image_1_Distinct Gene Profiles of Bone Marrow-Derived Macrophages and Microglia During Neurotropic Coronavirus-Induced Demyelination.TIF

<p>Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination and axonal loss. Demyelinating lesions are associated with infiltrating T lymphocytes, bone marrow-derived macrophages (BMDM), and activated resident microglia. Tissue damage is thought to be mediated by T cell produced cytokines and chemokines, which activate microglia and/or BMDM to both strip myelin and produce toxic factors, ultimately damaging axons and promoting disability. However, the relative contributions of BMDM and microglia to demyelinating pathology are unclear, as their identification in MS tissue is difficult due to similar morphology and indistinguishable surface markers when activated. The CD4 T cell-induced autoimmune murine model of MS, experimental autoimmune encephalitis (EAE), in which BMDM are essential for demyelination, has revealed pathogenic and repair-promoting phenotypes associated with BMDM and microglia, respectively. Using a murine model of demyelination induced by a gliatropic coronavirus, in which BMDM are redundant for demyelination, we herein characterize gene expression profiles of BMDM versus microglia associated with demyelination. While gene expression in CNS infiltrating BMDM was upregulated early following infection and subsequently sustained, microglia expressed a more dynamic gene profile with extensive mRNA upregulation coinciding with peak demyelination after viral control. This delayed microglia response comprised a highly pro-inflammatory and phagocytic profile. Furthermore, while BMDM exhibited a mixed phenotype of M1 and M2 markers, microglia repressed the vast majority of M2-markers. Overall, these data support a pro-inflammatory and pathogenic role of microglia temporally remote from viral control, whereas BMDM retained their gene expression profile independent of the changing environment. As demyelination is caused by multifactorial insults, our results highlight the plasticity of microglia in responding to distinct inflammatory settings, which may be relevant for MS pathogenesis.</p

Unusual Recognition and Separation of Hydrated Metal Sulfates [M₂(μ-SO₄)₂(H₂O)_<i>n</i>, M = Zn^II, Cd^II, Co^II, Mn^II] by a Ditopic Receptor

A ditopic receptor <b>L1</b>, having metal binding bis­(2-picolyl) donor and anion binding urea group, is synthesized and explored toward metal sulfate recognition via formation of dinuclear assembly, (<b>L1</b>)₂M₂(SO₄)₂. Mass spectrometric analysis, ¹H-DOSY NMR, and crystal structure analysis reveal the existence of a dinuclear assembly of MSO₄ with two units of <b>L1</b>. ¹H NMR study reveals significant downfield chemical shift of −NH protons of urea moiety of <b>L1</b> selectively with metal sulfates (e.g., ZnSO₄, CdSO₄) due to second-sphere interactions of sulfate with the urea moiety. Variable-temperature ¹H NMR studies suggest the presence of intramolecular hydrogen bonding interaction toward metal sulfate recognition in solution state, whereas intermolecular H-bonding interactions are observed in solid state. In contrast, anions in their tetrabutylammonium salts fail to interact with the urea −NH probably due to poor acidity of the tertiary butyl urea group of <b>L1</b>. Metal sulfate binding selectivity in solution is further supported by isothermal titration calorimetric studies of <b>L1</b> with different Zn salts in dimethyl sulfoxide (DMSO), where a binding affinity is observed for ZnSO₄ (<i>K</i>_a = 1.23 × 10⁶), which is 30- to 50-fold higher than other Zn salts having other counteranions in DMSO. Sulfate salts of Cd^II/Co^II also exhibit binding constants in the order of ∼1 × 10⁶ as in the case of ZnSO₄. Positive role of the urea unit in the selectivity is confirmed by studying a model ligand <b>L2</b>, which is devoid of anion recognition urea unit. Structural characterization of four MSO₄ [M = Zn^II, Cd^II, Co^II, Mn^II] complexes of <b>L1</b>, that is, complex <b>1</b>, [(<b>L1</b>)₂(Zn)₂(μ-SO₄)₂]; complex <b>2</b>, [(<b>L1</b>)₂(H₂O)₂(Cd)₂(μ-SO₄)₂]; complex <b>3</b>, [(<b>L1</b>)₂(H₂O)₂(Co)₂(μ-SO₄)₂]; and complex <b>4</b>, [(<b>L1</b>)₂(H₂O)₂(Mn)₂(μ-SO₄)₂], reveal the formation of sulfate-bridged eight-membered crownlike binuclear complexes, similar to one of the concentration-dependent dimeric forms of MSO₄ as observed in solid state. Finally, <b>L1</b> is found to be highly efficient in removing ZnSO₄ from both aqueous and semiaqueous medium as complex <b>1</b> in the presence of other competing Zn^II salts via precipitation through crystallization. Powder X-ray diffraction analysis has also confirmed bulk purity of complex <b>1</b> obtained from the above competitive crystallization experiment

