Publications by authors named "Hang Lu"

201 Publications

Graphical-model framework for automated annotation of cell identities in dense cellular images.

Elife 2021 Feb 24;10. Epub 2021 Feb 24.

Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, United States.

Although identifying cell names in dense image stacks is critical in analyzing functional whole-brain data enabling comparison across experiments, unbiased identification is very difficult, and relies heavily on researchers' experiences. Here we present a probabilistic-graphical-model framework, CRF_ID, based on Conditional Random Fields, for unbiased and automated cell identification. CRF_ID focuses on maximizing intrinsic similarity between shapes. Compared to existing methods, CRF_ID achieves higher accuracy on simulated and ground-truth experimental datasets, and better robustness against challenging noise conditions common in experimental data. CRF_ID can further boost accuracy by building atlases from annotated data in highly computationally efficient manner, and by easily adding new features (e.g. from new strains). We demonstrate cell annotation in images across strains, animal orientations, and tasks including gene-expression localization, multi-cellular and whole-brain functional imaging experiments. Together, these successes demonstrate that unbiased cell annotation can facilitate biological discovery, and this approach may be valuable to annotation tasks for other systems.
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http://dx.doi.org/10.7554/eLife.60321DOI Listing
February 2021

A Multicellular Network Mechanism for Temperature-Robust Food Sensing.

Cell Rep 2020 Dec;33(12):108521

Centre for Developmental Neurobiology, King's College London, London SE1 1UL, UK. Electronic address:

Responsiveness to external cues is a hallmark of biological systems. In complex environments, it is crucial for organisms to remain responsive to specific inputs even as other internal or external factors fluctuate. Here, we show how the nematode Caenorhabditis elegans can discriminate between different food levels to modulate its lifespan despite temperature perturbations. This end-to-end robustness from environment to physiology is mediated by food-sensing neurons that communicate via transforming growth factor β (TGF-β) and serotonin signals to form a multicellular gene network. Specific regulations in this network change sign with temperature to maintain similar food responsiveness in the lifespan output. In contrast to robustness of stereotyped outputs, our findings uncover a more complex robustness process involving the higher order function of discrimination in food responsiveness. This process involves rewiring a multicellular network to compensate for temperature and provides a basis for understanding gene-environment interactions. Together, our findings unveil sensory computations that integrate environmental cues to govern physiology.
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http://dx.doi.org/10.1016/j.celrep.2020.108521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773553PMC
December 2020

High-Temporal-Resolution smFISH Method for Gene Expression Studies in Embryos.

Anal Chem 2021 01 23;93(3):1369-1376. Epub 2020 Dec 23.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 313 Ferst Drive NW, Atlanta, Georgia 30332, United States.

Recent development in fluorescence-based molecular tools has contributed significantly to developmental studies, including embryogenesis. Many of these tools rely on multiple steps of sample manipulation, so obtaining large sample sizes presents a major challenge as it can be labor-intensive and time-consuming. However, large sample sizes are required to uncover critical aspects of embryogenesis, for example, subtle phenotypic differences or gene expression dynamics. This problem is particularly relevant for single-molecule fluorescence in situ hybridization (smFISH) studies in embryogenesis. Microfluidics can help address this issue by allowing a large number of samples and parallelization of experiments. However, performing efficient reagent exchange on chip for large numbers of embryos remains a bottleneck. Here, we present a microfluidic pipeline for large-scale smFISH imaging of embryos with minimized labor. We designed embryo traps and engineered a protocol allowing for efficient chemical exchange for hundreds of embryos simultaneously. Furthermore, the device design and small footprint optimize imaging throughput by facilitating spatial registration and enabling minimal user input. We conducted the smFISH protocol on chip and demonstrated that image quality is preserved. With one device replacing the equivalent of 10 glass slides of embryos mounted manually, our microfluidic approach greatly increases throughput. Finally, to highlight the capability of our platform to perform longitudinal studies with high temporal resolution, we conducted a temporal analysis of gene expression in early embryos. The method demonstrated here paves the way for systematic high-temporal-resolution studies that will benefit large-scale RNAi and drug screens and in systems beyond embryos.
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http://dx.doi.org/10.1021/acs.analchem.0c02966DOI Listing
January 2021

Electron beam-induced deposition of platinum from Pt(CO)Cl and Pt(CO)Br.

Beilstein J Nanotechnol 2020 27;11:1789-1800. Epub 2020 Nov 27.

Delft University of Technology, Fac. Applied Sciences, Dept. Imaging Physics, Lorentzweg 1, 2628CJ Delft, Netherlands.

Two platinum precursors, Pt(CO)Cl and Pt(CO)Br, were designed for focused electron beam-induced deposition (FEBID) with the aim of producing platinum deposits of higher purity than those deposited from commercially available precursors. In this work, we present the first deposition experiments in a scanning electron microscope (SEM), wherein series of pillars were successfully grown from both precursors. The growth of the pillars was studied as a function of the electron dose and compared to deposits grown from the commercially available precursor MeCpPtMe. The composition of the deposits was determined using energy-dispersive X-ray spectroscopy (EDX) and compared to the composition of deposits from MeCpPtMe, as well as deposits made in an ultrahigh-vacuum (UHV) environment. A slight increase in metal content and a higher growth rate are achieved in the SEM for deposits from Pt(CO)Cl compared to MeCpPtMe. However, deposits made from Pt(CO)Br show slightly less metal content and a lower growth rate compared to MeCpPtMe. With both Pt(CO)Cl and Pt(CO)Br, a marked difference in composition was found between deposits made in the SEM and deposits made in UHV. In addition to Pt, the UHV deposits contained halogen species and little or no carbon, while the SEM deposits contained only small amounts of halogen species but high carbon content. Results from this study highlight the effect that deposition conditions can have on the composition of deposits created by FEBID.
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http://dx.doi.org/10.3762/bjnano.11.161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705861PMC
November 2020

Enabling high-throughput single-animal gene-expression studies with molecular and micro-scale technologies.

