regenerating neurons revealed transcriptional programs that are correlated with either successful or failed regeneration in wild-type and
dlk-1 (0) animals, respectively. This method also allowed studying heterogeneity displayed by the same type of neuron and found gene modules with expression patterns correlated with axon regrowth rate. Our results establish fs-LM as a spatially resolved single-cell isolation method for phenotype-to-genotype mapping.
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J Biomed Opt,
2015]
The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.
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Kreeger, L., Arur, S., ZHAO, P., Ben-Yakar, A., Trimmer, K., Messing, R., Ma, K., Martin, C., Zemelman, B., Jiang, N., Maiya, R.
[
International Worm Meeting,
2019]
C. elegans has become a versatile system for studying in vivo nerve regeneration since the advent of precise laser axotomy method for severing specific axons. Through mutant and RNAi screening, a number of regeneration regulator genes have been identified. Nevertheless, their downstream effectors remain elusive. As a complementary approach, we propose to perform single-cell RNA-sequencing on regrowing neurons to capture the genome-wide dynamics underlying nerve regeneration. However, it has been technically unfeasible to isolate regrowing neurons from living C. elegans. The prevalent isolation method uses FACS to sort neurons of interest from chemo-mechanically dissociated animals, thus requires thousands of animals with synchronized nerve injury, which cannot be obtained even with state-of-the-art automated microfluidic systems. We developed a new femtosecond laser microdissection (fs-LM) method to rapidly and precisely isolate single cells directly from living tissue or organisms by leveraging femtosecond laser ablation as a high-precision cutting tool. Compared to traditional laser capture microdissection, our method provides a few crucial advantages. 1) fs-LM yields intact single cells without sample sectioning, freezing, or fixing, thus preventing sample degradation or contamination. 2) compared to the dissociation and sorting method, fs-LM induces less stress response in isolated cells. 3) fs-LM preserves the spatial and phenotypic information of the collected neurons. In addition, by correlating gene expression to the context-dependent regeneration phenotypes, it is possible to further dissect the genetic activities encoding nerve regeneration. 4) fs-LM does can isolate unlabeled cells. We isolated regrowing posterior lateral microtubule (PLM) neurons from larval 4 stage animals. Single cell RNA-sequencing on the isolated neurons identified gene expression patterns underlying axon regeneration. To demonstrate the versatility of our method, we have also dissected and sequenced single C. elegans oocytes and mammalian brain neurons.
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[
Methods Mol Biol,
2015]
While traditional chemical fixation methods for C. elegans electron microscopy (EM) have provided invaluable anatomical and structural information, the development of high-pressure freeze (HPF) and freeze substitution (FS) protocols offers advantages for high-resolution imaging. Specimens prepared using HPF methodology exhibit fewer distortion artifacts due to fixation and dehydration, have improved antigenicity, and result in a more physiologically accurate structural representation of the worm. In the HPF technique, freely moving worms are frozen at high-pressure (2100 bar) and low temperature (-180 C) within milliseconds. These conditions prevent the formation of ice crystals that can damage cellular structures. Samples then undergo FS, during which worms are slowly brought to room temperature while substituting amorphous ice with organic solvents to preserve tissue in its near native state and provide contrast for imaging. FS can be performed in an automatic freeze substitution (AFS) machine or in makeshift, temperature controlled chambers. Fixed worms can be embedded in plastic resin and further processed for a variety of imaging techniques. Samples then viewed using scanning (SEM) or transmission electron microscopy (TEM) will show enhanced preservation of organelles, cell morphology, and antigenicity for immunocytochemistry.
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[
Am J Trop Med Hyg,
1977]
Saline antigen extracts of microfilariae, adult worms and third-stage larvae of subperiodic Brugia malayi maintained in gerbils were prepared for use as skin test reagents. Patients were studied on three different islands in the Philippines, one endemic for Bancroftian filariasis (Sorsogon, Luzon), another endemic for Malayan filariasis (Palawan) and the third without endemic filariasis (Cebu). A dose-response curve was established initially in patients with Bancroftian filariasis: thereafter 1.0 microng of the B. malayi antigens and 0.05 microng of Dirofilaria immitis FST antigen (obtained from Dr. T. Sawada) were used. Sizes of reactions were measured by recording the diameters of wheals at 20 minutes, 24 and 48 hours. There was a very high correlation in immediate hypersensitivity reactions among the three B. malayi antigens. Reaction sizes followed a normal distribution. When an area of an antigen-induced wheal 3 X that of the saline control was considered a positive reaction, 99% of 150 patients with Bancroftian filariasis and 96% of 45 subjects with Malayan filariasis reacted to B. malayi larval antigen. Only 68% of patients with Bancroftian filariasis but 90% of those with Malayan filariasis reacted to D. immitis FST antigen. There was no relationship between skin reactivity and age, sex, microfilaremia or severity of clinical disease. Approximately half of 50 patients who lived in an endemic area for W. bancrofti but had neither patent infection nor clinical disease reacted to B. malayi antigens. A maximum of 7% of 120 age- and sex-matched controls from Cebu gave false positive reactions with any of the antigens. Only a small proportion of patients gave 24- and 48-hour reactions. It is concluded that the use of antigens prepared from a human parasite, subperiodic B. malayi, which is easily maintained in a laboratory animal host, improves the ability to diagnose filarial infections by immunological means.
