IL-17A Orchestrates Reactive Oxygen Species/HIF1α-Mediated Metabolic Reprogramming in Psoriasis.

Immune cell-derived IL-17A is one of the key pathogenic cytokines in psoriasis, an immunometabolic disorder. Although IL-17A is an established regulator of cutaneous immune cell biology, its functional and metabolic effects on nonimmune cells of the skin, particularly keratinocytes, have not been comprehensively explored. Using multiomics profiling and systems biology-based approaches, we systematically uncover significant roles for IL-17A in the metabolic reprogramming of human primary keratinocytes (HPKs). High-throughput liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy revealed IL-17A-dependent regulation of multiple HPK proteins and metabolites of carbohydrate and lipid metabolism. Systems-level MitoCore modeling using flux-balance analysis identified IL-17A-mediated increases in HPK glycolysis, glutaminolysis, and lipid uptake, which were validated using biochemical cell-based assays and stable isotope-resolved metabolomics. IL-17A treatment triggered downstream mitochondrial reactive oxygen species and HIF1α expression and resultant HPK proliferation, consistent with the observed elevation of these downstream effectors in the epidermis of patients with psoriasis. Pharmacological inhibition of HIF1α or reactive oxygen species reversed IL-17A-mediated glycolysis, glutaminolysis, lipid uptake, and HPK hyperproliferation. These results identify keratinocytes as important target cells of IL-17A and reveal its involvement in multiple downstream metabolic reprogramming pathways in human skin.

The Largest Subunit of Human TFIIIC Complex, TFIIIC220, a Lysine Acetyltransferase Targets Histone H3K18.

TFIIIC is a multi-subunit complex required for tRNA transcription by RNA polymerase III. Human TFIIIC holo-complex possesses lysine acetyltransferase activity that aids in relieving chromatin-mediated repression for RNA polymerase III-mediated transcription and chromatin assembly. Here we have characterized the acetyltransferase activity of the largest and DNA-binding subunit of TFIIIC complex, TFIIIC220. Purified recombinant human TFIIIC220 acetylated core histones H3, H4 and H2A in vitro. Moreover, we have identified the putative catalytic domain of TFIIIC220 that efficiently acetylates core histones in vitro. Mutating critical residues of the putative acetyl-CoA binding 'P loop' drastically reduced the catalytic activity of the acetyltransferase domain. Further analysis showed that the knockdown of TFIIIC220 in mammalian cell lines dramatically reduces global H3K18 acetylation level, which was rescued by overexpression of the putative acetyltransferase domain of human TFIIIC220. Our findings indicated a possibility of a crucial role for TFIIIC220 in maintaining acetylation homeostasis in the cell.

Translationally Controlled Tumor Protein-Mediated Stabilization of Host Antiapoptotic Protein MCL-1 Is Critical for Establishment of Infection by Intramacrophage Parasite Leishmania donovani.

In the early phase of infection, the intramacrophage pathogen Leishmania donovani protects its niche with the help of the antiapoptotic protein myeloid cell leukemia-1 (MCL-1). Whether Leishmania could exploit MCL-1, an extremely labile protein, at the late phase is still unclear. A steady translational level of MCL-1 observed up to 48 h postinfection and increased caspase-3 activity in MCL-1-silenced infected macrophages documented its importance in the late hours of infection. The transcript level of MCL-1 showed a sharp decline at 6 h postinfection, and persistent MCL-1 expression in cyclohexamide-treated cells negates the possibility of de novo protein synthesis, thereby suggesting infection-induced stability. Increased ubiquitination, a prerequisite for proteasomal degradation of MCL-1, was also found to be absent in the late hours of infection. Lack of interaction with its specific E3 ubiquitin ligase MULE (MCL-1 ubiquitin ligase E3) and specific deubiquitinase USP9X prompted us to search for blockade of the ubiquitin-binding site in MCL-1. To this end, TCTP (translationally controlled tumor protein), a well-known binding partner of MCL-1 and antiapoptotic regulator, was found to be strongly associated with MCL-1 during infection. Phosphorylation of TCTP, a requirement for MCL-1 binding, was also increased in infected macrophages. Knockdown of TCTP decreased MCL-1 expression and short hairpin RNA-mediated silencing of TCTP in an infected mouse model of visceral leishmaniasis showed decreased parasite burden and induction of liver cell apoptosis. Collectively, our investigation revealed a key mechanism of how L. donovani exploits TCTP to establish infection within the host.

Utilizing microblogs for optimized real-time resource allocation in post-disaster scenarios.