Role of Wingtip Substituents on Benzene-Platform-Based Tetrapodal Ligands toward the Formation of a Self-Assembled Silver Carbene Cage

Tetrapodal imidazolium ligands <b>L</b>^<b>1</b>–<b>L</b>^<b>3</b> as their PF₆^– salts are synthesized in good yields by reacting 1,2,4,5-tetrakis­(bromomethyl)­benzene with <i>N</i>-methylimidazole, <i>N</i>-benzylimidazole, and <i>N</i>-ethylimidazole, respectively. Single-crystal X-ray diffraction studies of <b>L</b>^<b>1</b>·4PF₆, <b>L</b>^<b>2</b>·4PF₆, and <b>L</b>^<b>3</b>·4PF₆ show the chair conformation of the tetrapodal imidazoliums (<b>L</b>^<b>1</b>–<b>L</b>^<b>3</b>), where 1,5- and 2,4-imidazolium moieties are oriented in opposite directions of the benzene plane. The PF₆^– salts of <b>L</b>^<b>1</b>–<b>L</b>^<b>3</b> are reacted with Ag₂O to synthesize different silver complexes of N-heterocyclic carbene (NHC), <b>1</b>–<b>3</b>, respectively, in good yields. Crystals of all three complexes suitable for single-crystal X-ray diffraction study are also isolated. Structural analysis of <b>1</b>, i.e., the complex of <b>L</b>^<b>1</b>·4PF₆ containing methyl as a wingtip substituent, and Ag₂O shows the formation of a bimetallic silver NHC (NHC-Ag) complex, [(<b>L</b>^<b>1</b>-4H)·2Ag]·2PF₆, which is rotationally disordered over an inversion of symmetry of the space group <i>P</i>2₁/<i>c</i>. Elemental analysis and solution-state ¹H and ¹³C NMR studies confirm the above molecular formula of complex <b>1</b>. When <b>L</b>^<b>2</b>·4PF₆ functionalized with the benzyl wingtip moiety is explored for similar complexation with Ag₂O, the isolated complex <b>2</b> shows the formation of a simple NHC-Ag complex with molecular formula [(<b>L</b>^<b>2</b>-4H)·2Ag]·2PF₆, as observed in the case of <b>1</b>. Interestingly, the reaction of <b>L</b>^<b>3</b>·4PF₆ containing ethyl as the wingtip substituent and Ag₂O shows the formation of a silver-ion-assisted tetranuclear molecular box of [Ag₄(<b>L</b>^<b>3</b>-4H)₂]⁴⁺ (<b>3</b>)

Role of Wingtip Substituents on Benzene-Platform-Based Tetrapodal Ligands toward the Formation of a Self-Assembled Silver Carbene Cage

