Synapses are sites of intercellular communication between neurons and from neurons to target organs, and of signal integration that underly physiological and behavioral responses. We have developed a modular platform, Interluminescence (Int), for experimental control of synaptic transmission: bioluminescent light, generated by a luciferase oxidizing a luciferin, from a presynaptic neuron is used to activate transsynaptic optogenetic ion channels in the postsynaptic neuron. Two strategies can modulate postsynaptic neurons in the presence of luciferin. In the 'Act-Int' approach, a luciferase is genetically expressed in synaptic vesicles and released during depolarization-induced presynaptic vesicle fusion and exocytosis. In the 'Persist-Int' approach, a luciferase is tethered to the presynaptic membrane where it can support sustained transsynaptic signaling. Both strategies can activate postsynaptic neurons with comparable efficacy under the conditions tested. By design, the modularity of the platform permits the use of luciferases and opsins ranging in brightness and light sensitivity, with the luciferase targeted to different subcellular regions of the presynaptic neuron, and the postsynaptic opsin being excitatory or inhibitory. Our results demonstrate the utility and versatility of Interluminescence to mediate synaptic transmission that is either activity-dependent or activity-independent.

Genetically encoded sensors and actuators have advanced the ability to observe and manipulate cellular activity, yet few non-invasive strategies enable cells to directly couple their intracellular states to user-defined outputs. We promote a bioluminescent activity-dependent (BLADe) platform that facilitates programmable feedback through genetically encoded light generation. Using calcium (Ca2+) flux as a model, we engineered a Ca2+-dependent luciferase that functions as an activity-gated light source capable of photoactivating light-sensing actuators. As an initial demonstration of the versatility of this platform we present two separate use cases in neurons. In the first application, the presence of luciferin triggers Ca2+ dependent local illumination that provides activity dependent gene expression by activating a light-sensitive transcription factor. In the second application, neuronal activity-driven Ca2+ fluctuations via locally generated bioluminescence control neural dynamics through opsin activation in single cells, populations and intact tissue. BLADe can be expanded to couple any signal that bioluminescent enzymes can be engineered to detect with the wide variety of photosensing actuators. This modular strategy of coupling an activity dependent light emitter to a light sensing actuator offers, in principle, a generalizable framework for state dependent cell-autonomous control across biological systems.

Monitoring intracellular calcium is central to understanding cell signaling across nearly all cell types and organisms. Fluorescent genetically encoded calcium indicators (GECIs) remain the standard tools for in vivo calcium imaging, but require intense excitation light, leading to photobleaching, background autofluorescence and phototoxicity. Bioluminescent GECIs, which generate light enzymatically, eliminate these artifacts but have been constrained by low dynamic range and suboptimal calcium affinities. Here we show that CaBLAM ('calcium bioluminescence activity monitor'), an engineered bioluminescent calcium indicator, achieves an order-of-magnitude improvement in signal contrast and a tunable affinity matched to physiological cytosolic calcium. CaBLAM enables single-cell and subcellular activity imaging at video frame rates in cultured neurons and sustained imaging over hours in awake, behaving animals. These capabilities establish CaBLAM as a robust and general alternative to fluorescent GECIs, extending calcium imaging to regimes where excitation light is undesirable or infeasible.

Early stages of Huntington's disease (HD) before the onset of motor and cognitive symptoms are characterized by imbalanced excitatory and inhibitory output from the cortex to striatal and subcortical structures. The window before the onset of symptoms presents an opportunity to adjust the firing rate within microcircuits with the goal of restoring the impaired E/I balance, thereby preventing or slowing down disease progression. Here, we investigated the effect of presymptomatic cell-type specific manipulation of activity of pyramidal neurons and parvalbumin interneurons in the M1 motor cortex on disease progression in the R6/2 HD mouse model. Our results show that dampening excitation of Emx1 pyramidal neurons or increasing activity of parvalbumin interneurons once daily for 3 weeks during the pre-symptomatic phase alleviated HD-related motor coordination dysfunction. Cell-type-specific modulation to normalize the net output of the cortex is a potential therapeutic avenue for HD and other neurodegenerative disorders.

