Roles of epidermal growth factor receptor, claudin-1 and occludin in multi-step entry of hepatitis C virus into polarized hepatoma spheroids.

The multi-step process of hepatitis C virus (HCV) entry is facilitated by various host factors, including epidermal growth factor receptor (EGFR) and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), which are thought to function at later stages of the HCV entry process. Using single particle imaging of HCV infection of polarized hepatoma spheroids, we observed that EGFR performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. We previously observed, and in this study confirmed, that EGFR is not required for HCV migration to the tight junction. EGFR is required for the recruitment of clathrin to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization at a stage following the recruitment of clathrin. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into APPL1- and EEA1-associated early endosomes, which form the site of virion uncoating. The tight junction proteins, CLDN1 and OCLN, function at two distinct stages of HCV entry. Despite its appreciated function as a "late receptor" in HCV entry, CLDN1 is required for efficient HCV virion accumulation at the tight junction. Huh-7.5 cells lacking CLDN1 accumulate HCV virions primarily at the initial basolateral surface. OCLN is required for the late stages of virion internalization. This study produced further insight into the unusually complex HCV endocytic process.

Industrial-era decline in subarctic Atlantic productivity.

Marine phytoplankton have a crucial role in the modulation of marine-based food webs1, fishery yields2 and the global drawdown of atmospheric carbon dioxide3. However, owing to sparse measurements before satellite monitoring in the twenty-first century, the long-term response of planktonic stocks to climate forcing is unknown. Here, using a continuous, multi-century record of subarctic Atlantic marine productivity, we show that a marked 10 ± 7% decline in net primary productivity has occurred across this highly productive ocean basin over the past two centuries. We support this conclusion by the application of a marine-productivity proxy, established using the signal of the planktonic-derived aerosol methanesulfonic acid, which is commonly identified across an array of Greenlandic ice cores. Using contemporaneous satellite-era observations, we demonstrate the use of this signal as a robust and high-resolution proxy for past variations in spatially integrated marine productivity. We show that the initiation of declining subarctic Atlantic productivity broadly coincides with the onset of Arctic surface warming4, and that productivity strongly covaries with regional sea-surface temperatures and basin-wide gyre circulation strength over recent decades. Taken together, our results suggest that the decline in industrial-era productivity may be evidence of the predicted5 collapse of northern Atlantic planktonic stocks in response to a weakened Atlantic Meridional Overturning Circulation6-8. Continued weakening of this Atlantic Meridional Overturning Circulation, as projected for the twenty-first century9,10, may therefore result in further productivity declines across this globally relevant region.

Low oxidation state and hydrido group 2 complexes: synthesis and applications in the activation of gaseous substrates.

Numerous industrial processes utilise gaseous chemical feedstocks to produce useful chemical products. Atmospheric and other small molecule gases, including anthropogenic waste products (e.g. carbon dioxide), can be viewed as sustainable building blocks to access value-added chemical commodities and materials. While transition metal complexes have been well documented in the reduction and transformation of these substrates, molecular complexes of the terrestrially abundant alkaline earth metals have also demonstrated promise with remarkable reactivity reported towards an array of industrially relevant gases over the past two decades. This review covers low oxidation state and hydrido group 2 complexes and their role in the reduction and transformation of a selection of important gaseous substrates towards value-added chemical products.

The effect of single mutations in Zika virus envelope on escape from broadly neutralizing antibodies.

IMPORTANCE: The wide endemic range of mosquito-vectored flaviviruses-such as Zika virus and dengue virus serotypes 1-4-places hundreds of millions of people at risk of infection every year. Despite this, there are no widely available vaccines, and treatment of severe cases is limited to supportive care. An avenue toward development of more widely applicable vaccines and targeted therapies is the characterization of monoclonal antibodies that broadly neutralize all these viruses. Here, we measure how single amino acid mutations in viral envelope protein affect neutralizing antibodies with both broad and narrow specificities. We find that broadly neutralizing antibodies with potential as vaccine prototypes or biological therapeutics are quantifiably more difficult to escape than narrow, virus-specific neutralizing antibodies.

Reductions of Arenes using a Magnesium-Dinitrogen Complex.