Tetrapodal imidazolium ligands <b>L</b>^<b>1</b>–<b>L</b>^<b>3</b> as their PF₆^– salts are synthesized in good yields by reacting 1,2,4,5-tetrakis­(bromomethyl)­benzene with <i>N</i>-methylimidazole, <i>N</i>-benzylimidazole, and <i>N</i>-ethylimidazole, respectively. Single-crystal X-ray diffraction studies of <b>L</b>^<b>1</b>·4PF₆, <b>L</b>^<b>2</b>·4PF₆, and <b>L</b>^<b>3</b>·4PF₆ show the chair conformation of the tetrapodal imidazoliums (<b>L</b>^<b>1</b>–<b>L</b>^<b>3</b>), where 1,5- and 2,4-imidazolium moieties are oriented in opposite directions of the benzene plane. The PF₆^– salts of <b>L</b>^<b>1</b>–<b>L</b>^<b>3</b> are reacted with Ag₂O to synthesize different silver complexes of N-heterocyclic carbene (NHC), <b>1</b>–<b>3</b>, respectively, in good yields. Crystals of all three complexes suitable for single-crystal X-ray diffraction study are also isolated. Structural analysis of <b>1</b>, i.e., the complex of <b>L</b>^<b>1</b>·4PF₆ containing methyl as a wingtip substituent, and Ag₂O shows the formation of a bimetallic silver NHC (NHC-Ag) complex, [(<b>L</b>^<b>1</b>-4H)·2Ag]·2PF₆, which is rotationally disordered over an inversion of symmetry of the space group <i>P</i>2₁/<i>c</i>. Elemental analysis and solution-state ¹H and ¹³C NMR studies confirm the above molecular formula of complex <b>1</b>. When <b>L</b>^<b>2</b>·4PF₆ functionalized with the benzyl wingtip moiety is explored for similar complexation with Ag₂O, the isolated complex <b>2</b> shows the formation of a simple NHC-Ag complex with molecular formula [(<b>L</b>^<b>2</b>-4H)·2Ag]·2PF₆, as observed in the case of <b>1</b>. Interestingly, the reaction of <b>L</b>^<b>3</b>·4PF₆ containing ethyl as the wingtip substituent and Ag₂O shows the formation of a silver-ion-assisted tetranuclear molecular box of [Ag₄(<b>L</b>^<b>3</b>-4H)₂]⁴⁺ (<b>3</b>)

A Cyanuric Acid Platform Based Tripodal Bis-heteroleptic Ru(II) Complex of Click Generated Ligand for Selective Sensing of Phosphates via C–H···Anion Interaction

A new bis-heteroleptic trinuclear Ru­(II) complex (<b>1</b>[PF₆]₆) has been synthesized from electron deficient cyanuric acid platform based copper-catalyzed azide–alkyne cycloaddition, i.e., CuAAC click generated ligand, 1,3,5-tris [(2-aminoethyl-1H-1,2,3-triazol-4-yl)-pyridine]-1,3,5-triazinane-2,4,6-trione (<b>L1)</b>. Complex <b>1</b>[PF₆]₆ displays weak luminescence (ϕ_f = 0.002) at room temperature with a short lifetime of ∼5 ns in acetonitrile. It shows selective sensing of hydrogen pyrophosphate (HP₂O₇^3–) through 20-fold enhanced emission intensity (ϕ_f = 0.039) with a 15 nm red shift in emission maxima even in the presence of a large excess of various competitive anions like F^–, Cl^–, AcO^–, BzO^–, NO₃^–, HCO₃^–, HSO₄^–, HO^–, and H₂PO₄^– in acetonitrile. Selective change in the decay profile as well as in the lifetime of <b>1</b>[PF₆]₆ in the presence of HP₂O₇^3– (108 ns) further supports its selectivity toward HP₂O₇^3–. UV–vis and photoluminescence titration profiles and corresponding Job’s plot analyses suggest 1:3 host–guest stoichiometric binding between <b>1</b>[PF₆]₆ and HP₂O₇^3–. High emission enhancement of <b>1</b>[PF₆]₆ in the presence of HP₂O₇^3– has resulted in the detection limit of the anion being as low as 0.02 μM. However, <b>1</b>[PF₆]₆ shows selectivity toward higher analogues of phosphates (e.g., ATP, ADP, and AMP) over HP₂O₇^3–/H₂PO₄^– in 10% Tris HCl buffer (10 mM)/acetonitrile medium. Downfield shifting of the triazole C–H in a ¹H NMR titration study confirms that the binding of HP₂O₇^3–/H₂PO₄^– is occurring via C–H···anion interaction. The single crystal X-ray structure of complex <b>1</b> having NO₃^– counteranion, <b>1</b>[NO₃]₆ shows binding of NO₃^– with complex <b>1</b> via C–H···NO₃^– interactions

Bis-Heteroleptic Ruthenium(II) Complex of a Triazole Ligand as a Selective Probe for Phosphates