Significance:Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research.Aim:We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs.Approach:We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array (MEAs) recordings in primary neurons.Results:Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical.Conclusions:Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer (FRET), the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

Background Tumor cell spheroids are organized multicellular structures that form during the expansive growth of carcinoma cells. Spheroids formation is thought to contribute to metastasis by supporting growth and survival of mobile tumor cell populations. Methods and results We investigated how spheroid architecture affects OXPHOS activity, microRNA expression, and intraperitoneal survival of an ovarian carcinoma cell line using high resolution respirometry, quantitative RT-PCR, and a rodent intraperitoneal growth model. Rates of oxidative phosphorylation/respiration per cell of cells growing as spheroids were nearly double those of a variant of the same cell type growing in suspension as loosely aggregated cells. Further, inhibition of spheroid formation by treatment with CDH2 (N-cadherin) siRNA reduced the rate of OXPHOS to that of the non-spheroid forming variant. Cells growing as spheroids showed greatly enhanced expression of miR-221/222, an oncomiR that targets multiple tumor suppressor genes and promotes invasion, and reduced expression of miR-9, which targets mitochondrial tRNA-modification enzymes and inhibits OXPHOS. Consistent with greater efficiency of ATP generation, tumor cells growing as spheroids injected into the nutrient-poor murine peritoneum survived longer than cells growing in suspension as loosely associated aggregates. Conclusions The data indicate that growth in spheroid form enhances the OXPHOS activity of constituent tumor cells. In addition, spheroid architecture affects expression of microRNA genes involved in growth control and mitochondrial function. During the mobile phase of metastasis, when ovarian tumor cells disperse through nutrient-poor environments such as the peritoneum, enhanced OXPHOS activity afforded by spheroid architecture would enhance survival and metastatic potential.

Tumor cell spheroids are three dimensional multicellular structures that form during the expansive growth of carcinoma cells. Spheroids support tumor metastasis as vehicles of dissemination, promoting growth and survival of bulk tumor and cancer stem cells within the mobile tumor cell population. Deciphering how spheroid architecture affects tumor cell phenotype will be essential for the development of therapeutics to inhibit transperitoneal metastasis and the development of peritoneal carcinomatosis by ovarian cancers. We investigated how spheroid formation directly affects OXPHOS activity and microRNA expression in a cultured ovarian carcinoma cell system. The rate of oxidative phosphorylation/respiration per cell in spheroids was nearly double that of the same cell type growing in suspension as single cells. Cells growing as spheroids showed greatly enhanced expression of miR-221/222, an oncomiR that targets multiple tumor suppressor genes, promotes invasion, as well as reduced expression of miR-9, which targets mitochondrial tRNA-modification enzymes and inhibits OXPHOS. Consistent with the greater efficiency of ATP generation afforded by OXPHOS phosphorylation, tumor cells growing as spheroids injected into the nutrient-poor environment of the murine peritoneum survived longer than the cells growing in suspension as loosely associated aggregates. The data suggest that in addition to the reported effects of spheroid formation on cancer cell growth and phenotype, including promotion of stem cell generation, spheroid architecture increases the OXPHOS activity of constituent tumor cells. During the mobile phase of metastasis, when ovarian tumor cells disperse through nutrient-poor environments such as the peritoneum, enhanced OXPHOS activity afforded by spheroid architecture would enhance survival and thereby contribute to metastatic potential.

AbstractCa2+plays many critical roles in cell physiology and biochemistry, leading researchers to develop a number of fluorescent small molecule dyes and genetically encodable probes that optically report changes in Ca2+concentrations in living cells. Though such fluorescence-based genetically encoded Ca2+indicators (GECIs) have become a mainstay of modern Ca2+sensing and imaging, bioluminescence-based GECIs—probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein—have several distinct advantages over their fluorescent counterparts. Bioluminescent tags do not photobleach, do not suffer from nonspecific autofluorescent background, and do not lead to phototoxicity since they do not require the extremely bright extrinsic excitation light typically required for fluorescence imaging, especially with 2-photon microscopy. Current BL GECIs perform poorly relative to fluorescent GECIs, producing small changes in bioluminescence intensity due to high baseline signal at resting Ca2+concentrations and suboptimal Ca2+affinities. Here, we describe the development of a new bioluminescent GECI, “CaBLAM,” which displays much higher contrast (dynamic range) than previously described bioluminescent GECIs and has a Ca2+affinity suitable for capturing physiological changes in cytosolic Ca2+concentration. Derived from a new variant ofOplophorus gracilirostrisluciferase with superior in vitro properties and a highly favorable scaffold for insertion of sensor domains, CaBLAM allows for single-cell and subcellular resolution imaging of Ca2+dynamics at high frame rates in cultured neurons andin vivo. CaBLAM marks a significant milestone in the GECI timeline, enabling Ca2+recordings with high spatial and temporal resolution without perturbing cells with intense excitation light.