In this contribution, we present "Birch-type", and other reductions of simple arenes by the potassium salt of an anionic magnesium dinitrogen complex, [{K(TCHPNON)Mg}2(µ-N2)] (TCHPNON=4,5-bis(2,4,6-tricyclohexylanilido)-2,7-diethyl-9,9-dimethyl-xanthene), which acts as a masked dimagnesium(I) diradical in these reactions. This reagent is non-hazardous, easy-to-handle, and in some cases provides access to 1,4-cyclohexadiene reduction products under relatively mild reaction conditions. This system works effectively to reduce benzene, naphthalene and anthracene through magnesium-bound "Birch-type" reduction intermediates. Cyclohexadiene products can be subsequently released from the magnesium centres by protonolysis with methanol. In contrast, the reduction of substituted arenes is less selective and involves competing reaction pathways. For toluene and 1,3,5-triphenylbenzene, the structural authentication of "Birch-type" reduction intermediates is conclusive, although the formation of corresponding 1,4-cyclohexadiene derivatives was low yielding. Reduction of anisole did not yield an isolable "Birch-type" intermediate, but instead gave a C-O activation product. Treating triphenylphosphine with [{K(TCHPNON)Mg}2(µ-N2)] resulted in the extrusion of both biphenyl and dinitrogen to afford a magnesium(II) phosphanide [{K(TCHPNON)Mg(µ-PPh2)}2]. Reduction of fluorobenzene proceeded via C-F activation of the arene, and isolation of the magnesium(II) fluoride [{K(TCHPNON)Mg(µ-F)}2]. Finally, the two-electron reduction of 1,3,5,7-cyclooctatetraene (COT) with [{K(TCHPNON)Mg}2(µ-N2)] yielded a complex, [{K(TCHPNON)Mg}2(µ-COT)], incorporating the aromatic dianion (COT2-).

Pinacol Cross-Coupling Promoted by an Aluminyl Anion.

A simple sequential addition protocol for the reductive coupling of ketones and aldehydes by a potassium aluminyl grants access to unsymmetrical pinacolate derivatives. Isolation of an aluminium ketyl complex presents evidence for the accessibility of radical species. Product release from the aluminium centre was achieved using an iodosilane, forming the disilylated 1,2-diol and a neutral aluminium iodide, thereby demonstrating the steps required to generate a closed synthetic cycle for pinacol (cross) coupling at an aluminyl anion.

Synthesis and Reactivity of Discrete Europium(II) Hydride Complexes.

The bulky ß-diketiminate ligand frameworks [BDIDCHP]- and [BDIDipp/Ar]- (BDI=[HC{C(Me)2N-Dipp/Ar}2]- (Dipp=2,6-diisopropylphenyl (Dipp); Ar=2,6-dicyclohexylphyenyl (DCHP) or 2,4,6-tricyclohexylphyenyl (TCHP)) have been developed for the kinetic stabilisation of the first europium (II) hydride complexes, [(BDIDCHP)Eu(µ-H)]2, [(BDIDipp/DCHP)Eu(µ-H)]2 and [(BDIDipp/TCHP)Eu(µ-H)]2, respectively. These complexes represent the first step beyond the current lanthanide(II) hydrides that are all based on ytterbium. Tuning the steric profile of ß-diketiminate ligands from a symmetrical to unsymmetrical disposition, enhanced solubility and stability in the solution-state. This provides the first opportunity to study the structure and bonding of these novel Eu(II) hydride complexes crystallographically, spectroscopically and computationally, with their preliminary reactivity investigated.

Enforcing Metal-Arene Interactions in Bulky p-Terphenyl Bis(anilide) Complexes of Group 2 Metals (Be-Ba): Potential Precursors for Low-Oxidation-State Alkaline Earth Metal Systems.