Authors:
Jason Wan Hang Lu

Lab Chip 2020 12;20(24):4528-4538

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA. and School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.

Gene expression and regulation play diverse and important roles across all living systems. By quantifying the expression, whether in a sample of single cells, a specific tissue, or in a whole animal, one can gain insights into the underlying biology. Many biological questions now require single-animal and tissue-specific resolution, such as why individuals, even within an isogenic population, have variations in development and aging across different tissues and organs. The popular techniques that quantify the transcriptome (e.g. RNA-sequencing) process populations of animals and cells together and thus, have limitations in both individual and spatial resolution. There are single-animal assays available (e.g. fluorescent reporters); however, they suffer other technical bottlenecks, such as a lack of robust sample-handling methods. Microfluidic technologies have demonstrated various improvements throughout the years, and it is likely they can enhance the impact of these single-animal gene-expression assays. In this perspective, we aim to highlight how the engineering/method-development field have unique opportunities to create new tools that can enable us to robustly answer the next set of important questions in biology that require high-density, high-quality gene expression data.
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http://dx.doi.org/10.1039/d0lc00881hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769683PMC
December 2020

The Role of Fairness in Early Characterization of New Technologies: Effects on Selective Exposure and Risk Perception.

Risk Anal 2020 Nov 10. Epub 2020 Nov 10.

Cornell University, Ithaca, NY, USA.

Previous research suggests that when individuals have limited knowledge to make sense of new or emerging technologies, they may rely more on available cues, such as the fairness of those managing the risks, when developing their attitudinal and behavioral responses to the technology. To examine this further, we designed an online experiment (N = 1,042) to test the effects of risk managers' nonoutcome fairness on individuals' selective exposure to additional information and perceived risk. As the study context, we used the development of enhanced geothermal systems (EGS), which uses drilling to tap deep underground sources of heat for district heating and electricity and remains low in familiarity among the U.S. public. The results suggest that participants who read about the fair risk manager were subsequently more likely to have positive attitudes toward EGS development. In turn, those with more positive attitudes were more likely to select and read positively valenced articles about EGS, resulting in an indirect effect of the fairness condition. Although this study also explored whether uncertainty moderated this fairness effect on information seeking, it found no evidence. Additionally, when participants were exposed to information featuring fair risk managers, perceived risk decreased, an effect that was mediated by beliefs that EGS was controllable and not dreadful. These results underscore the importance of using practices that will increase nonoutcome fairness in the introduction of new technologies.
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http://dx.doi.org/10.1111/risa.13633DOI Listing
November 2020

Time-Resolved Single-Cell Assay for Measuring Intracellular Reactive Oxygen Species upon Exposure to Ambient Particulate Matter.

Environ Sci Technol 2020 10 8;54(20):13121-13130. Epub 2020 Oct 8.

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States.

Health risks associated with exposure to ambient particulate matter (PM) are a major concern around the world. Adverse PM health effects have been proposed to be linked to oxidative stress through the generation of reactive oxygen species (ROS). cellular assays can provide insights into components or characteristics of PM that best account for its toxicity at a cellular level. However, most current assays report cell population averages and are mostly time endpoint measurements and thus provide no temporal information. This poses limitations on our understanding of PM health effects. In this study, we developed a microfluidic assay that can measure cellular ROS responses at the single-cell level and evaluate temporal dynamic behavior of single cells. We first established a protocol that enables culturing cells in our microfluidic platform and that can provide reproducible ROS readouts. We further examined the heterogeneous ROS responses of cell populations and tracked the dynamics of individual cellular responses upon exposure to different concentrations of PM extracts. Our results show that in an alveolar macrophage cell line, cellular ROS responses are highly heterogeneous. ROS responses from different cells can vary over an order of magnitude, and large coefficients of variation at each timepoint measurement indicate a high variability. The dynamic behavior of single-cell responses is strongly dependent on PM concentrations. Our work serves as a proof-of-principle demonstration of the capability of our microfluidic technology to study time-resolved single-cell responses upon PM exposure. We envision applying this high-resolution, high-content assay to investigate a wide array of single-cell responses (beyond ROS) upon exposure to different types of PM in the future.
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http://dx.doi.org/10.1021/acs.est.0c02889DOI Listing
October 2020

[Bacterial Community Shifts and Nitrogen Removal Characteristics for a SNAD Process Treating Anaerobic Digester Liquor of Swine Wastewater (ADLSW) in a Continuous-Flow Biofilm Reactor (CFBR)].

Huan Jing Ke Xue 2020 May;41(5):2349-2357

College of Resources and Environment, Chengdu University of Information Technology, Chengdu 610225, China.

To realize a simultaneous partial nitrification, ANAMMOX (anaerobic ammonium oxidation), and denitrification (SNAD) process treating anaerobic digester liquor of swine wastewater (ADLSW) in a continuous-flow biofilm reactor (CFBR), we first gradually increased the influent ammonium (NH-N) concentration, and then enhanced the ADLSW ratio in the influent during operation; dissolved oxygen (DO) was controlled at (0.4±0.1) mg·L by adjusting the air flow rate, and the temperature was kept at (30±1)℃. Meanwhile, high-throughput sequencing and quantitative PCR (polymerase chain reaction) techniques were used to analyze the bacterial community shifts and the amount of dominant nitrogen removal bacteria. The results demonstrated that a successful start-up of the SNAD process was accomplished in 150 d, and replacement of the actual biogas slurry was completed in 298 d. The effluent (NO-N+NO-N)/ΔNH-N value was less than 0.11, and the average removal rates of NH-N and TN (total nitrogen) increased to 63.26% and 55.71%, respectively. Moreover, high-throughput sequencing results demonstrated that the dominant microbial populations at phylum level were Chloroflexi (with a relative abundance of 50.78%), Proteobacteria (13.34%), and Planctomycetes (9.26%). The relative abundance of increased from 1.55% to 1.98%. In addition, the relative abundance of and increased from 0.01% and (<0.01%) to 4.66% and 4.18%, respectively, and the relative abundance of increased from (<0.01%) to 2.06%. Meanwhile, qPCR analysis showed that the amounts of ammonia-oxidizing bacteria, ANAMMOX, and denitrifying bacteria increased significantly compared with the inoculated sludge. An efficient and stable nitrogen removal rate can be achieved, and the follow-up processing cost can be reduced, by application of the SNAD treatment process for ADLSW.
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http://dx.doi.org/10.13227/j.hjkx.201910018DOI Listing
May 2020

Effect of phytase on nutrient digestibility and expression of intestinal tight junction and nutrient transporter genes in pigs.

