Structural basis of mitochondrial membrane bending by the I-II-III2-IV2 supercomplex.

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.

Long-range electron proton coupling in respiratory complex I - insights from molecular simulations of the quinone chamber and antiporter-like subunits.

Respiratory complex I is a redox-driven proton pump. Several high-resolution structures of complex I have been determined providing important information about the putative proton transfer paths and conformational transitions that may occur during catalysis. However, how redox energy is coupled to the pumping of protons remains unclear. In this article, we review biochemical, structural and molecular simulation data on complex I and discuss several coupling models, including the key unresolved mechanistic questions. Focusing both on the quinone-reductase domain as well as the proton-pumping membrane-bound domain of complex I, we discuss a molecular mechanism of proton pumping that satisfies most experimental and theoretical constraints. We suggest that protonation reactions play an important role not only in catalysis, but also in the physiologically-relevant active/deactive transition of complex I.

Tetrahydrocannabinols: potential cannabimimetic agents for cancer therapy.

Tetrahydrocannabinols (THCs) antagonize the CB1 and CB2 cannabinoid receptors, whose signaling to the endocannabinoid system is essential for controlling cell survival and proliferation as well as psychoactive effects. Most tumor cells express a much higher level of CB1 and CB2; THCs have been investigated as potential cancer therapeutic due to their cannabimimetic properties. To date, THCs have been prescribed as palliative medicine to cancer patients but not as an anticancer modality. Growing evidence of preclinical research demonstrates that THCs reduce tumor progression by stimulating apoptosis and autophagy and inhibiting two significant hallmarks of cancer pathogenesis: metastasis and angiogenesis. However, the degree of their anticancer effects depends on the origin of the tumor site, the expression of cannabinoid receptors on tumor cells, and the dosages and types of THC. This review summarizes the current state of knowledge on the molecular processes that THCs target for their anticancer effects. It also emphasizes the substantial knowledge gaps that should be of concern in future studies. We also discuss the therapeutic effects of THCs and the problems that will need to be addressed in the future. Clarifying unanswered queries is a prerequisite to translating the THCs into an effective anticancer regime.

Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism.

Mutations in mitochondrial DNA encoded subunit of ATP synthase, MT-ATP6, are frequent causes of neurological mitochondrial diseases with a range of phenotypes from Leigh syndrome and NARP to ataxias and neuropathies. Here we investigated the functional consequences of an unusual heteroplasmic truncating mutation m.9154C>T in MT-ATP6, which caused peripheral neuropathy, ataxia and IgA nephropathy. ATP synthase not only generates cellular ATP, but its dimerization is required for mitochondrial cristae formation. Accordingly, the MT-ATP6 truncating mutation impaired the assembly of ATP synthase and disrupted cristae morphology, supporting our molecular dynamics simulations that predicted destabilized a/c subunit subcomplex. Next, we modeled the effects of the truncating mutation using patient-specific induced pluripotent stem cells. Unexpectedly, depending on mutation heteroplasmy level, the truncation showed multiple threshold effects in cellular reprogramming, neurogenesis and in metabolism of mature motor neurons (MN). Interestingly, MN differentiation beyond progenitor stage was impaired by Notch hyperactivation in the MT-ATP6 mutant, but not by rotenone-induced inhibition of mitochondrial respiration, suggesting that altered mitochondrial morphology contributed to Notch hyperactivation. Finally, we also identified a lower mutation threshold for a metabolic shift in mature MN, affecting lactate utilization, which may be relevant for understanding the mechanisms of mitochondrial involvement in peripheral motor neuropathies. These results establish a critical and disease-relevant role for ATP synthase in human cell fate decisions and neuronal metabolism.

An RBM10 and NF-κB interacting host lncRNA promotes JEV replication and neuronal cell death.

IMPORTANCE: Central nervous system infection by flaviviruses such as Japanese encephalitis virus, Dengue virus, and West Nile virus results in neuroinflammation and neuronal damage. However, little is known about the role of long non-coding RNAs (lncRNAs) in flavivirus-induced neuroinflammation and neuronal cell death. Here, we characterized the role of a flavivirus-induced lncRNA named JINR1 during the infection of neuronal cells. Depletion of JINR1 during virus infection reduces viral replication and cell death. An increase in GRP78 expression by JINR1 is responsible for promoting virus replication. Flavivirus infection induces the expression of a cellular protein RBM10, which interacts with JINR1. RBM10 and JINR1 promote the proinflammatory transcription factor NF-κB activity, which is detrimental to cell survival.