An extremely bulky p-terphenyl bis(aniline), p-C6H4{C6H4[N(H)TCHP]-2}2 (TCHP = 2,4,6-tricyclohexylphenyl) TCHPTerphH2, has been developed. Deprotonation of a less bulky analogue, DipTerphH2 (Dip = 2,6-diisopropylphenyl), with BePh2 affords the bimetallic system, [(BePh)2(µ-DipTerph)] 1. Treating either TCHPTerphH2 or DipTerphH2 with Mg{CH2(SiMe3)}2 gives the monomeric bis(anilide) complexes [Mg(ArTerph)] (Ar = Dip 2, TCHP 3) which display rare examples of η6-arene coordination to the metal center. Treating 2 with THF leads to partial dissociation of the Mg···arene interaction and formation of [Mg(DipTerph)(THF)] 4. Reactions of the bis(aniline)s with the group 2 metal amides [M{N(SiMe3)2}2] afford dimeric, structurally analogous compounds [{M(ArTerph)}2] (Ar = Dip, M = Ca 5, Sr 6, Ba 7; Ar = TCHP, M = Ca 8, Sr 9, Ba 10) which display intermolecular M···arene interactions in the solid state. Computational studies have shown that the intramolecular M···Î·6-arene interactions in models of the ether-free metal bis(anilide) compounds are largely electrostatic in nature. Reductions of these compounds with alkali metals led to mixtures of unidentified products.

Nonlinear rise in Greenland runoff in response to post-industrial Arctic warming.

The Greenland ice sheet (GrIS) is a growing contributor to global sea-level rise1, with recent ice mass loss dominated by surface meltwater runoff2,3. Satellite observations reveal positive trends in GrIS surface melt extent4, but melt variability, intensity and runoff remain uncertain before the satellite era. Here we present the first continuous, multi-century and observationally constrained record of GrIS surface melt intensity and runoff, revealing that the magnitude of recent GrIS melting is exceptional over at least the last 350 years. We develop this record through stratigraphic analysis of central west Greenland ice cores, and demonstrate that measurements of refrozen melt layers in percolation zone ice cores can be used to quantifiably, and reproducibly, reconstruct past melt rates. We show significant (P < 0.01) and spatially extensive correlations between these ice-core-derived melt records and modelled melt rates5,6 and satellite-derived melt duration4 across Greenland more broadly, enabling the reconstruction of past ice-sheet-scale surface melt intensity and runoff. We find that the initiation of increases in GrIS melting closely follow the onset of industrial-era Arctic warming in the mid-1800s, but that the magnitude of GrIS melting has only recently emerged beyond the range of natural variability. Owing to a nonlinear response of surface melting to increasing summer air temperatures, continued atmospheric warming will lead to rapid increases in GrIS runoff and sea-level contributions.

Reductive Coupling of a Diazoalkane Derivative Promoted by a Potassium Aluminyl and Elimination of Dinitrogen to Generate a Reactive Aluminium Ketimide.

The reaction of 9-diazo-9H-fluorene (fluN2 ) with the potassium aluminyl K[Al(NON)] ([NON]2- =[O(SiMe2 NDipp)2 ]2- , Dipp=2,6-iPr2 C6 H3 ) affords K[Al(NON)(κN1 ,N3 -{(fluN2 )2 })] (1). Structural analysis shows a near planar 1,4-di(9H-fluoren-9-ylidene)tetraazadiide ligand that chelates to the aluminium. The thermally induced elimination of dinitrogen from 1 affords the neutral aluminium ketimide complex, Al(NON)(N=flu)(THF) (2) and the 1,2-di(9H-fluoren-9-yl)diazene dianion as the potassium salt, [K2 (THF)3 ][fluN=Nflu] (3). The reaction of 2 with N,N'-diisopropylcarbodiimide (iPrN=C=NiPr) affords the aluminium guanidinate complex, Al(NON){N(iPr)C(N=CMe2 )N(CHflu)} (4), showing a rare example of reactivity at a metal ketimide ligand. Density functional theory (DFT) calculations have been used to examine the bonding in the newly formed [(fluN2 )2 ]2- ligand in 1 and the ketimide bonding in 2. The mechanism leading to the formation of 4 has also been studied using this technique.

Three Oxidative Addition Routes of Alkali Metal Aluminyls to Dihydridoaluminates and Reactivity with CO2.

