TBK1 regulates regeneration of pancreatic β-cells

Small-molecule inhibitors of non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) have shown to stimulate β-cell regeneration in multiple species. Here we demonstrate that TBK1 is predominantly expressed in β-cells in mammalian islets. Proteomic and transcriptome analyses revealed that genetic silencing of TBK1 increased expression of proteins and genes essential for cell proliferation in INS-1 832/13 rat β-cells. Conversely, TBK1 overexpression decreased sensitivity of β-cells to the elevation of cyclic AMP (cAMP) levels and reduced proliferation of β-cells in a manner dependent on the activity of cAMP-hydrolyzing phosphodiesterase 3 (PDE3). While the mitogenic effect of (E)3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA) is derived from inhibition of TBK1, PIAA augmented glucose-stimulated insulin secretion (GSIS) and expression of β-cell differentiation and proliferation markers in human embryonic stem cell (hESC)-derived β-cells and human islets. TBK1 expression was increased in β-cells upon diabetogenic insults, including in human type 2 diabetic islets. PIAA enhanced expression of cell cycle control molecules and β-cell differentiation markers upon diabetogenic challenges, and accelerated restoration of functional β-cells in streptozotocin (STZ)-induced diabetic mice. Altogether, these data suggest the critical function of TBK1 as a β-cell autonomous replication barrier and present PIAA as a valid therapeutic strategy augmenting functional β-cells

Towards Goals to Refine Prophylactic and Therapeutic Strategies Against COVID-19 Linked to Aging and Metabolic Syndrome

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gave rise to the coronavirus disease 2019 (COVID-19) pandemic. A strong correlation has been demonstrated between worse COVID-19 outcomes, aging, and metabolic syndrome (MetS), which is primarily derived from obesity-induced systemic chronic low-grade inflammation with numerous complications, including type 2 diabetes mellitus (T2DM). The majority of COVID-19 deaths occurs in people over the age of 65. Individuals with MetS are inclined to manifest adverse disease consequences and mortality from COVID-19. In this review, we examine the prevalence and molecular mechanisms underlying enhanced risk of COVID-19 in elderly people and individuals with MetS. Subsequently, we discuss current progresses in treating COVID-19, including the development of new COVID-19 vaccines and antivirals, towards goals to elaborate prophylactic and therapeutic treatment options in this vulnerable population

Metabolic Variations, Antioxidant Potential, and Antiviral Activity of Different Extracts of Eugenia singampattiana (an Endangered Medicinal Plant Used by Kani Tribals, Tamil Nadu, India) Leaf

Eugenia singampattiana is an endangered medicinal plant used by the Kani tribals of South India. The plant had been studied for its antioxidant, antitumor, antihyperlipidemic, and antidiabetic activity. But its primary and secondary metabolites profile and its antiviral properties were unknown, and so this study sought to identify this aspect in Eugenia singampattiana plant through different extraction methods along with their activities against porcine reproductive and respiratory syndrome virus (PRRSV). The GC-MS analysis revealed that 11 primary metabolites showed significant variations among the extracts. Except for fructose all other metabolites were high with water extract. Among 12 secondary metabolites showing variations, the levels of 4-hydroxy benzoic acid, caffeic acid, rutin, ferulic acid, coumaric acid, epigallocatechin gallate, quercetin, myricetin, and kaempferol were high with methanol extract. Since the flavonoid content of methanol extracts was high, the antioxidant potential, such as ABTS, and phosphomolybdenum activity increased. The plants antiviral activity against PRRSV was for the first time confirmed and the results revealed that methanol 25 µg and 75 to 100 µg in case of water extracts revealed antiviral activity

Crimean-Congo hemorrhagic fever virus nucleocapsid protein has dual RNA binding modes

<div><p>Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5’ and 3’ termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely.</p></div

Comparison of binding affinities by filter binding analysis and biolayer interferometry.

<p>(A). Sequence and most probable secondary structure of wild type panhandle from S-segment vRNA. The thirty nucleotides from 5’ and 3’ termini of S-segment vRNA panhandle are shown below the wild type panhandle. (B). Interaction of purified N protein with the [α³²P] CTP labeled RNA molecules shown in panel A was carried out by filter binding analysis, as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184935#pone.0184935.g001" target="_blank">Fig 1</a> and also in materials and methods. The binding profiles from filter binding analysis are shown next to the respective RNA. Insets show the double reciprocal plots as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184935#pone.0184935.g001" target="_blank">Fig 1C</a>. (C). Binding analysis of purified N protein with RNA molecules shown in panel A was also carried out using Bio-layer interferometry (BLI). Representative BLI sensograms showing over time association and dissociation of N protein (0.22 μM or 0.44 μM) with the RNA of interest. The BLI sensograms are shown next to the representative RNA.</p

Modeled 3D structure of CCHFV N protein.