Fine mapping of a new common bean anthracnose resistance gene (Co-18) to the proximal end of Pv10 in Indian landrace KRC-5.

KEY MESSAGE: Mapping and fine mapping of bean anthracnose resistance genes is a continuous process. We report fine mapping of anthracnose resistance gene Co-18 which is the first anthracnose gene mapped to Pv10. The discovery of resistance gene is a major gain in the bean anthracnose pathosystem research. Among the Indian common bean landraces, KRC-5 exhibit high levels of resistance to the bean anthracnose pathogen Colletotrichum lindemuthianum. To precisely map the anthracnose resistance gene, we used a Recombinant Inbred Line (F2:9 RIL) population (KRC-5 × Jawala). The inheritance test revealed that KRC-5 carries a dominant resistance gene temporarily designated as Co-18. We discovered two RAPD markers linked to Co-18 among 287 RAPD markers. These RAPD markers were eventually developed into SCARs (Sc-OPR15 and Sc-OPF6) and flank Co-18 on chromosome Pv10 at a distance of 5.3 and 4.2 cM, respectively. At 4.0-4.1 Mb on Pv10, we detected a SNP (single-nucleotide polymorphism) signal. We synthesized 58 SSRs and 83 InDels from a pool of 135 SSRs and 1134 InDels, respectively. Five SSRs, four InDels, and two SCARs were used to generate the high-density linkage map, which led to the identification of two SSRs (SSR24 and SSR36) that are tightly linked to Co-18. These two SSRs flank the Co-18 to 178 kb genomic region with 13 candidate genes including five NLR (nucleotide-binding and leucine-rich repeat) genes. The closely linked markers SSR24 and SSR36 will be used in cloning and pyramiding of the Co-18 gene with other R genes to develop durable resistant bean varieties.

Spatiotemporal mapping of nanotopography and thickness transitions of ultrathin foam films.

Freshly formed soap films, soap bubbles, or foam films display iridescent colors due to thin film interference that changes as squeeze flow drives drainage and a progressive decrease in film thickness. Ultrathin (thickness <100 nm) freestanding films of soft matter containing micelles, particles, polyelectrolyte-surfactant complexes, or other supramolecular structures or liquid crystalline phases display drainage via stratification. A fascinating array of thickness variations and transitions, including stepwise thinning and coexistence of thick-thin flat regions, arise in micellar foam films that undergo drainage via stratification. In this tutorial, we describe the IDIOM (interferometry digital imaging optical microscopy) protocols that combine the conventional interferometry principle with digital filtration and image analysis to obtain nanometer accuracy for thickness determination while having high spatial and temporal resolution. We provide fully executable image analysis codes and algorithms for the analysis of nanotopography and summarize some of the unique insights obtained for stratified micellar foam films.

Dynamic duos: the building blocks of dimensional mechanics.

Mechanics studies the relationships between space, time, and matter. These relationships can be expressed in terms of the dimensions of length , time , and mass . Each dimension broadens the scope of mechanics. Historically, mechanics emerged from geometry, which considers quantities like lengths or areas, with dimensions of the form . With the Renaissance, quantities combining space and time were considered, like speed, acceleration and later diffusivity, all of the form . Eventually, mechanics reached its full potential by including "mass-carrying" quantities such as mass, force, momentum, energy, action, power, viscosity, etc. These standard mechanical quantities have dimensions of the form where x and y are integers. In this contribution, we show that, thanks to this dimensional structure, these mass-carrying quantities can be readily arranged into a table such that x and y increase along the row and column, respectively. Ratios of quantities in the same rows provide characteristic lengths, while those in the same columns yield characteristic times, encompassing a great variety of physical phenomena from atomic to astronomical scales. Most generally, we show that selecting duos of mechanical quantities that are neither on the same row nor column of the table yields dynamics, where one mechanical quantity is understood as impelling motion, while the other impedes it. The force and the mass are the prototypes of impelling and impeding factors, but many other duos are possible. We present examples from the physical and biological realms, including planetary motion, sedimentation, explosions, fluid flows, turbulence, diffusion, cell mechanics, capillary and gravity waves, and spreading, pinching, and coalescence of drops and bubbles. This review provides a novel synthesis revealing the power of scaling or dimensional analysis, to understand processes governed by the interplay of two mechanical quantities. This elementary decomposition of space, time and motion into pairs of mechanical factors is the foundation of "dimensional mechanics", a method that this review wishes to promote and advance. Pairs are the fundamental building blocks, but they are only a starting point. Beyond this simple world of mechanical duos, we envision a richer universe that beckons with an interplay of three, four, or more quantities, yielding multiple characteristic lengths, times, and kinematics. This review is complemented by online video lectures, which initiate a discussion on the elaborate interplay of two or more mechanical quantities.