J Anim Sci 2020 Jul;98(7)

Department of Animal Sciences, Purdue University, West Lafayette, IN.

The study was conducted to determine the effects of high levels of phytase on growth performance, nutrient digestibility, phytate breakdown, and expression of mucosal tight junction and nutrient transporter genes in weanling pigs. A total of 128 barrows were penned in groups of four and used in a randomized completely block design and assigned to four treatments for a 28-d study. A two-phase feeding was implemented (phase 1: day 1 to 14; phase 2: day 15 to 28). The diets differed in dietary calcium (Ca) and phosphorus (P) levels (positive control [PC]: 8.1 to 7.1 g/kg Ca and 6.5 to 6.8 g/kg P; negative control [NC]: 6.6 to 5.5 g/kg Ca and 5.6 to 5.3 g/kg P) from phase 1 to phase 2, respectively. NC diets were supplemented with phytase at 0 (NC), 1,500 (NC + 1,500), or 3,000 (NC + 3,000) phytase units (FTU)/kg. Blood was collected after fasting (day 27) or feeding (day 28) for the measurement of plasma inositol concentrations. On day 28, two pigs per pen were euthanized. Duodenal-jejunal and ileal digesta samples and feces were collected to determine inositol phosphates (InsP3-6) concentrations. Phytase supplementation increased the body weight on days 14 and 28 (P < 0.05). Average daily gain and feed efficiency compared with NC were increased by phytase with the majority of its effect in phase 1 (P < 0.05). The apparent ileal digestibility and apparent total tract digestibility of P were increased in piglets fed phytase-supplemented diets (P < 0.01) compared with NC piglets. Disappearance of InsP6 and total InsP3-6 up to the duodenum-jejunum, ileum, and in feces was increased by both phytase application rates (P < 0.01). Plasma concentrations of myo-inositol were higher (P < 0.001) in the phytase-supplemented diets than PC and NC diets, irrespective of whether pigs were fed or fasted. Expression of claudin 3 was higher in pigs fed both phytase-supplemented diets in the duodenum and jejunum compared with PC and NC. Mucin 2 expression was lower in the ileum of NC + 3,000 fed piglets compared with PC (P < 0.05), whereas expression of GLUT2 (solute carrier family 2-facilitated glucose transporter member 2) was increased (P < 0.05) by the NC + 3,000 treatment in all sections. In summary, high phytase supplementation increased the growth performance of nursery pigs. The increased expression of GLUT2 by phytase may indicate an upregulation of glucose absorption from the intestine by phytase.
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http://dx.doi.org/10.1093/jas/skaa206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357581PMC
July 2020

An automated platform to monitor long-term behavior and healthspan in Caenorhabditis elegans under precise environmental control.

Commun Biol 2020 Jun 10;3(1):297. Epub 2020 Jun 10.

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, 30332, USA.

Health and longevity in all organisms are strongly influenced by the environment. To fully understand how environmental factors interact with genetic and stochastic factors to modulate the aging process, it is crucial to precisely control environmental conditions for long-term studies. In the commonly used model organism Caenorhabditis elegans, existing assays for healthspan and lifespan have inherent limitations, making it difficult to perform large-scale longitudinal aging studies under precise environmental control. To address these constraints, we developed the Health and Lifespan Testing Hub (HeALTH), an automated, microfluidic-based system for robust longitudinal behavioral monitoring. Our system provides long-term (i.e. entire lifespan) spatiotemporal environmental control. We demonstrate healthspan and lifespan studies under a variety of genetic and environmental perturbations while observing how individuality plays a role in the aging process. This system is generalizable beyond aging research, particularly for short- or long-term behavioral assays, and could be adapted for other model systems.
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http://dx.doi.org/10.1038/s42003-020-1013-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7287092PMC
June 2020

Microfluidic perfusion modulates growth and motor neuron differentiation of stem cell aggregates.

Analyst 2020 Jul 9;145(14):4815-4826. Epub 2020 Jun 9.

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA.

Microfluidic technologies provide many advantages for studying differentiation of three-dimensional (3D) stem cell aggregates, including the ability to control the culture microenvironment, isolate individual aggregates for longitudinal tracking, and perform imaging-based assays. However, applying microfluidics to studying mechanisms of stem cell differentiation requires an understanding of how microfluidic culture conditions impact cell phenotypes. Conventional cell culture techniques cannot directly be applied to the microscale, as microscale culture varies from macroscale culture in multiple aspects. Therefore, the objective of this work was to explore key parameters in microfluidic culture of 3D stem cell aggregates and to understand how these parameters influence stem cell behavior and differentiation. These studies were done in the context of differentiation of embryonic stem cells (ESCs) to motor neurons (MNs). We assessed how media exchange frequency modulates the biochemical microenvironment, including availability of exogenous factors (e.g. nutrients, small molecule additives) and cell-secreted molecules, and thereby impacts differentiation. The results of these studies provide guidance on how key characteristics of 3D cell cultures can be considered when designing microfluidic culture parameters. We demonstrate that discontinuous perfusion is effective at supporting stem cell aggregate growth. We find that there is a balance between the frequency of media exchange, which is needed to ensure that cells are not nutrient-limited, and the need to allow accumulation of cell-secreted factors to promote differentiation. Finally, we show how microfluidic device geometries can influence transport of biomolecules and potentially promote asymmetric spatial differentiation. These findings are instructive for future work in designing devices and experiments for culture of cell aggregates.
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http://dx.doi.org/10.1039/d0an00491jDOI Listing
July 2020

Ankyrin Is An Intracellular Tether for TMC Mechanotransduction Channels.

Neuron 2020 07 22;107(1):112-125.e10. Epub 2020 Apr 22.

Neurobiology Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK; Department of Biology, KU Leuven, 3000 Leuven, Belgium. Electronic address:

Mechanotransduction channels have been proposed as force sensors in various physiological processes, such as hearing and touch. In particular, TMC1 has been shown to constitute the pore of hair cell mechanotransduction channels, but little is known about how force is sensed by TMC channels. Here, we identify UNC-44/ankyrin as an essential component of the TMC-1 mechanotransduction channel complex in the sensory cilia of Caenorhabditis elegans mechanoreceptor neurons. Ankyrin binds indirectly to TMC-1 via evolutionarily conserved CIB proteins, which are required for TMC-1-mediated mechanosensation in C. elegans OLQ neurons and body wall muscles. Mechanosensory activity conferred by ectopically expressed TMCs in mechanoinsensitive neurons depends on both ankyrin and CIB proteins, indicating that the ankyrin-CIB subcomplex is required for TMC mechanosensitivity. Our work indicates that ankyrin is a long-sought intracellular tether that transmits force to TMC mechanotransduction channels.
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http://dx.doi.org/10.1016/j.neuron.2020.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343241PMC
July 2020

A portable, low-cost device for precise control of specimen temperature under stereomicroscopes.

PLoS One 2020 11;15(3):e0230241. Epub 2020 Mar 11.

School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, United States of America.

To facilitate precise and convenient control of biological sample temperature, we developed a low-cost device that can be used independently or with any stereomicroscope. The purpose of the device is to control the thermal environment during experimental intervals in which a specimen must be manipulated outside of an incubator, e.g. for dissection or slide-mounting in preparation for imaging. Sample temperatures can be both cooled to below and heated to above room temperatures, and stably maintained at a precision of +/- 0.1˚C. To demonstrate the utility of this device, we report improved characterization of the penetrance of a short-acting temperature-sensitive allele in C. elegans embryos, and identification of the upper temperature threshold for embryonic viability for six Caenorhabditis species. By controlling the temperature environment even as a specimen is manipulated, this device offers consistency and flexibility, reduces environmental noise, and enables precision timing in experiments requiring temperature shifts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230241PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065815PMC
June 2020

Molecular epidemiology and hematologic characterization of δβ-thalassemia and hereditary persistence of fetal hemoglobin in 125,661 families of greater Guangzhou area, the metropolis of southern China.

BMC Med Genet 2020 02 28;21(1):43. Epub 2020 Feb 28.

The Second School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.

Background: Individuals with δβ-thalassemia/HPFH and β-thalassemia usually present with intermedia or thalassemia major. No large-scale survey on HPFH/δβ-thalassemia in southern China has been reported to date. The purpose of this study was to examine the molecular epidemiology and hematologic characteristics of these disorders in Guangzhou, the largest city in Southern China, to offer advice for thalassemia screening programs and genetic counseling.

Methods: A total of 125,661 couples participated in pregestational thalassemia screening. 654 subjects with fetal hemoglobin (HbF) level ≥ 5% were selected for further investigation. Gap-PCR combined with Multiplex ligation dependent probe amplification (MLPA) was used to screen for β-globin gene cluster deletions. Gene sequencing for the promoter region of HBG1 /HBG2 gene was performed for all those subjects.

Results: A total of 654 individuals had hemoglobin (HbF) levels≥5, and 0.12% of the couples were found to be heterozygous for HPFH/δβ-thalassemia, including Chinese γ (γδβ)-thal, Southeast Asia HPFH (SEA-HPFH), Taiwanese deletion and Hb Lepore-Boston-Washington. The highest prevalence was observed in the Huadu district and the lowest in the Nansha district. Three cases were identified as carrying β-globin gene cluster deletions, which had not been previously reported. Two at-risk couples (0.0015%) were required to receive prenatal diagnosis. We also found 55cases of nondeletional-HPFH (nd-HPFH), including 54 with Italian nd-HPFH and one with the γ-197C-T heterozygous state. It is difficult to discriminate between Chinese γ (γδβ)-thal and Italian nd-HPFH carriers using hemoglobin (Hb) analysis.

Conclusions: This study is the first to describe the familial prevalence of HPFH/δβ-thalassemia and the high-risk rate in Greater Guangzhou Area, and the findings will support the implementation of thalassemia screening for three common deletions by gap-PCR. We also presented a systematic description of genotype-phenotype relationships which will be useful for genetic counseling and prenatal diagnostic services for β-thalassemia intermedia.
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http://dx.doi.org/10.1186/s12881-020-0981-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049201PMC
February 2020

A spontaneous complex structural variant in rcan-1 increases exploratory behavior and laboratory fitness of Caenorhabditis elegans.

PLoS Genet 2020 02 24;16(2):e1008606. Epub 2020 Feb 24.