<p>Potential RNA-binding regions of CCHFV N protein. The electrostatic surface potential of N protein was visualized by PyMol. The positive surface is colored blue and the negative surface is colored red. The isolated positively charged surfaces constituted by residues (K339, K343, K346, R384, K411, H453) and (R134, R140, Q467) and (H195, H197, K222, R225, K282, K286) may be involved in the formation of hypothetical binding pockets for the vRNA panhandle and single strand RNA. A cartoon of CCHFV N protein in which tryptophan residues are highlighted as pink spheres is shown at the right.</p

Binding parameters calculated by filter binding analysis for the association of CCHFV N protein with different RNA molecules in RNA binding buffer at two NaCl concentrations.

<p>Binding parameters calculated by filter binding analysis for the association of CCHFV N protein with different RNA molecules in RNA binding buffer at two NaCl concentrations.</p

Filter binding analysis.

<p>(A). SDS-PAGE of bacterially expressed and purified CCHFV N protein. Lane 1 shows the protein ladder. Lane 2 shows the bacterial lysate before the purification of N protein. Lane 3 shows the CCHFV N protein purified by NiNTA column chromatography using native purification protocol. (B). The RNA molecules shown in this panel were synthesized by T7 transcription and tested for binding with purified N protein. The prediction of secondary structures by mFold revealed that complementary nucleotides from 5’ and 3’ termini undergo base pairing and form hairpin structure, referred as panhandle structure in this manuscript. The nucleotides deleted at the 5’ terminus (G<i>luc</i> RNA 5’ del) and 3’ terminus (G<i>luc</i> RNA 3’ del) are not shown. (C). Binding profiles for the purified CCHFV N protein with the RNA molecules from panel B are shown. Increasing concentrations of N protein were added to a fixed concentration of [α³²P] CTP labeled RNA and the mixture was incubated at room temperature for 45 minutes, followed by filtration through nitrocellulose filter. The RNA-N protein complex retained on the filter was quantified by scintillation counter and the radioactive signal was used to calculate the percent RNA bound at each input concentration of N protein using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184935#pone.0184935.e001" target="_blank">Eq 1</a>, to generate the binding profile. Inset shows the double reciprocal plot for the calculation of ΔR_max, used to calculate the percent bound RNA in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184935#pone.0184935.e001" target="_blank">Eq 1</a>. The dissociation constant (K_d) was calculated as mentioned in materials and methods. Standard deviation was calculated from three independent experiments.</p

CCHFV N protein does not have RNA unwinding activity.

<p>An RNA heteroduplex formed between radiolabeled 3’ and cold 5’ UTR sequences of CCHFV S-segment vRNA is shown at the top. Both the UTR sequences contained a stretch of “U” residues. SNV N protein unwinds the heterodupplex from 5’ to 3’ direction and requires a single strand RNA sequence to initiate unwinding. The heteroduplex shown at the top was incubated with either SNV N protein of CCHFV N protein for increasing time intervals. The mixture was fractionated on 10% SDS-PAGE to examine the release of radiolabled 3’ UTR sequence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184935#sec002" target="_blank">materials and methods</a> for details).</p

RNA binding competition experiment.

<p>(A). As previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184935#pone.0184935.ref033" target="_blank">33</a>], a fixed concentration of CCHFV N protein (200 nM) was incubated with a constant concentration of [α³²P] CTP labeled wild type CCHFV S-segment vRNA panhandle (0.01nM) and increasing concentrations (0.1 nM, 1 nM, and 10 nM) of competitor cold vRNA panhandle (open circle), thirty-nucleotide long 5’ NCR sequence (filled square) and thirty nucleotide long 3’ NCR sequence (filled triangle). (B and C). The competition experiment performed in these panels was carried out similar to panel A, except CCHV V N protein (350 nM) was incubated with the constant concentration (0.01nM) of P³²-CTP labeled 5’ NCR (panel B) or P³²-CTP labeled 3’ NCR (Panel C) and increasing concentrations of competitor RNA as mentioned in panel A. (D). To determine whether N protein can simultaneously bind both vRNA panhandle and 5’ UTR sequence, a filter binding assay was performed in which a fixed concentration of radiolabeled vRNA panhandle was incubated with increasing concentrations of N protein-5’ NCR complex. The complex was generated by incubating N protein with saturating concentrations of cold 5’ NCR sequence, as mentioned in the text. Reaction mixtures were filtered through a nitrocellulose filter, and the percentage of hot S segment RNA retained on the filter was plotted <i>versus</i> input concentrations N-5’ NCR complex to generate the binding profile for the calculation of <i>Kd</i>. In a similar experiment increasing concentrations of preformed complex between N protein and cold vRNA panhandle were added to a fixed concentration of radiolabeled 5’ NCR (E) and 3’ NCR (F). Reaction mixtures were filtered through a nitrocellulose filter as mentioned above for the generation of binding profiles.</p