Three distinct routes are reported to the soluble, dihydridoaluminate compounds, AM[Al(NONDipp )(H)2 ] (AM=Li, Na, K, Rb, Cs; [NONDipp ]2- =[O(SiMe2 NDipp)2 ]2- ; Dipp=2,6-iPr2 C6 H3 ) starting from the alkali metal aluminyls, AM[Al(NONDipp )]. Direct H2 hydrogenation of the heavier analogues (AM=Rb, Cs) produced the first examples of structurally characterized rubidium and caesium dihydridoaluminates, although harsh conditions were required for complete conversion. Using 1,4-cyclohexadiene (1,4-CHD) as an alternative hydrogen source in transfer hydrogenation reactions provided a lower energy pathway to the full series of products for AM=Li-Cs. A further moderation in conditions was noted for the thermal decomposition of the (silyl)(hydrido)aluminates, AM[Al(NONDipp )(H)(SiH2 Ph)]. Probing the reaction of Cs[Al(NONDipp )] with 1,4-CHD provided access to a novel inverse sandwich complex, [{Cs(Et2 O)}2 {Al(NONDipp )(H)}2 (C6 H6 )], containing the 1,4-dialuminated [C6 H6 ]2- dianion and representing the first time that an intermediate in the commonly utilized oxidation process of 1,4-CHD to benzene has been trapped. The synthetic utility of the newly installed Al-H bonds has been demonstrated by their ability to reduce CO2 under mild conditions to form the bis-formate AM[Al(NONDipp )(O2 CH)2 ] compounds, which exhibit a diverse series of eyecatching bimetallacyclic structures.

Synthesis and Reactivity of Alkali Metal Hydrido-Magnesiate Complexes which Exhibit Group 1 Metal Counter-Cation Specific Stability.

Reactions of the series of alkali metal amides M(HMDS) (M = Li-Cs; HMDS = [N(SiMe3)2]-) with the neutral magnesium(II) hydride compound [Mg(BDIDipp)(µ-H)]2 (BDIDipp = [CH{C(Me)NDipp}2], Dipp = 2,6-iPr2-C6H3) have been carried out. When M = Li or Na, the reactions yielded Mg(BDIDipp)(HMDS) and MH as the primary products. In the sodium amide reaction, [Na2(HMDS)][{Mg(BDIDipp)}2(H)3] was obtained as a low-yield by-product. When M = K-Cs, the reactions gave the group 1 metal hydrido-magnesiates, M2[Mg(BDIDipp)(HMDS)(H)]2·(benzene)n (n = 0 or 1), the thermal stability of which increases with the increasing molecular weight of the alkali metal involved. Reactions of Cs2[Mg(BDIDipp)(HMDS)(H)]2·(benzene) with 18-crown-6 and CO gave the first monomeric alkali metal hydrido-magnesiate [Cs(18-crown-6)][Mg(BDIDipp)(HMDS)(H)] and the ethenediolate complex Cs2[{Mg(BDIDipp)(HMDS)}2(µ-C2H2O2)], respectively. The new synthetic route to alkali metal hydrido-magnesiates described herein may facilitate further reactivity studies of this rare compound class.

Coordination and Activation of N2 at Low-Valent Magnesium using a Heterobimetallic Approach: Synthesis and Reactivity of a Masked Dimagnesium Diradical.

The activation of dinitrogen (N2 ) by transition metals is central to the highly energy intensive, heterogeneous Haber-Bosch process. Considerable progress has been made towards more sustainable homogeneous activations of N2 with d- and f-block metals, though little success has been had with main group metals. Here we report that the reduction of a bulky magnesium(II) amide [(TCHP NON)Mg] (TCHP NON=4,5-bis(2,4,6-tricyclohexylanilido)-2,7-diethyl-9,9-dimethyl-xanthene) with 5 % w/w K/KI yields the magnesium-N2 complex [{K(TCHP NON)Mg}2 (µ-N2 )]. DFT calculations and experimental data show that the dinitrogen unit in the complex has been reduced to the N2 2- dianion, via a transient anionic magnesium(I) radical. The compound readily reductively activates CO, H2 and C2 H4 , in reactions in which it acts as a masked dimagnesium(I) diradical.

Synthesis, Characterization, and Structural Analysis of AM[Al(NONDipp)(H)(SiH2Ph)] (AM = Li, Na, K, Rb, Cs) Compounds, Made Via Oxidative Addition of Phenylsilane to Alkali Metal Aluminyls.