School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

Over long evolutionary timescales, major changes to the copy number, function, and genomic organization of genes occur, however, our understanding of the individual mutational events responsible for these changes is lacking. In this report, we study the genetic basis of adaptation of two strains of C. elegans to laboratory food sources using competition experiments on a panel of 89 recombinant inbred lines (RIL). Unexpectedly, we identified a single RIL with higher relative fitness than either of the parental strains. This strain also displayed a novel behavioral phenotype, resulting in higher propensity to explore bacterial lawns. Using bulk-segregant analysis and short-read resequencing of this RIL, we mapped the change in exploration behavior to a spontaneous, complex rearrangement of the rcan-1 gene that occurred during construction of the RIL panel. We resolved this rearrangement into five unique tandem inversion/duplications using Oxford Nanopore long-read sequencing. rcan-1 encodes an ortholog to human RCAN1/DSCR1 calcipressin gene, which has been implicated as a causal gene for Down syndrome. The genomic rearrangement in rcan-1 creates two complete and two truncated versions of the rcan-1 coding region, with a variety of modified 5' and 3' non-coding regions. While most copy-number variations (CNVs) are thought to act by increasing expression of duplicated genes, these changes to rcan-1 ultimately result in the reduction of its whole-body expression due to changes in the upstream regions. By backcrossing this rearrangement into a common genetic background to create a near isogenic line (NIL), we demonstrate that both the competitive advantage and exploration behavioral changes are linked to this complex genetic variant. This NIL strain does not phenocopy a strain containing an rcan-1 loss-of-function allele, which suggests that the residual expression of rcan-1 is necessary for its fitness effects. Our results demonstrate how colonization of new environments, such as those encountered in the laboratory, can create evolutionary pressure to modify gene function. This evolutionary mismatch can be resolved by an unexpectedly complex genetic change that simultaneously duplicates and diversifies a gene into two uniquely regulated genes. Our work shows how complex rearrangements can act to modify gene expression in ways besides increased gene dosage.
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http://dx.doi.org/10.1371/journal.pgen.1008606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058356PMC
February 2020

Intentions to Seek Information About the Influenza Vaccine: The Role of Informational Subjective Norms, Anticipated and Experienced Affect, and Information Insufficiency Among Vaccinated and Unvaccinated People.

Risk Anal 2020 10 19;40(10):2040-2056. Epub 2020 Feb 19.

Department of Psychology, University of Illinois at Urbana-Champaign, Champaign, IL, USA.

When deciding whether to vaccinate, people often seek information through consequential processes that are not currently well understood. A survey of a nationally representative sample of U.S. adults (N = 2,091) explored the factors associated with intentions to seek influenza vaccine information in the 2018-2019 influenza season. This survey shed light on what motivates intentions to seek information about the influenza vaccine through the lens of the risk information seeking and processing (RISP) model. The model explained information-seeking intentions well among both unvaccinated and vaccinated respondents. Key findings show that informational subjective norms, information insufficiency, and different types of affect are strong predictors of information-seeking intentions. Theoretical insights on extending the RISP model and practical guidance on designing interventions are provided.
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http://dx.doi.org/10.1111/risa.13459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434697PMC
October 2020

Effects of biochar on soil microbial community and functional genes of a landfill cover three years after ecological restoration.

Sci Total Environ 2020 May 6;717:137133. Epub 2020 Feb 6.

Guangdong Provincial Key Laboratory of Soil and Groundwater Pollution Control, School of Environmental Science and Engineering, Southern University of Science and Technology, Shenzhen 518055, China; State Environmental Protection Key Laboratory of Integrated Surface Water Groundwater Pollution Control, School of Environmental Science and Engineering, Southern University of Science and Technology, Shenzhen 518055, China. Electronic address:

Landfills, as the most common approach to disposing of municipal solid waste worldwide, disturb native ecosystems and create a need for ecological restoration. The restoration of landfill cover with biochar has shown immediate potential to improve soil microbial functions within one year. However, such characteristics could change after a longer period of time. Here, soil properties, microbial communities, and microbial functional genes related to nutrient cycling were investigated three years after the biochar amendment of the topsoil of a subtropical landfill cover. The results showed that the levels of soil organic matter, water content, total carbon (C), total nitrogen (N) and total phosphorus (P) of biochar-amended soils were higher than those of control soil. Different soil microbial community structures were observed in the biochar-amended and control soils. Nine phyla, including Proteobacteria and Acidobacteria, but not Actinobacteria or Chloroflexi, were enriched in the biochar-amended soil. Although the impact of biochar on shaping microbial communities increased after a longer period of restoration, no differences were observed in soils that were amended using different biochar:soil ratios. The abundances of functional genes related to C and N cycling decreased, whereas those of genes related to P cycling were higher in soils that received biochar amendment. This finding suggests that compared with the control soil, biochar-amended soils were less active in processes involved in C and N cycling but enhanced in processes related to P cycling. This study can serve as a reference for future ecological restoration of degraded lands using biochar.
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http://dx.doi.org/10.1016/j.scitotenv.2020.137133DOI Listing
May 2020

Dissociation of the FEBID precursor cis-Pt(CO)Cl driven by low-energy electrons.

Phys Chem Chem Phys 2020 Mar 6;22(11):6100-6108. Epub 2020 Feb 6.

CEFITEC, Departamento de Física, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal.

In this study, we present experimental and theoretical results on dissociative electron attachment and dissociative ionisation for the potential FEBID precursor cis-Pt(CO)Cl. UHV surface studies have shown that high purity platinum deposits can be obtained from cis-Pt(CO)Cl. The efficiency and energetics of ligand removal through these processes are discussed and experimental appearance energies are compared to calculated thermochemical thresholds. The present results demonstrate the potential effectiveness of electron-induced reactions in the deposition of this FEBID precursor, and these are discussed in conjunction with surface science studies on this precursor and the design of new FEBID precursors.
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http://dx.doi.org/10.1039/c9cp06633kDOI Listing
March 2020

Multimodal Stimulation in a Microfluidic Device Facilitates Studies of Interneurons in Sensory Integration in C. elegans.

Small 2020 03 31;16(10):e1905852. Epub 2020 Jan 31.

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA.

Animals' perception and behavior involve integration of multiple sensory modalities. Caenorhabditis elegans is a useful model for studying multimodal sensory integration, as it has well-characterized neuronal circuits in a relatively simple nervous system. However, most studies based on functional imaging have only been conducted on single modal stimuli, because well-controlled multimodal experiments for C. elegans are technically difficult. For instance, no single systems currently deliver precise stimuli with spatial, temporal, and intensity control, despite prior hypotheses that interneurons do integrate these sensory inputs to control behavior. Here, a microfluidic platform that can easily deliver spatially and temporally controlled combination stimuli to C. elegans is presented. With this platform, both sensory and interneuron activity is measured in response to mechanical and chemical stimulations in a quantitative and high-throughput manner. It is found that the activity of command interneuron PVC can be modulated by prior stimulation both within the same and across different modalities. The roles of monoaminergic and peptidergic signaling are further examined on the process of multimodal integration through PVC activity. The approach exemplified here is envisioned to be broadly applicable in different contexts to elucidate underlying mechanisms and identify genes affecting multisensory integration.
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http://dx.doi.org/10.1002/smll.201905852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720847PMC
March 2020

miR-492 Promotes Cancer Progression by Targeting GJB4 and Is a Novel Biomarker for Bladder Cancer.

Onco Targets Ther 2019 24;12:11453-11464. Epub 2019 Dec 24.

Department of Urology Surgery, Liaoning Province Cancer Hospital and Institute (Cancer Hospital of China Medical University), Shenyang 110004, People's Republic of China.

Background: Bladder cancer is the most common urinary system malignancy in the United States and is characterized by its diverse prognosis and high recurrence rate. However, the molecular mechanisms underlying its progression remain unknown. Accumulating evidence suggests a critical role for miRNAs in bladder cancer progression.

Methods And Results: In this study, we found that miR-492 expression levels were significantly higher in bladder cancer tissue and the serum of bladder cancer patients by bioinformatics analysis and a panel of clinical samples. The results of receiver operating characteristic curve analysis suggested the potential diagnostic value of serum miR-492 for bladder cancer. In vitro and in vivo functional assays showed that knockdown of miR-492 suppressed proliferation and metastasis of bladder cancer cells. Gap junction beta-4 protein was predicted to be a direct target of miR-492, which was validated using a luciferase reporter assay. Further cellular functional assays showed that suppression of miR-492 abrogated bladder cancer cell proliferation and metastasis by targeting gap junction beta-4 protein.

Conclusion: miR-492 promotes cancer progression by targeting GJB4 and is a novel biomarker for bladder cancer.
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http://dx.doi.org/10.2147/OTT.S223448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935362PMC
December 2019

A Multifunctional Imidazolium-Based Silicone Material with Conductivity, Self-Healing, Fluorescence, and Stretching Sensitivity.

Macromol Rapid Commun 2019 Dec 5;40(23):e1900469. Epub 2019 Nov 5.

Key Laboratory of Special Functional Aggregated Materials & Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, P. R. China.

Wearable devices have gained substantial interest for a wide range of applications, including biomonitoring and entertainment. They are basically composed of sensors and substrate materials. Recently, silicone materials have been extensively used in wearable devices because of their unique properties. Silicone materials, which possess remarkable insulation, predominantly serve as a substrate instead of a signaling material due to the indispensable electrical conductivity in wearable devices. Herein, a novel kind of silicone material, with both good conductivity and excellent self-healing efficiency, is designed by introducing imidazolium into the silicone polymer in one step. The free ions afford an ionic conductivity as high as 2.79 × 10 S m , representing a significant improvement over traditional silicone materials. Because of the good conductivity, the silicone material is sensitive to stretching and can be applied as a flexible sensor. On the other hand, the material exhibits a high healing efficiency, reaching 89% in 6 h, due to the dynamic supramolecular interaction of the ion crosslink sites at the crack surface. Furthermore, the silicone material emits a yellow-green fluorescence under UV light.
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http://dx.doi.org/10.1002/marc.201900469DOI Listing
December 2019

smFISH in chips: a microfluidic-based pipeline to quantify in situ gene expression in whole organisms.

Lab Chip 2020 01 2;20(2):266-273. Epub 2019 Dec 2.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA. and School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.

Gene expression and genetic regulatory networks in multi-cellular organisms control complex physiological processes ranging from cellular differentiation to development to aging. Traditional methods to investigate gene expression relationships rely on using bulk, pooled-population assays (e.g. RNA-sequencing and RT-PCR) to compare gene expression levels in hypo- or hyper-morphic mutant animals (e.g. gain-of-function or knockout). This approach is limited, especially in complex gene networks, as these genetic mutations may affect the expressions of related genes in unforseen ways. In contrast, we developed a microfluidic-based pipeline to discover gene relationships in a single genetic background. The microfluidic device provides efficient reagent exchange and the ability to track individual animals. By automating a robust microfluidic reagent exchange strategy, we adapted and validated single molecule fluorescent in situ hybridization (smFISH) on-chip and combined this technology with live-imaging of fluorescent transcriptional reporters. Together, this multi-level information enabled us to quantify a gene expression relationship with single-animal resolution. While this microfluidic-based pipeline is optimized for live-imaging and smFISH C. elegans studies, the strategy is highly-adaptable to other biological models as well as combining other live and end-point biological assays, such as behavior-based toxicology screening and immunohistochemistry.
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http://dx.doi.org/10.1039/c9lc00896aDOI Listing
January 2020

Parallel Processing of Two Mechanosensory Modalities by a Single Neuron in C. elegans.

Dev Cell 2019 12 14;51(5):617-631.e3. Epub 2019 Nov 14.

Howard Hughes Medical Institute, Department of Biology, Stanford University, Stanford, CA, USA. Electronic address:

Neurons convert synaptic or sensory inputs into cellular outputs. It is not well understood how a single neuron senses, processes multiple stimuli, and generates distinct neuronal outcomes. Here, we describe the mechanism by which the C. elegans PVD neurons sense two mechanical stimuli: external touch and proprioceptive body movement. These two stimuli are detected by distinct mechanosensitive DEG/ENaC/ASIC channels, which trigger distinct cellular outputs linked to mechanonociception and proprioception. Mechanonociception depends on DEGT-1 and activates PVD's downstream command interneurons through its axon, while proprioception depends on DEL-1, UNC-8, and MEC-10 to induce local dendritic Ca increase and dendritic release of a neuropeptide NLP-12. NLP-12 directly modulates neuromuscular junction activity through the cholecystokinin receptor homolog on motor axons, setting muscle tone and movement vigor. Thus, the same neuron simultaneously uses both its axon and dendrites as output apparatus to drive distinct sensorimotor outcomes.
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http://dx.doi.org/10.1016/j.devcel.2019.10.008DOI Listing
December 2019

Rapid and multi-cycle smFISH enabled by microfluidic ion concentration polarization for profiling of tissue-specific gene expression in whole .

Biomicrofluidics 2019 Nov 1;13(6):064101. Epub 2019 Nov 1.

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.

Understanding gene regulation networks in multicellular organisms is crucial to decipher many complex physiological processes ranging from development to aging. One technique to characterize gene expression with tissue-specificity in whole organisms is single-molecule fluorescence hybridization (smFISH). However, this protocol requires lengthy incubation times, and it is challenging to achieve multiplexed smFISH in a whole organism. Multiplexing techniques can yield transcriptome-level information, but they require sequential probing of different genes. The inefficient macromolecule exchange through diffusion-dominant transport across dense tissues is the major bottleneck. In this work, we address this challenge by developing a microfluidic/electrokinetic hybrid platform to enable multicycle smFISH in an intact model organism, . We integrate an ion concentration polarization based ion pump with a microfluidic array to rapidly deliver and remove gene-specific probes and stripping reagents on demand in individual animals. Using our platform, we can achieve rapid smFISH, an order of magnitude faster than traditional smFISH protocols. We also demonstrate the capability to perform multicycle smFISH on the same individual samples, which is impossible to do off-chip. Our method hence provides a powerful tool to study individual-specific, spatially resolvable, and large-scale gene expression in whole organisms.
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http://dx.doi.org/10.1063/1.5124827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824911PMC
November 2019

Controlled release of lysozyme based core/shells structured alginate beads with CaCO microparticles using Pickering emulsion template and in situ gelation.

Colloids Surf B Biointerfaces 2019 Nov 31;183:110410. Epub 2019 Jul 31.

Faculty of Fisheries and Protection of Waters, University of South Bohemian in Ceske Budejovice, Institute of Aquaculture and Protection of Waters, České Budějovice, 37005, Czech Republic.

To employ dual advantages of emulsion and gel, a facile approach was investigated to fabricate core/shells structured hydrogel beads based on sodium alginate (SA) via Pickering emulsion template and in situ gelation. The encapsulation and controlled release behavior were further studied using lysozyme (Ly) as the model protein. The optical micrographs and SEM images indicated the SA beads could well disperse with the size about 150 μm. CaCO microparticles were strong adhesive onto SA gel. It showed that 96.51 ± 0.62% Ly was loaded into the hydrogel beads. The released behavior of Ly could be regulated by external pH condition, and displayed highest release rate at pH 5.0. Whereas the lowest release rate was recorded at pH 7.0. The released behavior well followed the Hixcon-Crowell model which indicated that the release mechanism of Ly followed the corrosion diffusion law. The worth-while endeavor provide an artful and facile approach using Pickering emulsion template and in situ gelation to fabricate core/shells structured SA beads with high load capacity and controlled regulation of the entrapped functional component.
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http://dx.doi.org/10.1016/j.colsurfb.2019.110410DOI Listing
November 2019

Effect of phytase on intestinal phytate breakdown, plasma inositol concentrations, and glucose transporter type 4 abundance in muscle membranes of weanling pigs1.

J Anim Sci 2019 Sep;97(9):3907-3919

Department of Animal Sciences, Purdue University, West Lafayette, IN.

The objective of this present study was to determine the effects of phytase dosing on growth performance, mineral digestibility, phytate breakdown, and the level of glucose transporter type 4 (GLUT4) in muscle plasma membranes of weanling pigs. A total of 160 barrows were used in a randomized completely block design and assigned to 4 treatments for a 7-wk study. Depending on the feeding phase, diets differed in dietary calcium (Ca) and phosphorus (P) levels (positive control [PC]: 8 to 6.8g/kg Ca; 7.3 to 6.3 g/kg P; negative control [NC]: 5.5 to 5.2 g/kg Ca; 5.4 to 4.7 g/kg P). NC diets were supplemented with phytase at 0 (NC); 500 (NC + 500 FTU); or 2,000 FTU/kg (NC + 2,000 FTU) phytase units/kg. Blood was collected after fasting (day 48) or feeding (day 49) for measurement of plasma inositol concentrations. On day 49, 2 pigs per pen were euthanized, and duodenal and ileal digesta samples were collected to determine inositol phosphates (InsP6-2) concentrations. High phytase supplementation increased BW on days 21, 35, and 49 (P < 0.05). Over the entire feeding period, ADG, ADFI, and feed efficiency were increased by NC + 2,000 FTU compared with the other treatments (P < 0.05). Postprandial plasma inositol concentration was increased in NC + 2,000 (P < 0.01), but there was only a tendency (P = 0.06) of a higher fasting plasma inositol concentration in this group. Inositol concentrations in the portal vein plasma (day 49) were not different among treatments. Duodenal digesta InsP5 and InsP6 concentrations were similar in PC and NC, but higher in these 2 treatments (P < 0.05) than those supplemented with phytase. Phytase supplementation decreased InsP6-4, resulting in increased InsP3-2 and myo-inositol concentrations. Similar effects were found in ileal contents. Compared with NC, phytase supplementation resulted in greater cumulative InsP6-2 disappearance (93.6% vs. 72.8% vs. 25.0%, for NC + 2,000 FTU, NC + 500 FTU and NC, respectively, P < 0.01) till the distal ileum. Longissimus dorsi muscle plasma membrane GLUT4 concentration was increased by NC + 2,000 FTU (P < 0.01) compared with NC. In summary, high phytase supplementation increased growth performance of nursery pigs. The higher myo-inositol release from phytate could contribute to the increased expression of GLUT4 in muscle plasma membranes. Further investigation is needed to determine whether this is associated with enhanced cellular glucose uptake and utilization.
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http://dx.doi.org/10.1093/jas/skz234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735906PMC
September 2019

Extra-phosphoric effects of super dosing phytase on growth performance of pigs is not solely due to release of myo-inositol.