Foam film stratification, viscosity, and small-angle X-ray scattering of micellar SDS solutions over an extended concentration range (1< c/CMC < 75).

Ultrathin foam films (thickness, h < 100 nm) containing micelles undergo drainage via stratification manifested as coexisting thick-thin flat regions, nanoscopic non-flat topography, and the stepwise decrease in film thickness that yields a characteristic step-size. Most studies characterize the variation in step size and stratification kinetics in micellar foam films in a limited concentration range, c/CMC < 12.5 (c < 100 mM). Likewise, most scattering studies characterize micelle dimensions, intermicellar distance, and volume fraction in bulk aqueous SDS solutions in this limited concentration range. In this contribution, we show drainage via stratification can be observed for concentrations up to c/CMC < 75 (c < 600 mM). Understanding the stratification behavior of freely draining micellar films with sodium dodecyl sulfate (SDS) concentration varying in the range 10 mM ≤ cSDS ≤ 600 mM is essential for molecular engineering, consumer product formulations, and controlling foaming in industrial processes. Here, we visualize and analyze nanoscopic thickness variations and transitions in stratifying foam films using Interferometry Digital Imaging Optical Microscopy (IDIOM) protocols. We compare step size obtained from foam stratification to micelle dimension, micelle volume fraction, and intermicellar distance obtained from small angle X-ray scattering studies. Even though the volume fraction increases and approaches 25% at c = 600 mM, the solution viscosity only increases by a factor of four compared to the solvent, consistent with the findings from both stratification and scattering studies. These comparisons allow us to explore the effect of micelle size, morphology, and intermicellar interactions on supramolecular oscillatory structural disjoining pressure, which influences the stratification behavior of draining foam films containing micelles under confinement.

Pinching dynamics, extensional rheology, and stringiness of saliva substitutes.

Saliva substitutes are human-made formulations extensively used in medicine, food, and pharmaceutical research to emulate human saliva's biochemical, tribological, and rheological properties. Even though extensional flows involving saliva are commonly encountered in situations such as swallowing, coughing, sneezing, licking, drooling, gleeking, and blowing spit bubbles, rheological evaluations of saliva and its substitutes in most studies rely on measured values of shear viscosity. Natural saliva possesses stringiness or spinnbarkeit, governed by extensional rheology response, which cannot be evaluated or anticipated from the knowledge of shear rheology response. In this contribution, we comprehensively examine the rheology of twelve commercially available saliva substitutes using torsional rheometry for rate-dependent shear viscosity and dripping-onto-substrate (DoS) protocols for extensional rheology characterization. Even though most formulations are marketed as having suitable rheology, only three displayed measurable viscoelasticity and strain-hardening. Still, these too, failed to emulate the viscosity reduction with the shear rate observed for saliva or match perceived stringiness. Finally, we explore the challenges in creating saliva-like formulations for dysphagia patients and opportunities for using DoS rheometry for diagnostics and designing biomimetic fluids.

Absolute pitch: neurophysiological evidence for early brain activity in prefrontal cortex.

Absolute pitch (AP) is the ability to rapidly label pitch without an external reference. The speed of AP labeling may be related to faster sensory processing. We compared time needed for auditory processing in AP musicians, non-AP musicians, and nonmusicians (NM) using high-density electroencephalographic recording. Participants responded to pure tones and sung voice. Stimuli evoked a negative deflection peaking at ~100 ms (N1) post-stimulus onset, followed by a positive deflection peaking at ~200 ms (P2). N1 latency was shortest in AP, intermediate in non-AP musicians, and longest in NM. Source analyses showed decreased auditory cortex and increased frontal cortex contributions to N1 for complex tones compared with pure tones. Compared with NM, AP musicians had weaker source currents in left auditory cortex but stronger currents in left inferior frontal gyrus (IFG) during N1, and stronger currents in left IFG during P2. Compared with non-AP musicians, AP musicians exhibited stronger source currents in right insula and left IFG during N1, and stronger currents in left IFG during P2. Non-AP musicians had stronger N1 currents in right auditory cortex than nonmusicians. Currents in left IFG and left auditory cortex were correlated to response times exclusively in AP. Findings suggest a left frontotemporal network supports rapid pitch labeling in AP.