Aedes aegypti is the primary vector of dengue virus (DENV) and other arboviruses. Previous literature suggests that vertebrate and invertebrate lipids and the nutritional status of mosquitoes modify virus infection. Here, we developed a vertebrate lipid-depleted Ae. aegypti cell line to investigate if chronic depletion of vertebrate lipids normally present in a blood meal and insect cell culture medium would impact cell growth and virus infection. Chronic depletion of vertebrate lipids reduced cell size and proliferation, although cells retained equivalent total intracellular lipids per cell by reducing lipolysis and modifying gene expression related to sugar and lipid metabolism. Downregulation of innate immunity genes was also observed. We hypothesized that chronic depletion of vertebrate lipids would impact virus infection; however, the same amount of DENV was produced per cell. This study reveals how Ae. aegypti cells adapt in the absence of vertebrate lipids, and how DENV can replicate equally well in cells that contain predominately vertebrate or invertebrate lipids.

In higher eukaryotic cells, mitochondria are essential subcellular organelles for energy production, cell signaling, and the biosynthesis of biomolecules. The mitochondrial DNA (mtDNA) genome is indispensable for mitochondrial function because it encodes protein subunits of the electron transport chain and a full set of transfer and ribosomal RNAs. MtDNA degradation has emerged as an essential quality control measure to maintain mtDNA and to cope with mtDNA damage resulting from endogenous and environmental factors. Among all types of DNA damage known, abasic (AP) sites, sourced from base excision repair and spontaneous base loss, are the most abundant endogenous DNA lesions in cells. In mitochondria, AP sites trigger rapid DNA loss; however, the mechanism and molecular factors involved in the process remain elusive. Herein, we demonstrate that the stability of AP sites is reduced dramatically upon binding to a major mtDNA packaging protein, mitochondrial transcription factor A (TFAM). The half-life of AP lesions within TFAM-DNA complexes is 2 to 3 orders of magnitude shorter than that in free DNA, depending on their position. The TFAM-catalyzed AP-DNA destabilization occurs with nonspecific DNA or mitochondrial light-strand promoter sequence, yielding DNA single-strand breaks and DNA-TFAM cross-links. TFAM-DNA cross-link intermediates prior to the strand scission were also observed upon treating AP-DNA with mitochondrial extracts of human cells. In situ trapping of the reaction intermediates (DNA-TFAM cross-links) revealed that the reaction proceeds via Schiff base chemistry facilitated by lysine residues. Collectively, our data suggest a novel role of TFAM in facilitating the turnover of abasic DNA.

Aedes aegypti is the primary vector of a number of viruses pathogenic to humans including dengue virus (DENV). DENV infection leads to widespread transcriptomic and proteomic alterations in mosquito cells. Here we identified alterations to the mosquito cell secretome during DENV infection by performing liquid chromatography tandem mass spectrometry. We found that an extracellular fragment of low-density lipoprotein receptor-related protein 1(LRP-1) was present during infection. Previous literature suggests that LRP-1 regulates cholesterol homeostasis. Therefore, we hypothesized that DENV modifies LRP-1 protein expression to maintain host-derived intracellular cholesterol, which would facilitate virus replication within membrane-associated replication compartments. Accordingly, stimuli that are present during flavivirus infection reduced LRP-1 protein expression. We also found that dsRNA knockdown of LRP-1 increased intracellular cholesterol and DENV viral RNA. Further, depletion of intracellular lipids reduced infection. Together, these data suggest that DENV reduces LRP-1 protein expression, possibly through regulated intramembrane proteolysis (RIP), to increase intracellular cholesterol and facilitate replication in Ae. aegypti.

PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn2+, a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas in the presence of Mg2+, the chemical step becomes rate limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn2+ concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase.

Aedes aegypti is the primary vector of dengue virus (DENV), and acquires this virus from a vertebrate host during blood feeding. Previous literature has shown that vertebrate blood factors such as complement protein C5a and low-density lipoprotein (LDL) influence DENV acquisition in the mosquito. Here, we show that extracellular vesicles in cell culture medium inhibit DENV infection in mosquito cells. Specifically, extracellular vesicles enter into mosquito cells and inhibit an early stage of infection. Extracellular vesicles had no effect on virus cell attachment or entry. Instead, extracellular vesicles restricted virus membrane fusion. Extracellular vesicles only inhibited DENV infection in mosquito cells and not vertebrate cells. These data highlight a novel virus-vector-host interaction that limits virus infection in mosquito cells by restricting virus membrane fusion.

Infection with Zika virus (ZIKV) is of growing concern since infection is associated with the development of congenital neurological disease. Quantitative reverse transcription PCR (qRT-PCR) has been the standard for ZIKV detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper testing. Studies have suggested that ZIKV detection in urine is more sensitive and has a longer window of detection compared to serum and saliva. The objective of this study was to develop a urine diagnostic test that could be completed in under 30 minutes. Urine samples spiked with ZIKV or dengue virus were tested using RT-LAMP as well as by conventional quantitative qRT-PCR. These techniques were then validated using crude lysates made from ZIKV infected mosquitoes in addition to urine and serum samples from ZIKV infected patients. RT-LAMP specifically detected ZIKV in urine and serum for ZIKV infected patients and crude mosquito lysates. This test was performed in under 30 minutes and did not require RNA extraction from urine nor mosquitos. This approach could be used for monitoring of exposed individuals, especially pregnant women, couples wanting to conceive, or individuals with suspicious symptoms as well as surveillance of mosquito populations.

Zika virus (ZIKV) is a rapidly emerging flavivirus that has been associated with a number of congenital neurological manifestations. Here, we show that ZIKV replicated efficiently in mouse neural stem cells (mNSCs). ZIKV infection caused a cytopathic effect without affecting cell viability, yet led to a significant decrease in the number of proteins secreted into mNSC supernatants. A gene expression array of neural stem cell progenitor and differentiation markers suggested that infection reduced the number of neuronal and oligodendrocyte progenitors while increasing the number of astrocyte progenitors. Infection in astrocytes increased transcription of key genes involved in the antiviral response. These data provide molecular and cellular evidence that ZIKV significantly alters neural development in the vertebrate host and that astrocyte differentiation may be a protective response that limits neuropathogenesis.

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder II1 Apr 2017MP23-03 URINE BASED RAPID MOLECULAR DIAGNOSIS OF ZIKA VIRUS Laura Lamb, Sarah Bartolone, Michael Conway, Maya Tree, Olivia Lossia, Gary Dunbar, Julien Rossignol, Christopher Smith, and Michael Chancellor Laura LambLaura Lamb More articles by this author , Sarah BartoloneSarah Bartolone More articles by this author , Michael ConwayMichael Conway More articles by this author , Maya TreeMaya Tree More articles by this author , Olivia LossiaOlivia Lossia More articles by this author , Gary DunbarGary Dunbar More articles by this author , Julien RossignolJulien Rossignol More articles by this author , Christopher SmithChristopher Smith More articles by this author , and Michael ChancellorMichael Chancellor More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.731AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Infection with Zika virus (ZIKV) is of growing concern since it is suspected with causing brain defects in newborns including microcephaly, and having potential and severe neurological and autoimmune complications. ZIKV is anticipated to spread globally within the next year, therefore a rapid and ideally non-invasive diagnostic test is urgently needed. Studies have suggested that ZIKV detection in urine is more sensitive and has a longer window of detection as ZIKV may be shed in the urine. The objective of this study was to develop a urine diagnostic test that could be completed under 30 minutes. METHODS Urine samples spiked with ZIKV or related other related arboviruses including Dengue fever were tested using conventional real time PCR. Samples were also tested using a new methodology we developed that utilized reverse transcription loop mediated isothermal amplification (RT-LAMP). These techniques were also validated using samples from ZIKV infected patients and mosquitoes. RESULTS RT-LAMP could specifically detect ZIKV in ZIKV positive samples. This could be done in under 30 minutes and did not require timely RNA extraction from the urine, unlike real time PCR. CONCLUSIONS Here we describe a technique by which ZIKV can be rapidly detected in a non-invasive urine sample. This may allow for easy monitoring of potentially exposed individuals, especially pregnant women, couples wanting to conceive, or individuals with suspicious symptoms. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e293-e294 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Laura Lamb More articles by this author Sarah Bartolone More articles by this author Michael Conway More articles by this author Maya Tree More articles by this author Olivia Lossia More articles by this author Gary Dunbar More articles by this author Julien Rossignol More articles by this author Christopher Smith More articles by this author Michael Chancellor More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Arboviruses are a large group of viruses that are transmitted by arthropods including ticks and mosquitoes. The global diversity of arboviruses is unknown; however, theoretical studies have estimated that over 2,000 mosquito-borne flaviviruses may exist. An increasing number of flaviviruses can only infect insect cells. We hypothesize that insect-specific flaviviruses (ISFVs) represent model genetic precursors to pathogenic flaviviruses, although the genetic mechanisms required for adaptation to vertebrate hosts are unclear. In this study, we determined that Kamiti River virus (KRV) infection was inhibited by innate immunity pathways in vertebrate cells. KRV infection of IRF3,5,7-/- mouse embryonic fibroblasts led to low levels of viral protein production and shedding of infectious progeny. These data suggest that ISFVs cannot evade vertebrate innate immune pathways. Identifying cellular pathways and genetic changes that are required for adaptation of arthropod-specific arboviruses to vertebrate hosts is critical to understanding emerging infectious disease.