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[
Traffic,
2011]
Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.
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[
J Vis Exp,
2012]
Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques. Microfluidic devices have been used for sub-cellular imaging, in vivo laser microsurgery and cellular imaging. In vivo imaging requires immobilization of organisms. This has been achieved using suction, tapered channels, deformable membranes, suction with additional cooling anesthetic gas, temperature sensitive gels, cyanoacrylate glue and anesthetics such as levamisole. Commonly used anesthetics influence synaptic transmission and are known to have detrimental effects on sub-cellular neuronal transport. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from -10 m to -800 m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min. We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F). Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in -30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.
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[
Methods Mol Biol,
2014]
Miniature devices are powerful new tools that can be used to address multiple questions in biology especially in investigating an individual cell or organism. The primary step forward has been the ease of soft lithography fabrication which has allowed researchers from different disciplines, with incomplete technical knowledge, to develop and use new devices for their own research problems. In this chapter, we describe a simple fabrication process that will allow investigators to make microfluidic devices for in vivo imaging studies using genetic model organisms such as C. elegans, Drosophila larvae, and zebrafish larvae. This microfluidic technology enables detailed studies on multiple cellular and subcellular phenomena including intracellular vesicle trafficking in living organisms over different developmental stages in an anesthetic free environment.
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[
Nat Commun,
2016]
Next generation drug screening could benefit greatly from in vivo studies, using small animal models such as Caenorhabditis elegans for hit identification and lead optimization. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans, we developed an automated microfluidic platform. This platform can image 15 z-stacks of 4,000 C. elegans from 96 different populations using a large-scale chip with a micron resolution in 16min. Using this platform, we screened 100,000 animals of the poly-glutamine aggregation model on 25 chips. We tested the efficacy of 1,000 FDA-approved drugs in improving the aggregation phenotype of the model and identified four confirmed hits. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays.
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Martin SF, Martin C, Ben-Yakar A, Satarasinghe PN, Hegarty E, Mondal S, Gokce SK, Sahn JJ, Iyer S, Sae-Lee W, Hodges T, Scott LL, Ghorashian N, Pierce JT
[
ACS Chem Neurosci,
2018]
The nematode Caenorhabditis elegans, with tractable genetics and a well-defined nervous system, provides a unique whole-animal model system to identify novel drug targets and therapies for neurodegenerative diseases. Large-scale drug or target screens in models that recapitulate the subtle age- and cell-specific aspects of neurodegenerative diseases are limited by a technological requirement for high-throughput analysis of neuronal morphology. Recently we developed a model of amyloid precursor protein-induced neurodegeneration that exhibits progressive degeneration of select cholinergic neurons. Our previous work with this model suggests that small molecule ligands of the sigma 2 receptor (2R), which was recently cloned and identified as transmembrane protein 97 (TMEM97), are neuroprotective. To determine structure-activity relationships for unexplored chemical space in our 2R/Tmem97 ligand collection, we developed an in vivo high-content screening (HCS) assay to identify potential drug leads. The HCS assay uses our recently developed large-scale microfluidic immobilization chip and automated imaging platform. We discovered norbenzomorphans that reduced neurodegeneration in our C. elegans model, including two compounds that demonstrated significant neuroprotective activity at multiple doses. These findings provide further evidence that 2R/Tmem97-binding norbenzomorphans may represent a new drug class for treating neurodegenerative diseases.
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[
PLoS One,
2015]
BACKGROUND: Acinetobacter baumannii is becoming an increasing menace in health care settings especially in the intensive care units due to its ability to withstand adverse environmental conditions and exhibit innate resistance to different classes of antibiotics. Here we describe the biological contributions of abeD, a novel membrane transporter in bacterial stress response and antimicrobial resistance in A. baumannii. RESULTS: The abeD mutant displayed ~ 3.37 fold decreased survival and >5-fold reduced growth in hostile osmotic (0.25 M; NaCl) and oxidative (2.631 M-6.574 M; H2O2) stress conditions respectively. The abeD inactivated cells displayed increased susceptibility to ceftriaxone, gentamicin, rifampicin and tobramycin (~ 4.0 fold). The mutant displayed increased sensitivity to the hospital-based disinfectant benzalkonium chloride (~3.18-fold). In Caenorhabditis elegans model, the abeD mutant exhibited (P<0.01) lower virulence capability. Binding of SoxR on the regulatory fragments of abeD provide strong evidence for the involvement of SoxR system in regulating the expression of abeD in A. baumannii. CONCLUSION: This study demonstrates the contributions of membrane transporter AbeD in bacterial physiology, stress response and antimicrobial resistance in A. baumannii for the first time.