In the aftermath of a disaster event, it is of utmost important to ensure efficient allocation of emergency resources (e.g. food, water, shelter, medicines) to locations where the resources are needed (need-locations). There are several challenges in this goal, including the identification of resource-needs and resource-availabilities in real time, and deciding a policy for allocating the available resources from where they are available (availability-locations) to the need-locations. In recent years, social media, and especially microblogging sites such as Twitter, have emerged as important sources of real-time information on disasters. There have been some attempts to identify resource-needs and resource-availabilities from microblogging sites. However, there has not been much work on having a policy for optimized and real-time resource allocation based on the information obtained from microblogs. Specifically, the allocation of critical resources must be done in an optimal way by understanding the utility of emergency resources at various need-locations at a given point of time. This paper attempts to develop such a utility-driven model for optimized resource allocation in a post-disaster scenario, based on information extracted from microblogs in real time. Experiments show that the proposed model achieves much better allocation of resources than baseline models-the allocation by the proposed model is not only more efficient in terms of quickly bringing down resource-deficits at various need-locations, but also more fair in distributing the available resources among the various need-locations.

Increased host ATP efflux and its conversion to extracellular adenosine is crucial for establishing Leishmania infection.

Intracellular survival of Leishmania donovani demands rapid production of host ATP for its sustenance. However, a gradual decrease in intracellular ATP in spite of increased glycolysis suggests ATP efflux during infection. Accordingly, upon infection, we show here that ATP is exported and the major exporter was pannexin-1, leading to raised extracellular ATP levels. Extracellular ATP shows a gradual decrease after the initial increase, and analysis of cell surface ATP-degrading enzymes revealed induction of the ectonucleotidases CD39 and CD73. Ectonucleotidase-mediated ATP degradation leads to increased extracellular adenosine (eADO), and inhibition of CD39 and CD73 in infected cells decreased adenosine concentration and parasite survival, documenting the importance of adenosine in infection. Inhibiting adenosine uptake by cells did not affect parasite survival, suggesting that eADO exerts its effect through receptor-mediated signalling. We also show that Leishmania induces the expression of adenosine receptors A2AR and A2BR, both of which are important for anti-inflammatory responses. Treating infected BALB/c mice with CD39 and CD73 inhibitors resulted in decreased parasite burden and increased host-favourable cytokine production. Collectively, these observations indicate that infection-induced ATP is exported, and after conversion into adenosine, propagates infection via receptor-mediated signalling.

Spectrophotometric Assessment of Heme Oxygenase-1 Activity in Leishmania-infected Macrophages.

Heme oxygenase-1 (HO-1) is a stress responsive enzyme that metabolizes heme and releases free iron, carbon monoxide (CO), and biliverdin (BV), which rapidly undergoes conversion to bilirubin (BL). Estimation of bilirubin is the basis of HO-1 assay. HO-1 activity is widely employed to determine antioxidant response of cells under different physiological stress environment. Intra-macrophage infection often acts as such a stress inducer and measurement of HO-1 activity in infected cells indicates the ability of pathogens towards modulating oxidative response of host. The present protocol describes analysis of HO-1 activity in infected macrophages by spectrophotometric method, which is much less complex and therefore advantageous over other methods like high-performance liquid chromatography, radiochemical methods and detection of CO by gas chromatography. The main steps include: (1) Preparation of macrophage microsomal fraction containing HO-1 (2) Isolation of rat liver cytosolic fraction containing biliverdin reductase and (3) Assessment of heme oxygenase-1 activity by spectrophotometric detection of bilirubin. This method provides a simple and sensitive approach to measure cellular antioxidant response under infected condition.

Corrigendum: The Dietary Flavonoid, Luteolin, Negatively Affects Neuronal Differentiation.

[This corrects the article DOI: 10.3389/fnmol.2019.00041.].

The Dietary Flavonoid, Luteolin, Negatively Affects Neuronal Differentiation.

Luteolin, a polyphenolic plant flavonoid, has been attributed with numerous beneficial properties like anti-cancer, antioxidant, and anti-inflammatory action. Luteolin has been reported earlier to be neuroprotective in models of spinal cord injury and traumatic brain injury and also induces neurite outgrowth in PC12 cells. However, the effect of luteolin on early differentiation, which might be important for its beneficial effects, is unknown. In this report, we show that luteolin negatively affects early differentiation of embryonic stem cells, hampering the formation of embryoid bodies. At later stages of differentiation, luteolin specifically inhibits neuronal differentiation, where the expression of early neuronal markers is suppressed, whereas luteolin treatment does not inhibit expression of meso- and endodermal markers. Further, in a developing zebrafish model, luteolin treatment leads to fewer numbers of mitotic cells in the brain. These specific effects of luteolin on neuronal differentiation could possibly be due to its ability to inhibit the lysine acetyltransferase, p300, since the structurally closely related p300 non-inhibitor flavonoid, apigenin, does not inhibit neuronal differentiation. These results show that luteolin perturbs neuronal differentiation of embryonic stem cells.

Leishmania donovani Exploits Macrophage Heme Oxygenase-1 To Neutralize Oxidative Burst and TLR Signaling-Dependent Host Defense.

Suppression of host oxidative burst is essential for survival of the intracellular parasite Leishmania donovani Screening of macrophage antioxidant enzymes during infection revealed marked upregulation of the heme-degrading enzyme, heme oxygenase-1 (HO-1). Moreover, HO-1-silenced RAW macrophages depicted increased superoxide production and decreased parasite survival. HO-1 induction decreased cellular heme content, thereby inhibiting the heme-dependent maturation of gp91phox, a catalytic component of major reactive oxygen species-producing enzyme NAD(P)H oxidase. Decreased gp91phox expression resulted in reduced stability of p22phox, another component of the catalytic center of NAD(P)H oxidase. Replenishing infected cells with exogenous heme reversed these effects and restored NAD(P)H oxidase activity. Persistent HO-1 expression at late hour of infection prompted us to investigate its effect on other host defense parameters, and inhibition study revealed a reciprocal relationship of HO-1 with host proinflammatory responses. Among all the HO-1-mediated heme degradation products (CO, Fe, and biliverdin), only CO documented potent anti-inflammatory effects. Quenching of CO during infection increased the production of disease-resolving cytokines IL-12 and TNF-α. Coimmunoprecipitation experiments revealed that CO inhibited the interaction of TLR4 with MyD88 and TIR domain-containing adapter-inducing IFN-ß, thereby dampening the activation of NF-κB and IFN regulatory factor 3-mediated production of proinflammatory cytokines. Administration of HO-1 inhibitor tin protoporphyrin IX dichloride in infected BALB/c mice led to a decrease in liver and spleen parasite burden along with increased production of IL-12 and TNF-α. These results suggest that HO-1 on one hand inhibits reactive oxygen species generation and on the other hand downregulates host favorable cytokine responses, thereby facilitating intramacrophage parasite survival.

Recent advances in understanding Leishmania donovani infection: The importance of diverse host regulatory pathways.

Invasion of host cell by pathogens induce various intracellular signalling pathways. The host cell through the initiation of these signalling circuits desperately wants to get rid of the pathogen, whereas the pathogen tries to subvert these defence strategies to create an environment for their successful survival. Leishmania spp. is not an exception. Leishmania have to evolve a range of strategic mechanisms to neutralize macrophage defensive arsenals which enable the parasite to replicate within the phagolysosome of infected host. Understanding these signalling mechanisms in detail will not only improve our basic knowledge of host-pathogen interaction but will also help us to develop effective drug targets not only against leishmaniasis but also for many other macrophage associated diseases. © 2018 IUBMB Life, 70(7):593-601, 2018.

Chemical and genetic rescue of an ep300 knockdown model for Rubinstein Taybi Syndrome in zebrafish.

EP300 is a member of the EP300/CBP family of lysine acetyltransferases (KATs) with multiple roles in development and physiology. Loss of EP300/CBP activity in humans causes a very rare congenital disorder called Rubinstein Taybi Syndrome (RSTS). The zebrafish genome has two co-orthologs of lysine acetyltransferase EP300 (KAT3B) in zebrafish viz. ep300a and ep300b. Chemical inhibition of Ep300 with C646, a competitive inhibitor and morpholino-based genetic knockdown of ep300a and ep300b cause defects in embryonic development reminiscent of the human RSTS syndrome. Remarkably, overexpression of Ep300a KAT domain results in near complete rescue of the jaw development defects, a characteristic feature of RSTS in human suggesting the dispensability of the protein-interaction and DNA-binding domains for at least some developmental roles of Ep300. We also perform a chemical screen and identify two inhibitors of deacetylases, CHIC35 and HDACi III, that can partially rescue the RSTS-like phenotypes. Thus, modeling rare human genetic disorders in zebrafish allows for functional understanding of the genes involved and can also yield small molecule candidates towards therapeutic goals.

Recombinant Approaches for Microbial Xylanases: Recent Advances and Perspectives.

Xylanases are crucial enzymes to hydrolyse the xylan of plant hemicellulose in order to complete the carbon cycle. Xylanases have been used widely in a variety of industries ranging from food and feed industry to pulp and paper industry. Most of the industrial processes which using xylanase requires a thermostable and alkali stable enzyme. Therefore it is desired to produce high thermostable and alkali stable xylanase with high activity. In this review a number of molecular techniques are used in this genomic era have been utilized to enhance physiological properties of xylanases for greater commercial application in the industries. A brief outline of diverse molecular techniques such as genome-walking PCR, thermal asymmetric interlaced PCR (TAIL-PCR), staggered extension process (StEP) recombination method, site-directed mutagenesis together with metagenomic approaches have been discussed which are used to improve the charactestics of xylanases and its production. Metagenomic studies along with directed evolution by mutant creation have also been reported as an effective tool in improvement of xylanase activity and its properties. This review comprehensively describes the recent reports and different combinatorial approaches towards production of efficient xylanases.

The role of PD-1 in regulation of macrophage apoptosis and its subversion by Leishmania donovani.

Programmed death-1 receptor (PD-1) expressed in many immune cells is known to trigger T-cell exhaustion but the significance of macrophage-associated PD-1 in relevance to macrophage apoptosis is not known. This study is aimed to delineate whether PD-1 pathway has any role in eliciting macrophage apoptosis and, if so, then how the intra-macrophage parasite, Leishmania donovani modulates PD-1 pathway for protecting its niche. Resting macrophages when treated with H2O2 showed increased PD-1 expression and apoptosis, which was further enhanced on PD-1 agonist treatment. The administration of either PD-1 receptor or PD-1 ligand-blocking antibodies reversed the process thus documenting the involvement of PD-1 in macrophage apoptosis. On the contrary, L. donovani-infected macrophages showed decreased PD-1 expression concurrent with inhibition of apoptosis. The activation of PD-1 pathway was found to negatively regulate the phosphorylation of pro-survival AKT, which was reversed during infection. Infection-induced PD-1 downregulation led to the activation of AKT resulting in phosphorylation and subsequent inhibition of proapoptotic protein BAD. Strong association of SHP2 (a SH2-containing ubiquitously expressed tyrosine-specific protein phosphatase) with PD-1 along with AKT deactivation observed in H2O2-treated macrophages was reversed by L. donovani infection. Kinetic analysis coupled with inhibitor-based approach and knockdown experiments demonstrated that L. donovani infection actively downregulated the PD-1 by deactivating NFATc1 as revealed by its reduced nuclear translocation. The study thus elucidates the detailed mechanism of the role of PD-1 in macrophage apoptosis and its negative modulation by Leishmania for their intracellular survival.

Medical Requirements During a Natural Disaster: A Case Study on WhatsApp Chats Among Medical Personnel During the 2015 Nepal Earthquake.

OBJECTIVE: The objective of this study was to explore a log of WhatsApp messages exchanged among members of the health care group Doctors For You (DFY) while they were providing medical relief in the aftermath of the Nepal earthquake in April 2015. Our motivation was to identify medical resource requirements during a disaster in order to help government agencies and other responding organizations to be better prepared in any upcoming disaster. METHODS: A large set of WhatsApp (WhatsApp Inc, Mountain View, CA) messages exchanged among DFY members during the Nepal earthquake was collected and analyzed to identify the medical resource requirements during different phases of relief operations. RESULTS: The study revealed detailed phase-wise requirements for various types of medical resources, including medicines, medical equipment, and medical personnel. The data also reflected some of the problems faced by the medical relief workers in the earthquake-affected region. CONCLUSIONS: The insights from this study may help not only the Nepalese government, but also authorities in other earthquake-prone regions of the world to better prepare for similar disasters in the future. Moreover, real-time analysis of such online data during a disaster would aid decision-makers in dynamically formulating resource-mapping strategies. (Disaster Med Public Health Preparedness. 2017;11:652-655).

Leishmania donovani Exploits Myeloid Cell Leukemia 1 (MCL-1) Protein to Prevent Mitochondria-dependent Host Cell Apoptosis.

Apoptosis is one of the mechanisms used by host cells to remove unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Among all the anti-apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels and compared with infected control, MCL-1-silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection-induced MCL-1 expression was regulated by transcription factor CREB (cAMP-response element-binding protein) and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70-silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release-mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited the macrophage anti-apoptotic protein MCL-1 to prevent BAK-mediated mitochondria-dependent apoptosis thereby protecting its niche, which is essential for disease progression.