A new bis-heteroleptic ruthenium­(II) complex (<b>1</b>) of 2-(1-methyl-1<i>H</i>-1,2,3-triazol-4-yl) pyridine (<b>L</b>) ligand was extensively explored for anion sensing studies. <b>1</b>[PF₆]₂ shows selective sensing of dihydrogen phosphate (H₂PO₄^–)/hydrogen pyrophosphate (HP₂O₇^3–) among halides, HCO₃^–, AcO^–, NO₃^–, ClO₄^–, HSO₄^–, OH^–, BzO^–, H₂PO₄^–, and HP₂O₇^3– in acetonitrile. Enhancement of emission intensity of <b>1</b>[PF₆]₂ along with a 10 nm red shift of the emission maximum is observed in the presence of H₂PO₄^–/HP₂O₇^3– selectively. The photoluminescence (PL) titration experiment of <b>1</b>[PF₆]₂ results in binding constants (<i>K</i>_a) of 5.28 × 10⁴ M^–1 and 4.67 × 10⁴ M^–1 for H₂PO₄^– and HP₂O₇^3–, respectively, which is in good agreement with the <i>K</i>_a values obtained from UV–vis titration experiments (2.97 × 10⁴ M^–1 and 2.45 × 10⁴ M^–1 for H₂PO₄^– and HP₂O₇^3–, respectively). High selectivity of <b>1</b>[PF₆]₂ toward these two anions in acetonitrile is further confirmed by PL intensity measurement of <b>1</b>[PF₆]₂ upon addition of these two anions in the presence of a large excess of other competitive anions. Further, considerable changes in the lifetime (τ) as well as in the decay pattern of <b>1</b>[PF₆]₂ in the presence of H₂PO₄^–/HP₂O₇^3– among all tested anions support the selective binding property of <b>1</b>[PF₆]₂ toward these two anions. Significant downfield shift of the triazole −CH proton of <b>1</b>[PF₆]₂ with 1 equiv of H₂PO₄^– (Δδ = 0.26 ppm) and HP₂O₇^3– (Δδ = 0.23 ppm) in deuterated dimethyl sulfoxide proclaim binding mechanism via C–H···anion interaction in solution state. Finally, single-crystal X-ray structural analysis confirms the first example of dihydrogen pyrophosphate (H₂P₂O₇^2–) recognition via solitary C–H···anion interactions

Identification of VHY/Dusp15 as a Regulator of Oligodendrocyte Differentiation through a Systematic Genomics Approach

<div><p>Multiple sclerosis (MS) is a neuroinflammatory disease characterized by a progressive loss of myelin and a failure of oligodendrocyte (OL)-mediated remyelination, particularly in the progressive phases of the disease. An improved understanding of the signaling mechanisms that control differentiation of OL precursors may lead to the identification of new therapeutic targets for remyelination in MS. About 100 mammalian Protein Tyrosine Phosphatases (PTPs) are known, many of which are involved in signaling both in health and disease. We have undertaken a systematic genomic approach to evaluate PTP gene activity in multiple sclerosis autopsies and in related <em>in vivo</em> and <em>in vitro</em> models of the disease. This effort led to the identification of Dusp15/VHY, a PTP previously believed to be expressed only in testis, as being transcriptionally regulated during OL differentiation and in MS lesions. Subsequent RNA interference studies revealed that Dusp15/VHY is a key regulator of OL differentiation. Finally, we identified PDGFR-beta and SNX6 as novel and specific Dusp15 substrates, providing an indication as to how this PTP might exert control over OL differentiation.</p> </div

Ten-point standardized rating scale for EAE clinical score monitoring.

<p>The final score for each animal is determined by the addition of all the above mentioned categories. Note: Score  =  15 for dead animal.</p

PTPs the most strongly modulated during EAE in mice spinal cord and cerebellum.

<p>Number of PTP genes significantly modulated during the EAE time course in the spinal cord and in the cerebellum has been monitored and represented in two graphics. The number of PTP genes modulated increases dramatically over time. At day 28, the number of PTP genes modulated decreases until a basal level in cerebellum but remains high in the spinal cord. The highest fold changes in gene expression versus Sham animals have been reported in the table. Most of these PTPs have already been described in inflammatpry processes. Statistical analysis were performed using student <i>t-</i>test.</p

Schematic overview of the experimental approach.

<p>Schematic overview of the experimental approach.</p