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[
Microbiology,
2013]
Klebsiella pneumoniae is a Gram-negative bacillus that causes serious infections in immunocompromised human hosts and exhibits significant multidrug resistance. In this study, we identified a novel lysR-family regulator (designated oxyR(KP)) in the genome of K. pneumoniae NTUH-K2044 whose functions have remained enigmatic so far. Functional characterization of the putative lysR regulator oxyR(KP) with respect to cellular physiology and antimicrobial susceptibility was performed by generating an isogenic mutant, oxyR(KP) in a hypervirulent clinical isolate of K. pneumoniae. The K. pneumoniae oxyR(KP) mutant was sensitive to hyperosmotic and bile conditions. Disruption of oxyR(KP) increased the susceptibility of K. pneumoniae to oxidative (0.78947 mM hydrogen peroxide) and nitrosative (30 mM acidified nitrite) stress by ~1.4-fold and ~10-fold, respectively. Loss of the Klebsiella regulator led to a decrease in the minimum inhibitory concentrations for chloramphenicol (10-fold), erythromycin (6-fold), nalidixic acid (>50-fold) and trimethoprim (10-fold), which could be restored following complementation. The relative change in expression of resistance-nodulation-cell division super family (RND) efflux gene acrB was decreased by approximately fivefold in the oxyR(KP) mutant as evidenced by qRT-PCR. In a Caenorhabditis elegans model, the oxyR(KP) mutant exhibited significantly (P<0.01) lower virulence. Overall, results detailed in this report reflect the pleiotropic role of the oxyR(KP) signalling system and diversity of the resistance determinants in hypervirulent K1 serotype K. pneumoniae NTUH-K2044.
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[
PLoS One,
2012]
BACKGROUND: The diffusion of antibiotics through the outer membrane is primarily affected by the porin super family, changes contribute to antibiotic resistance. Recently we demonstrated that the CpxAR two-component signaling system alters the expression of an uncharacterized porin OmpC(KP), to mediate antimicrobial resistance in K. pneumoniae. PRINCIPAL FINDINGS: In this study, functional characterization of the putative porin OmpC(KP) (denoted kpnO) with respect to antimicrobial susceptibility and virulence was evaluated by generating an isogenic mutant, kpnO in a clinical isolate of K. pneumoniae. Estimation of uronic acid content confirmed that kpnO produced 2.0 fold lesser capsular polysaccharide than the wild-type. The kpnO displayed higher sensitivity to hyper osmotic and bile conditions. Disruption of kpnO increased the susceptibility of K. pneumoniae to oxidative and nitrostative stress by 1.6 fold and >7 fold respectively. The loss of the Klebsiella porin led to an increase in the minimum inhibitory concentration of tetracycline (3-fold), nalidixic acid (4-fold), tobramycin (4-fold), streptomycin (10-fold), and spectinomycin (10-fold), which could be restored following complementation. The single deletion of kpnO reduced the survival of the pathogen by 50% when exposed to disinfectants. In Caenorhabditis elegans model, the kpnO mutant exhibited significantly (P<0.01) lower virulence. To dissect the role of PhoBR signaling system in regulating the expression of the kpnO, a phoB(KP) isogenic mutant was constructed. The phoB(KP) mutant exhibited impaired gastrointestinal stress response and decreased antimicrobial susceptibility. The mRNA levels of kpnO were found to be 4-fold less in phoB(KP) mutant compared to wild type. A regulatory role of PhoB(KP) for the expression of kpnO was further supported by the specific binding of PhoB(KP) to the putative promoter of kpnO. CONCLUSIONS AND SIGNIFICANCE: Loss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044.
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[
Cell Biosci,
2022]
BACKGROUND: Bone morphogenetic protein (BMP) is a phylogenetically conserved signaling pathway required for development that is aberrantly expressed in several age-related diseases including cancer, Alzheimer's disease, obesity, and cardiovascular disease. Aberrant BMP signaling in mice leads to obesity, suggesting it may alter normal metabolism. The role of BMP signaling regulating cancer metabolism is not known. METHODS: To examine BMP regulation of metabolism, C. elegans harboring BMP gain-of-function (gof) and loss-of-function (lof) mutations were examined for changes in activity of catabolic and anabolic metabolism utilizing Western blot analysis and fluorescent reporters. AMP activated kinase (AMPK) gof and lof mutants were used to examine AMPK regulation of BMP signaling. H1299 (LKB1 wild-type), A549 (LKB1 lof), and A549-LKB1 (LKB1 restored) lung cancer cell lines were used to study BMP regulation of catabolic and anabolic metabolism. Studies were done using recombinant BMP ligands to activate BMP signaling, and BMP receptor specific inhibitors and siRNA to inhibit signaling. RESULTS: BMP signaling in both C. elegans and cancer cells is responsive to nutrient conditions. In both C. elegans and lung cancer cell lines BMP suppressed AMPK, the master regulator of catabolism, while activating PI3K, a regulator of anabolism. In lung cancer cells, inhibition of BMP signaling by siRNA or small molecules increased AMPK activity, and this increase was mediated by activation of LKB1. BMP2 ligand suppressed AMPK activation during starvation. BMP2 ligand decreased expression of TCA cycle intermediates and non-essential amino acids in H1299 cells. Furthermore, we show that BMP activation of PI3K is mediated through BMP type II receptor. We also observed feedback signaling, as AMPK suppressed BMP signaling, whereas PI3K increased BMP signaling. CONCLUSION: These studies show that BMP signaling suppresses catabolic metabolism and stimulates anabolic metabolism. We identified feedback mechanisms where catabolic induced signaling mediated by AMPK negatively regulates BMP signaling, whereas anabolic signaling produces a positive feedback regulation of BMP signing through Akt. These mechanisms were conserved in both lung cancer cells and C. elegans. These studies suggest that aberrant BMP signaling causes dysregulation of metabolism that is a potential mechanism by which BMP promotes survival of cancer cells.
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Campbell BE, Schwarz EM, Gasser RB, Hofmann A, Pell J, Brown CT, Williams BA, Gregory TR, Boag PR, Jex AR, Jabbar A, Sternberg PW, Young ND, Hall RS, Zhu XQ, Howe AC, Mondal A, Antoshechkin I, Loukas A, Korhonen PK
[
Genome Biol,
2013]
BACKGROUND: The barber's pole worm, Haemonchus contortus, is one of the most economically important parasites of small ruminants worldwide. Although this parasite can be controlled using anthelmintic drugs, resistance against most drugs in common use has become a widespread problem. We provide a draft of the genome and the transcriptomes of all key developmental stages of H. contortus to support biological and biotechnological research areas of this and related parasites. RESULTS: The draft genome of H. contortus is 320 Mb in size and encodes 23,610 protein-coding genes. On a fundamental level, we elucidate transcriptional alterations taking place throughout the life cycle, characterize the parasite's gene silencing machinery, and explore molecules involved in development, reproduction, host-parasite interactions, immunity, and disease. The secretome of H. contortus is particularly rich in peptidases linked to blood-feeding activity and interactions with host tissues, and a diverse array of molecules is involved in complex immune responses. On an applied level, we predict drug targets and identify vaccine molecules. CONCLUSIONS: The draft genome and developmental transcriptome of H. contortus provide a major resource to the scientific community for a wide range of genomic, genetic, proteomic, metabolomic, evolutionary, biological, ecological, and epidemiological investigations, and a solid foundation for biotechnological outcomes, including new anthelmintics, vaccines and diagnostic tests. This first draft genome of any strongylid nematode paves the way for a rapid acceleration in our understanding of a wide range of socioeconomically important parasites of one of the largest nematode orders.