We report the oxidative addition of phenylsilane to the complete series of alkali metal (AM) aluminyls [AM{Al(NONDipp)}]2 (AM = Li, Na, K, Rb, and Cs). Crystalline products (1-AM) have been isolated as ether or THF adducts, [AM(L)n][Al(NONDipp)(H)(SiH2Ph)] (AM = Li, Na, K, Rb, L = Et2O, n = 1; AM = Cs, L = THF, n = 2). Further to this series, the novel rubidium rubidiate, [{Rb(THF)4}2(Rb{Al(NONDipp)(H)(SiH2Ph)}2)]+ [Rb{Al(NONDipp)(H)(SiH2Ph)}2]-, was isolated during an attempted recrystallization of Rb[Al(NONDipp)(H)(SiH2Ph)] from a hexane/THF mixture. Structural and spectroscopic characterizations of the series 1-AM confirm the presence of µ-hydrides that bridge the aluminum and alkali metals (AM), with multiple stabilizing AM···π(arene) interactions to either the Dipp- or Ph-substituents. These products form a complete series of soluble, alkali metal (hydrido) aluminates that present a platform for further reactivity studies.

Potassium Aluminyl Promoted Carbonylation of Ethene.

The potassium aluminyl [K{Al(NONDipp )}]2 ([NONDipp ]2- =[O{SiMe2 NDipp}2 ]2- , Dipp=2,6-iPr2 C6 H3 ) activates ethene towards carbonylation with CO under mild conditions. An isolated bis-aluminacyclopropane compound reacted with CO via carbonylation of an Al-C bond, followed by an intramolecular hydrogen shift to form K2 [Al(NONDipp )(µ-CH2 CH=CO-1κ2 C1,3 -2κO)Al(NONDipp )Et]. Restricting the chemistry to a mono-aluminium system allowed isolation of [Al(NONDipp )(CH2 CH2 CO-κ2 C1,3 )]- , which undergoes thermal isomerisation to form the [Al(NONDipp )(CH2 CH=CHO-κ2 C,O)]- anion. DFT calculations highlight the stabilising influence of incorporated benzene at multiple steps in the reaction pathways.

Oxidative Addition of Hydridic, Protic, and Nonpolar E-H Bonds (E = Si, P, N, or O) to an Aluminyl Anion.

The aluminyl anion K[Al(NONDipp)] {NONDipp = [O(SiMe2NDipp)2]2-; Dipp = 2,6-iPr2C6H3} engages in oxidative additions with the E-H (E = Si, P, N, or O) bonds of phenylsilane (PhSiH3), mesityl phosphane (MesPH2; Mes = 2,4,6-Me3C6H2), 2,6-di-iso-propylaniline (DippNH2), and 2,6-di-tert-butyl-4-methylphenol (ArOH). The resulting (hydrido)aluminate salts are formed regardless of the E-H bond polarity. All of the products were characterized by nuclear magnetic resonance and infrared spectroscopic techniques and single-crystal X-ray diffraction. This study highlights the versatility of aluminyl anions to activate hydridic, acidic, and (essentially) nonpolar E-H bonds.

Synthesis and Reactivity of Acyclic Germanimines: Silyl Rearrangement and Cycloadditions.

We report the synthesis of aromatic germanimines [(HMDS)2Ge═NAr] (Ar = Ph, Mes, Dipp; Mes = 2,4,6-Me3C6H2, Dipp = 2,6-iPr2C6H3) and an investigation into their associated reactivity. [(HMDS)2Ge═NPh] decomposes above -30 °C, while [(HMDS)2Ge═NDipp] engages in an intramolecular reaction at 60 °C. [(HMDS)2Ge═NMes] was shown to rearrange via a 1,3-silyl migration to give [(HMDS){(SiMe3)(Mes)N}Ge(NSiMe3)] in a 1:7 equilibrium mixture at room temperature. These latter germanimines react with unsaturated polar substrates such as CO2, ketones, and arylisocyanate via a [2 + 2] cycloaddition pathway.