Foam film stratification studies probe intermicellar interactions.

Ultrathin foam films containing supramolecular structures like micelles in bulk and adsorbed surfactant at the liquid-air interface undergo drainage via stratification. At a fixed surfactant concentration, the stepwise decrease in the average film thickness of a stratifying micellar film yields a characteristic step size that also describes the quantized thickness difference between coexisting thick-thin flat regions. Even though many published studies claim that step size equals intermicellar distance obtained using scattering from bulk solutions, we found no reports of a direct comparison between the two length scales. It is well established that step size is inversely proportional to the cubic root of surfactant concentration but cannot be estimated by adding micelle size to Debye length, as the latter is inversely proportional to the square root of surfactant concentration. In this contribution, we contrast the step size obtained from analysis of nanoscopic thickness variations and transitions in stratifying foam films using Interferometry Digital Imaging Optical Microscopy (IDIOM) protocols, that we developed, with the intermicellar distance obtained using small-angle X-ray scattering. We find that stratification driven by the confinement-induced layering of micelles within the liquid-air interfaces of a foam film provides a sensitive probe of non-DLVO (Derjaguin-Landau-Verwey-Overbeek) supramolecular oscillatory structural forces and micellar interactions.

Hydrogen bonding rearrangement by a mitochondrial disease mutation in cytochrome bc1 perturbs heme bH redox potential and spin state.

Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease-related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme bH of cytochrome bc1, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme bH caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial heme-containing protein maquettes.

Development of a thin layer chromatography-based method to detect sorbitol presence in milk and its applicability in formalin preserved milk samples.

Sorbitol has been the new and emerging adulterant in dairy industry. The main aim of the study was to develop a method to detect sorbitol in milk, which is not affected by other sugars, polyols and formalin. Hence, a thin layer chromatographic (TLC) method was standardized to detect the sorbitol in milk. In the study 90 s duration for the impregnation of Silica gel 60F TLC plates with Cu- ions was found suitable to resolve sorbitol as a distinct spot. The standardized conditions were (1) developing solvent system consisting of n-propanol: ethyl acetate: water (7:1:2), (2) 0.5% of potassium permanganate in 0.1 M NaOH as color developing reagent. (3) Drying temperature (65°C/ 10 min.) after spraying the color developing reagent. The limit of detection was 0.2% of added sorbitol in milk. The standardized method could also detect the sorbitol in the presence of sucrose, glucose and polyols like mannitol and maltitol. In both cow and buffalo milk samples the standardized methodology performed well in detection of sorbitol. The method also performed well in sorbitol spiked formalin preserved milk samples. This method can be an alternative to the other methods involving costly equipment in detecting adulteration of milk with sorbitol.

Microbial polysaccharides: An emerging family of natural biomaterials for cancer therapy and diagnostics.

Microbial polysaccharides (MPs) offer immense diversity in structural and functional properties. They are extensively used in advance biomedical science owing to their superior biodegradability, hemocompatibility, and capability to imitate the natural extracellular matrix microenvironment. Ease in tailoring, inherent bio-activity, distinct mucoadhesiveness, ability to absorb hydrophobic drugs, and plentiful availability of MPs make them prolific green biomaterials to overcome the significant constraints of cancer chemotherapeutics. Many studies have demonstrated their application to obstruct tumor development and extend survival through immune activation, apoptosis induction, and cell cycle arrest by MPs. Synoptic investigations of MPs are compulsory to decode applied basics in recent inclinations towards cancer regimens. The current review focuses on the anticancer properties of commercially available and newly explored MPs, and outlines their direct and indirect mode of action. The review also highlights cutting-edge MPs-based drug delivery systems to augment the specificity and efficiency of available chemotherapeutics, as well as their emerging role in theranostics.

Aging Enhances Neural Activity in Auditory, Visual, and Somatosensory Cortices: The Common Cause Revisited.

In humans, age-related declines in vision, hearing, and touch coincide with changes in amplitude and latency of sensory-evoked potentials. These age-related differences in neural activity may be related to a common deterioration of supra-modal brain areas (e.g., PFC) that mediate activity in sensory cortices or reflect specific sensorineural impairments that may differ between sensory modalities. To distinguish between these two possibilities, we measured neuroelectric brain activity while 37 young adults (18-30 years, 18 males) and 35 older adults (60-88 years, 20 males) were presented with a rapid randomized sequence of lateralized auditory, visual, and somatosensory stimuli. Within each sensory domain, we compared amplitudes and latencies of sensory-evoked responses, source activity, and functional connectivity (via phase-locking value) between groups. We found that older adults' early sensory-evoked responses were greater in amplitude than those of young adults in all three modalities, which coincided with enhanced source activity in auditory, visual, and somatosensory cortices. Older adults also showed stronger neural synchrony than young adults between superior prefrontal and sensory cortices; and in older adults, the degree of phase synchrony was positively correlated with the magnitude of source activity in sensory areas. Critically, older adults who showed enhanced neural activity in one sensory domain also showed enhanced activity in other modalities. Together, these findings support the common cause hypothesis of aging and highlight the role of prefrontal regions in exerting top-down control over sensory cortices.SIGNIFICANCE STATEMENT A prominent theory of aging posits that age-related declines in sensory processing across domains are related to a single common neurobiological mechanism. However, the neural evidence supporting this common cause hypothesis has remained elusive. Our study revealed robust age-related changes in three sensory domains across a range of neural metrics. Importantly, older adults who showed increased neural activity within one sensory domain also showed enhanced neural activity in the other two sensory modalities. No such relation among activity in sensory cortices was observed in young adults. Age-related increases in neural activity in sensory cortices coincided with enhanced neural synchrony between the PFC and sensory cortices, underlining the importance of the PFC in regulating sensory processing.

Unexpected structures formed by the kinase RET C634R mutant extracellular domain suggest potential oncogenic mechanisms in MEN2A.

The RET receptor tyrosine kinase plays a pivotal role in cell survival, proliferation, and differentiation, and its abnormal activation leads to cancers through receptor fusions or point mutations. Mutations that disrupt the disulfide network in the extracellular domain (ECD) of RET drive multiple endocrine neoplasia type 2A (MEN2A), a hereditary syndrome associated with the development of thyroid cancers. However, structural details of how specific mutations affect RET are unclear. Here, we present the first structural insights into the ECD of the RET(C634R) mutant, the most common mutation in MEN2A. Using electron microscopy, we demonstrate that the C634R mutation causes ligand-independent dimerization of the RET ECD, revealing an unusual tail-to-tail conformation that is distinct from the ligand-induced signaling dimer of WT RET. Additionally, we show that the RETC634R ECD dimer can form complexes with at least two of the canonical RET ligands and that these complexes form very different structures than WT RET ECD upon ligand binding. In conclusion, this structural analysis of cysteine-mutant RET ECD suggests a potential key mechanism of cancer induction in MEN2A, both in the absence and presence of its native ligands, and may offer new targets for therapeutic intervention.

Diverse reaction behaviors of artificial ubiquinones in mitochondrial respiratory complex I.

The ubiquinone (UQ) reduction step catalyzed by NADH-UQ oxidoreductase (mitochondrial respiratory complex I) is key to triggering proton translocation across the inner mitochondrial membrane. Structural studies have identified a long, narrow, UQ-accessing tunnel within the enzyme. We previously demonstrated that synthetic oversized UQs, which are unlikely to transit this narrow tunnel, are catalytically reduced by native complex I embedded in submitochondrial particles but not by the isolated enzyme. To explain this contradiction, we hypothesized that access of oversized UQs to the reaction site is obstructed in the isolated enzyme because their access route is altered following detergent solubilization from the inner mitochondrial membrane. In the present study, we investigated this using two pairs of photoreactive UQs (pUQm-1/pUQp-1 and pUQm-2/pUQp-2), with each pair having the same chemical properties except for a ∼1.0 Å difference in side-chain widths. Despite this subtle difference, reduction of the wider pUQs by the isolated complex was significantly slower than of the narrower pUQs, but both were similarly reduced by the native enzyme. In addition, photoaffinity-labeling experiments using the four [125I]pUQs demonstrated that their side chains predominantly label the ND1 subunit with both enzymes but at different regions around the tunnel. Finally, we show that the suppressive effects of different types of inhibitors on the labeling significantly changed depending on [125I]pUQs used, indicating that [125I]pUQs and these inhibitors do not necessarily share a common binding cavity. Altogether, we conclude that the reaction behaviors of pUQs cannot be simply explained by the canonical UQ tunnel model.

Asymmetric information flow in brain networks supporting expressive language in childhood.

Low-beta (13-23 Hz) event-related desynchrony (ERD), a neural signature of expressive language, lateralizes from bilateral to left hemisphere in development. In contrast, low-beta event-related synchrony (ERS), thought to reflect inhibition, lateralizes from bilateral to the right hemisphere across development. Using whole-brain directed connectivity analyses, we aimed to characterize hemispheric and regional contributions to expressive language, in childhood. We studied 80 children and adolescents, 4 to less than 19 years of age, performing covert auditory verb generation in magnetoencephalography. Outdegree, indegree, and betweenness centrality were used to differentiate regions acting as drivers, receivers, and bridging hubs, respectively. The number of suprathreshold connections significantly increased with age for delta band (p < .01). Delta outflow was mapped to left inferior frontal gyrus (IFG), while regions of right hemisphere, including right IFG, showed significant inflow. The right parietal cortex showed significant ERS, but without corresponding outdegree or indegree. Betweenness mapped to midline cortical and subcortical structures. Results suggest Broca's area develops a driving role in the language network, while Broca's homologue receives information without necessarily propagating it. Subcortical and midline hubs act as intrahemispheric relays. Findings suggest that Broca's homologue is inhibited during expressive language, in development.

Regulation of microglia-mediated inflammation by host lncRNA Gm20559 upon flaviviral infection.

BACKGROUND: Japanese Encephalitis Virus (JEV) and West Nile Viruses (WNV) are neurotropic flaviviruses which cause neuronal death and exaggerated glial activation in the central nervous system. Role of host long non coding RNAs in shaping microglial inflammation upon flavivirus infections has been unexplored. This study attempted to decipher the role of lncRNA Gm20559 in regulating microglial inflammatory response in context of flaviviruses. METHODS: Antisense oligonucleotide LNA Gapmers designed against lncRNA Gm20559 and non-specific site (negative control) were used for Gm20559 knockdown in JEV and WNV-infected N9 microglial cells. Upon establishing successful Gm20559 knockdown, expression of various proinflammatory cytokines, chemokines, interferon-stimulated genes (ISGs) and RIG-I were checked by qRT-PCR and cytometric bead array. Western Blotting was done to analyse the phosphorylation level of various inflammatory markers and viral non-structural protein expression. Plaque Assays were employed to quantify viral titres in microglial supernatant upon knocking down Gm20559. Effect of microglial supernatant on HT22 neuronal cells was assessed by checking expression of apoptotic protein and viral non-structural protein by Western Blotting. RESULTS: Upregulation in Gm20559 expression was observed in BALB/c pup brains, primary microglia as well as N9 microglia cell line upon both JEV and WNV infection. Knockdown of Gm20559 in JEV and WNV-infected N9 cell led to the reduction of major proinflammatory cytokines - IL-1ß, IL-6, IP-10 and IFN-ß. Inhibition of Gm20559 upon JEV infection in N9 microglia also led to downregulation of RIG-I and OAS-2, which was not the case in WNV-infected N9 microglia. Phosphorylation level of P38 MAPK was reduced in case of JEV-infected N9 microglia and not WNV-infected N9 microglia. Whereas phosphorylation of NF-κB pathway was unchanged upon Gm20559 knockdown in both JEV and WNV-infected N9 microglia. However, treating HT22 cells with JEV and WNV-infected microglial supernatant with and without Gm20559 could not trigger cell death or influence viral replication. CONCLUSION: Knockdown studies on lncRNA Gm20559 suggests its pivotal role in maintaining the inflammatory milieu of microglia in flaviviral infection by modulating the expression of various pro-inflammatory cytokines. However, Gm20559-induced increased microglial proinflammatory response upon flavivirus infection fails to trigger neuronal death.