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[
J Microsc,
2008]
The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser-induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time-resolved fixation to arrest development at specific stages. The second method uses high-pressure freezing of whole worms followed by freeze-substitution (HPF-FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high-resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF-FS and electron tomography. This procedure combines the advantages of time-resolved fixation and superior ultrastructural preservation by high-pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.
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[
BMC Bioinformatics,
2007]
BACKGROUND: Protein-protein interaction has been used to complement traditional sequence homology to elucidate protein function. Most existing approaches only make use of direct interactions to infer function, and some have studied the application of indirect interactions for functional inference but are unable to improve prediction performance. We have previously proposed an approach, FS-Weighted Averaging, which uses topological weighting and level-2 indirect interactions (protein pairs connected via two interactions) for predicting protein function from protein interactions and have found that it yields predictions with superior precision on yeast proteins over existing approaches. Here we study the use of this technique to predict functional annotations from the Gene Ontology for seven genomes: Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Rattus norvegicus, Mus musculus, and Homo sapiens. RESULTS: Our analysis shows that protein-protein interactions provide supplementary coverage over sequence homology in the inference of protein function and is definitely a complement to sequence homology. We also find that FS-Weighted Averaging consistently outperforms two classical approaches, Neighbor Counting and Chi-Square, across the seven genomes for all three categories of the Gene Ontology. By randomly adding and removing interactions from the interactions, we find that Weighted Averaging is also rather robust against noisy interaction data. CONCLUSION: We have conducted a comprehensive study over seven genomes. We conclude that FS-Weighted Averaging can effectively make use of indirect interactions to make the inference of protein functions from protein interactions more effective. Furthermore, the technique is general enough to work over a variety of genomes.
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[
International Worm Meeting,
2005]
Understanding the basics of nerve regeneration can be used to identify novel therapies for neurological diseases. Animal models of nerve regeneration are mostly limited to vertebrate organisms. Establishment of axotomy models in invertebrate organisms such as C. elegans is highly desired because of remarkable research potential promised by genetics and screening methods. We reported a surgical technique based on femtosecond (fs) laser pulses to severe nerve processes (0.2-0.4 ? in radius) in the worm (Nature 432:822 (2004)). In this technique, amplified fs pulses were used in a low-energy processing regime to achieve axotomy with minimal surrounding damage. Anesthetized animals were mounted on a slide and 100 laser pulses with 10-40 nanojoule energy and 200 fs short duration at 1 kHz repetition rate were tightly focused on the fluorescent nerve processes. By cutting circumferential axons of D-type motor neurons, we were able to establish a nerve injury and regeneration model. Operated animals showed uncoordinated backward motion, a manifestation of D neuron deficiency (Nature 364:337-41 (1993)). Lesioned axons recovered in one day and the behavioral defect improved over the same time period. Invertebrate animals have differing capacities for natural recovery after nerve injury, probably regulated by permissive environment and intrinsic qualities of neurons. Since the energy deposited by laser pulses induces structural damage, the assessment of damage extent in the tissue after laser operation becomes important to understand tissue permissivity factors regarding nerve regeneration. To assess surrounding damage by laser pulses, we used a strain where both nerve processes and muscle cell membrane were labeled by GFP. We severed the nerve process and measured ensuing surrounding damage by loss of GFP signal in the lesion periphery. Unwanted damage by laser pulses seemed to be proportional to laser pulse intensity which provides a controllable parameter to curb damage spread, and, thus, facilitate the investigation on the permissive environmental factors. We extended the axotomy model to sensory neurons to determine if intrinsic qualities of neurons confer differential responses to nerve injury. The results, technique, and its potential applications will be discussed.
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[
Methods Cell Biol,
2010]
The roundworm Caenorhabditis elegans is one of the major model organisms in modern cell and developmental biology. Here, we present methods for the three-dimensional (3D) reconstruction of the worm ultrastructure. We describe the use of (1) serial-section analysis, (2) electron tomography, and (3) serial block face imaging by scanning electron microscopy (SEM). Sample preparation for high-pressure freezing/freeze substitution (HPF/FS) has been extensively covered in a previous volume of this "Methods in Cell Biology" series and will only be described briefly. We will discuss these 3D methods in light of recent research activities related to worm and early embryo biology.