J Anim Sci 2019 Sep;97(9):3898-3906

Department of Animal Sciences, Purdue University, West Lafayette, IN.

Two experiments were conducted to determine the effects of myo-inositol and phytase on growth performance, plasma metabolites, and nutrient digestibility of growing pigs. In experiment 1, 96 growing pigs with average initial body weight (BW) of 26.2 kg were used in a 25-d growth performance study. Pigs were assigned to four dietary treatments with three pigs per pen and eight replicate pens per treatment in a randomized complete block design. The four treatments were control diet (CD); CD + 2 g/kg inositol; CD + 1,000 FYT/kg phytase and CD + 3,000 FYT/kg phytase. Pigs were weighed individually every week. On day 25, blood sample was collected from one pig per pen to measure plasma metabolites concentrations. In experiment 2, 16 barrows (initial BW 34.8 ± 8.2 kg) were surgically fitted with T-cannulas. Pigs were allotted to four blocks based on BW and assigned to a quadruplicate 4 × 2 incomplete Latin square design with same four dietary treatments and two periods. Ileal digesta samples were collected from each pig on days 6 and 7 of each period to determine apparent ileal digestibility (AID) of nutrients. Phytase supplementation increased final BW and average daily gain (ADG) compared with CD (P < 0.05) with no effects on average daily feed intake (ADFI) and gain to feed (G:F) was higher in 3,000 FYT/kg phytase (P < 0.05). Inositol supplementation had no effects on growth performance. Plasma myo-inositol concentration was increased by inositol supplementation, and 3,000 FYT/kg phytase increased myo-inositol in the plasma by 97.2% (P < 0.05). Plasma P concentration was increased by 1,000 or 3,000 FYT/kg phytase with no effects on alkaline phosphatase (ALP), glucose, triglycerides (TAG), calcium (Ca), and urea concentrations. Phytase supplementation reduced (P < 0.05) the phytate-P concentration in the ileal digesta and increased the digestibility of phytate-P and total P with no effects on the AID of dry matter (DM), gross energy (GE), nitrogen (N), and Ca. In conclusion, the beneficial effects of 3,000 FYT/kg phytase on feed efficiency may due to the increased release of both myo-inositol and phosphorus (P), and may not be solely due to myo-inositol release by this level of phytase.
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http://dx.doi.org/10.1093/jas/skz232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735830PMC
September 2019

Digging deeper: methodologies for high-content phenotyping in Caenorhabditis elegans.

Lab Anim (NY) 2019 Jul 19;48(7):207-216. Epub 2019 Jun 19.

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA.

Deep phenotyping is an emerging conceptual paradigm and experimental approach aimed at measuring and linking many aspects of a phenotype to understand its underlying biology. To date, deep phenotyping has been applied mostly in cultured cells and used less in multicellular organisms. However, in the past decade, it has increasingly been recognized that deep phenotyping could lead to a better understanding of how genetics, environment and stochasticity affect the development, physiology and behavior of an organism. The nematode Caenorhabditis elegans is an invaluable model system for studying how genes affect a phenotypic trait, and new technologies have taken advantage of the worm's physical attributes to increase the throughput and informational content of experiments. Coupling of these technical advancements with computational and analytical tools has enabled a boom in deep-phenotyping studies of C. elegans. In this Review, we highlight how these new technologies and tools are digging into the biological origins of complex, multidimensional phenotypes.
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http://dx.doi.org/10.1038/s41684-019-0326-6DOI Listing
July 2019

Dynamic Mitochondrial Migratory Features Associated with Calcium Responses during T Cell Antigen Recognition.

J Immunol 2019 08 14;203(3):760-768. Epub 2019 Jun 14.

School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332;

A T cell clone is able to distinguish Ags in the form of peptide-MHC complexes with high specificity and sensitivity; however, how subtle differences in peptide-MHC structures translate to distinct T cell effector functions remains unknown. We hypothesized that mitochondrial positioning and associated calcium responses play an important role in T cell Ag recognition. We engineered a microfluidic system to precisely manipulate and synchronize a large number of cell-cell pairing events, which provided simultaneous real-time signaling imaging and organelle tracking with temporal precision. In addition, we developed image-derived metrics to quantify calcium response and mitochondria movement. Using myelin proteolipid altered peptide ligands and a hybridoma T cell line derived from a mouse model of experimental autoimmune encephalomyelitis, we observed that Ag potency modulates calcium response at the single-cell level. We further developed a partial least squares regression model, which highlighted mitochondrial positioning as a strong predictor of calcium response. The model revealed T cell mitochondria sharply alter direction within minutes following exposure to agonist peptide Ag, changing from accumulation at the immunological synapse to retrograde movement toward the distal end of the T cell body. By quantifying mitochondria movement as a highly dynamic process with rapidly changing phases, our result reconciles conflicting prior reports of mitochondria positioning during T cell Ag recognition. We envision applying this pipeline of methodology to study cell interactions between other immune cell types to reveal important signaling phenomenon that is inaccessible because of data-limited experimental design.
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http://dx.doi.org/10.4049/jimmunol.1800299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650333PMC
August 2019