Controlling Al-M Interactions in Group 1 Metal Aluminyls (M = Li, Na, and K). Facile Conversion of Dimers to Monomeric and Separated Ion Pairs.

The aluminyl compounds [M{Al(NONDipp)}]2 (NONDipp = [O(SiMe2NDipp)2]2-, Dipp = 2,6-iPr2C6H3), which exist as contacted dimeric pairs in both the solution and solid states, have been converted to monomeric ion pairs and separated ion pairs for each of the group 1 metals, M = Li, Na, and K. The monomeric ion pairs contain discrete, highly polarized Al-M bonds between the aluminum and the group 1 metal and have been isolated with monodentate (THF, M = Li and Na) or bidentate (TMEDA, M = Li, Na, and K) ligands at M. The separated ion pairs comprise group 1 cations that are encapsulated by polydentate ligands, rendering the aluminyl anion, [Al(NONDipp)]- "naked". For M = Li, this structure type was isolated as the [Li(TMEDA)2]+ salt directly from a solution of the corresponding contacted dimeric pair in neat TMEDA, while the polydentate [2.2.2]cryptand ligand was used to generate the separated ion pairs for the heavier group 1 metals M = Na and K. This work shows that starting from the corresponding contacted dimeric pairs, the extent of the Al-M interaction in these aluminyl systems can be readily controlled with appropriate chelating reagents.

Dihydrogen Activation by Lithium- and Sodium-Aluminyls.

To date, aluminyl anions have been exclusively isolated as their potassium salts. We report herein the synthesis of the lithium and sodium aluminyls, M2 [Al(NONDipp )]2 (M=Li, Na. NONDipp =[O(SiMe2 NDipp)2 ]2- ; Dipp=2,6-iPr2 C6 H3 ). Both compounds crystallize from non-coordinating solvent as "slipped" contacted dimeric pairs with strong M⋅⋅⋅π(aryl) interactions. Isolation from Et2 O solution affords the monomeric ion pairs (NONDipp )Al-M(Et2 O)2 , which contain discrete Al-Li and Al-Na bonds. The ability of the full series of Li, Na and K aluminyls to activate dihydrogen is reported.

Zika Virus Subverts Stress Granules To Promote and Restrict Viral Gene Expression.

Flaviviruses limit the cell stress response by preventing the formation of stress granules (SGs) and modulate viral gene expression by subverting different proteins involved in the stress granule pathway. In this study, we investigated the formation of stress granules during Zika virus (ZIKV) infection and the role stress granule proteins play during the viral life cycle. Using immunofluorescence and confocal microscopy, we determined that ZIKV disrupted the formation of arsenite-induced stress granules and changed the subcellular distribution, but not the abundance or integrity, of stress granule proteins. We also investigated the role of different stress granule proteins in ZIKV infection by using target-specific short interfering RNAs to deplete Ataxin2, G3BP1, HuR, TIA-1, TIAR, and YB1. Knockdown of TIA-1 and TIAR affected ZIKV protein and RNA levels but not viral titers. Conversely, depletion of Ataxin2 and YB1 decreased virion production despite having only a small effect on ZIKV protein expression. Notably, however, depletion of G3BP1 and HuR decreased and increased ZIKV gene expression and virion production, respectively. Using an MR766 Gaussia Luciferase reporter genome together with knockdown and overexpression assays, G3BP1 and HuR were found to modulate ZIKV replication. These data indicate that ZIKV disrupts the formation of stress granules by sequestering stress granule proteins required for replication, where G3BP1 functions to promote ZIKV infection while HuR exhibits an antiviral effect. The results of ZIKV relocalizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis and contribute to cellular gene dysregulation and ZIKV pathogenesis.IMPORTANCE Many viruses inhibit SGs. In this study, we observed that ZIKV restricts SG assembly, likely by relocalizing and subverting specific SG proteins to modulate ZIKV replication. This ZIKV-SG protein interaction is interesting, as many SG proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different SG proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of ZIKV modulating SG assembly and subverting specific SG proteins are alterations to cellular mRNA transcription, splicing, stability, and translation. Such changes in cellular ribostasis could profoundly affect neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine ZIKV infection. Our study provides new insights into virus-host interactions and the identification of the SG proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus.