Direct Measurement of Intermolecular Mechanical Force for Nonspecific Interactions between Small Molecules.

Mechanical force can evaluate intramolecular interactions in macromolecules. Because of the rapid motion of small molecules, it is extremely challenging to measure mechanical forces of nonspecific intermolecular interactions. Here, we used optical tweezers to directly examine the intermolecular mechanical force (IMMF) of nonspecific interactions between two cholesterols. We found that IMMFs of dimeric cholesterol complexes were dependent on the orientation of the interaction. The surprisingly high IMMF in cholesterol dimers (∼30 pN) is comparable to the mechanical stability of DNA secondary structures. Using Hess-like cycles, we quantified that changes in free energy of solubilizing cholesterol (ΔGsolubility) by ß-cyclodextrin (ßCD) and methylated ßCD (Me-ßCD) were as low as -16 and -27 kcal/mol, respectively. Compared to the ΔGsolubility of cholesterols in water (5.1 kcal/mol), these values indicated that cyclodextrins can easily solubilize cholesterols. Our results demonstrated that the IMMF can serve as a generic and multipurpose variable to dissect nonspecific intermolecular interactions among small molecules into orientational components.

Culturomics Approaches Expand the Diagnostic Accuracy for Sexually Transmitted Infections.

Sexually transmitted infections (STIs) are a major health concern with clinical manifestations being acknowledged to cause severe reproductive impairment. Research in infectious diseases has been centered around the known major pathogens for decades. However, we have just begun to understand that the microbiota of the female genital tract is of particular importance for disease initiation, infection progression, and pathological outcome. Thus, we are now aware that many poorly described, partially not yet known, or cultured bacteria may pave the way for an infection and/or contribute to disease severity. While sequencing-based methods are an important step in diagnosing STIs, culture-based methods are still the gold-standard method in diagnostic routine, providing the opportunity to distinguish phenotypic traits of bacteria. However, current diagnostic culture routines suffer from several limitations reducing the content of information about vaginal microbiota. A detailed characterization of microbiota-associated factors is needed to assess the impact of single-bacterial isolates from the vaginal community on vaginal health and the containment of STIs. Here we provide current concepts to enable modern culture routines and create new ideas to improve diagnostic approaches with a conjunct usage of bioinformatics. We aim to enable scientists and physicians alike to overcome long-accepted limitations in culturing bacteria of interest to the human health. Eventually, this may improve the quality of culture-based diagnostics, facilitate a research interface, and lead to a broader understanding of the role of vaginal microbiota in reproductive health and STIs.

Regulation of the Mitochondrion-Fatty Acid Axis for the Metabolic Reprogramming of Chlamydia trachomatis during Treatment with ß-Lactam Antimicrobials.

Infection with the obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. Since no vaccine is available to date, antimicrobial therapy is the only alternative in C. trachomatis infection. However, changes in chlamydial replicative activity and the occurrence of chlamydial persistence caused by diverse stimuli have been proven to impair treatment effectiveness. Here, we report the mechanism for C. trachomatis regulating host signaling processes and mitochondrial function, which can be used for chlamydial metabolic reprogramming during treatment with ß-lactam antimicrobials. Activation of signal transducer and activator of transcription 3 (STAT3) is a well-known host response in various bacterial and viral infections. In C. trachomatis infection, inactivation of STAT3 by host protein tyrosine phosphatases increased mitochondrial respiration in both the absence and presence of ß-lactam antimicrobials. However, during treatment with ß-lactam antimicrobials, C. trachomatis increased the production of citrate as well as the activity of host ATP-citrate lyase involved in fatty acid synthesis. Concomitantly, chlamydial metabolism switched from the tricarboxylic acid cycle to fatty acid synthesis. This metabolic switch was a unique response in treatment with ß-lactam antimicrobials and was not observed in gamma interferon (IFN-γ)-induced persistent infection. Inhibition of fatty acid synthesis was able to attenuate ß-lactam-induced chlamydial persistence. Our findings highlight the importance of the mitochondrion-fatty acid interplay for the metabolic reprogramming of C. trachomatis during treatment with ß-lactam antimicrobials.IMPORTANCE The mitochondrion generates most of the ATP in eukaryotic cells, and its activity is used for controlling the intracellular growth of Chlamydia trachomatis Furthermore, mitochondrial activity is tightly connected to host fatty acid synthesis that is indispensable for chlamydial membrane biogenesis. Phospholipids, which are composed of fatty acids, are the central components of the bacterial membrane and play a crucial role in the protection against antimicrobials. Chlamydial persistence that is induced by various stimuli is clinically relevant. While one of the well-recognized inducers, ß-lactam antimicrobials, has been used to characterize chlamydial persistence, little is known about the role of mitochondria in persistent infection. Here, we demonstrate how C. trachomatis undergoes metabolic reprogramming to switch from the tricarboxylic acid cycle to fatty acid synthesis with promoted host mitochondrial activity in response to treatment with ß-lactam antimicrobials.

Naphthoquinone-derivative as a synthetic compound to overcome the antibiotic resistance of methicillin-resistant S. aureus.

The treatment of Staphylococcus aureus (S. aureus) infections has become more difficult due to the emergence of multidrug resistance in the bacteria. Here, we report the synthesis of a lawsone (2-hydroxy-1,4-naphthoquinone)-based compound as an antimicrobial agent against methicillin-resistant S. aureus (MRSA). A series of lawsone-derivative compounds were synthesized by means of tuning the lipophilicity of lawsone and screened for minimum inhibitory concentrations against MRSA to identify a candidate compound that possesses a potent antibacterial activity. The identified lawsone-derivative compound exhibited significantly improved drug resistance profiles against MRSA compared to conventional antibiotics. The therapeutic efficacy of the compound was validated using murine models of wound infection as well as non-lethal systemic infection induced by MRSA. Our study further revealed the multifaceted modes of action of the compound, mediated by three distinctive mechanisms: (1) cell membrane damage, (2) chelation of intracellular iron ions, and (3) generation of intracellular reactive oxygen species.

Crosstalk Between Autophagy and Hypoxia-Inducible Factor-1α in Antifungal Immunity.

Modern medicine is challenged by several potentially severe fungal pathogens such as Aspergillus fumigatus, Candida albicans, or Histoplasma capsulatum. Though not all fungal pathogens have evolved as primary pathogens, opportunistic pathogens can still cause fatal infections in immuno-compromised patients. After infection with these fungi, the ingestion and clearance by innate immune cells is an important part of the host immune response. Innate immune cells utilize two different autophagic pathways, the canonical pathway and the non-canonical pathway, also called microtubule-associated protein 1A/1B-light chain 3 (LC3) -associated pathway (LAP), to clear fungal pathogens from the intracellular environment. The outcome of autophagy-related host immune responses depends on the pathogen and cell type. Therefore, the understanding of underlying molecular mechanisms of autophagy is crucial for the development and improvement of antifungal therapies. One of those molecular mechanisms is the interaction of the transcription-factor hypoxia-inducible factor 1α (HIF-1α) with the autophagic immune response. During this review, we will focus on a comprehensive overview of the role of autophagy and HIF-1α on the outcome of fungal infections.

The HIF-1α/LC3-II Axis Impacts Fungal Immunity in Human Macrophages.

The fungal pathogen Histoplasma capsulatum causes a spectrum of disease, ranging from local pulmonary infection to disseminated disease. The organism seeks residence in macrophages, which are permissive for its survival. Hypoxia-inducible factor 1α (HIF-1α), a principal regulator of innate immunity to pathogens, is necessary for macrophage-mediated immunity to H. capsulatum in mice. In the present study, we analyzed the effect of HIF-1α in human macrophages infected with this fungus. HIF-1α stabilization was detected in peripheral blood monocyte-derived macrophages at 2 to 24 h after infection with viable yeast cells. Further, host mitochondrial respiration and glycolysis were enhanced. In contrast, heat-killed yeasts induced early, but not later, stabilization of HIF-1α. Since the absence of HIF-1α is detrimental to host control of infection, we asked if large amounts of HIF-1α protein, exceeding those induced by H. capsulatum, altered macrophage responses to this pathogen. Exposure of infected macrophages to an HIF-1α stabilizer significantly reduced recovery of H. capsulatum from macrophages and produced a decrement in mitochondrial respiration and glycolysis compared to those of controls. We observed recruitment of the autophagy-related protein LC3-II to the phagosome, whereas enhancing HIF-1α reduced phagosomal decoration. This finding suggested that H. capsulatum exploited an autophagic process to survive. In support of this assertion, inhibition of autophagy activated macrophages to limit intracellular growth of H. capsulatum Thus, enhancement of HIF-1α creates a hostile environment for yeast cells in human macrophages by interrupting the ability of the pathogen to provoke host cell autophagy.

Impact of HIF-1α and hypoxia on fungal growth characteristics and fungal immunity.

Human pathogenic fungi are highly adaptable to a changing environment. The ability to adjust to low oxygen conditions is crucial for colonization and infection of the host. Recently, the impact of mammalian hypoxia-inducible factor-1α (HIF-1α) on fungal immunity has emerged. In this review, the role of hypoxia and HIF-1α in fungal infections is discussed regarding the innate immune response.

Inverse Correlation between IL-10 and HIF-1α in Macrophages Infected with Histoplasma capsulatum.

Hypoxia-inducible factor (HIF)-1α is a transcription factor that regulates metabolic and immune response genes in the setting of low oxygen tension and inflammation. We investigated the function of HIF-1α in the host response to Histoplasma capsulatum because granulomas induced by this pathogenic fungus develop hypoxic microenvironments during the early adaptive immune response. In this study, we demonstrated that myeloid HIF-1α-deficient mice exhibited elevated fungal burden during the innate immune response (prior to 7 d postinfection) as well as decreased survival in response to a sublethal inoculum of H. capsulatum The absence of myeloid HIF-1α did not alter immune cell recruitment to the lungs of infected animals but was associated with an elevation of the anti-inflammatory cytokine IL-10. Treatment with mAb to IL-10 restored protective immunity to the mutant mice. Macrophages (Mϕs) constituted most IL-10-producing cells. Deletion of HIF-1α in neutrophils or dendritic cells did not alter fungal burden, thus implicating Mϕs as the pivotal cell in host resistance. HIF-1α was stabilized in Mϕs following infection. Increased activity of the transcription factor CREB in HIF-1α-deficient Mϕs drove IL-10 production in response to H. capsulatum IL-10 inhibited Mϕ control of fungal growth in response to the activating cytokine IFN-γ. Thus, we identified a critical function for Mϕ HIF-1α in tempering IL-10 production following infection. We established that transcriptional regulation of IL-10 by HIF-1α and CREB is critical for activation of Mϕs by IFN-γ and effective handling of H. capsulatum.

Efficient synthesis of triarylamine-based dyes for p-type dye-sensitized solar cells.

The class of triarylamine-based dyes has proven great potential as efficient light absorbers in inverse (p-type) dye sensitized solar cells (DSSCs). However, detailed investigation and further improvement of p-type DSSCs is strongly hindered by the fact that available synthesis routes of triarylamine-based dyes are inefficient and particularly demanding with regard to time and costs. Here, we report on an efficient synthesis strategy for triarylamine-based dyes for p-type DSSCs. A protocol for the synthesis of the dye-precursor (4-(bis(4-bromophenyl)amino)benzoic acid) is presented along with its X-ray crystal structure. The dye precursor is obtained from the commercially available 4(diphenylamino)benzaldehyde in a yield of 87% and serves as a starting point for the synthesis of various triarylamine-based dyes. Starting from the precursor we further describe a synthesis protocol for the dye 4-{bis[4'-(2,2-dicyanovinyl)-[1,1'-biphenyl]-4-yl]amino}benzoic acid (also known as dye P4) in a yield of 74%. All synthesis steps are characterized by high yields and high purities without the need for laborious purification steps and thus fulfill essential requirements for scale-up.

Synthesis of CuInS2 nanocrystals from a molecular complex - characterization of the orthorhombic domain structure.

CuInS2 nanocrystals were synthesized by thermal decomposition of the molecular precursor [(Me3P)3Cu(SC2H4S)In(i)Pr2] in the presence of oleylamine in dioctyl phthalate. According to X-ray diffraction patterns, the as-synthesized CuInS2 nanocrystals crystallize in the wurtzite type structure. High-resolution transmission electron images and selected area electron diffraction patterns reveal a nanodomain structure. The individual domains are approximately 5-10 nm in size and characterized by short-range cation ordering, which assuming hypothetical long-range order, corresponds to an orthorhombic superstructure (space group Pmc21, a = 4.09 ± 0.01 Å, b = 7.16 ± 0.02 Å and c = 6.56 ± 0.03 Å). The domains are separated by twin and antiphase boundaries.

Tetranuclear organometallic complexes based on 1,2-ethanedithiolate ligands as potential precursors for CuMS2 (M = Ga, In).

The synthesis and characterization of a series of homologous tetranuclear complexes [((i)Pr3PCu)2(MR2)2(SCH2CH2S)2] (MR2 = GaMe2 (2), GaEt2 (3), Ga(i)Pr2 (4), Ga(n)Bu2 (5) and InMe2 (6), InEt2 (7), In(i)Pr2 (8), In(n)Bu2 (9)) and of related compounds [((i)Pr3PCu)3(MR2)(SCH2CH2S)2] (MR2 = GaMe2 (10), GaEt2 (11), Ga(i)Pr2 (12), Ga(n)Bu2 (13) and InMe2 (14), InEt2 (15), In(i)Pr2 (16), In(n)Bu2 (17)) are presented. The molecular structures of these were determined by single crystal X-ray diffraction. Thermolysis processes of all the representatives of 2-9 were investigated by simultaneous thermal analysis and thermal decomposition in a nitrogen atmosphere in a quartz glass tube. According to powder X-ray diffraction studies these thermolysis residues predominantly consist of CuMS2 (M = Ga, In), which mainly occurred in the well known tetragonal chalcopyrite-type structure and partially in the hexagonal wurtzite-type structure. As expected from their composition, representatives of 10-17 form mixtures of CuMS2 and large amounts of Cu(2-x)S upon thermolysis.

New organometallic single-source precursors for CuGaS(2)-polytypism in gallite nanocrystals obtained by thermolysis.

The complex [((i)Pr(3)PCu)(2)(Me(2)Ga)(2)(SCH(2)CH(2)S)(2)] (4) was synthesized from trimethylgallium, [((i)Pr(3)PCu)(4)(SCH(2)CH(2)S)(2)] (1) and ethanedithiol by elimination of methane. The related monomethyl compound [((i)Pr(3)PCu)(2)(MeGaSPh)(2)(SCH(2)CH(2)S)(2)] (5) has been prepared from [((i)Pr(3)PCuSPh)(3)] (2) and [(MeGaSCH(2)CH(2)S)(2)] (3) by a ligand exchange reaction in tetrahydrofuran solution. The molecular structures of 1 and 3-5 were determined by single crystal X-ray diffraction. Thermolysis of 4 and 5 results in the formation of the ternary semiconductor CuGaS(2), gallite. The residue of 5 was characterized using X-ray powder diffraction, energy dispersive X-ray spectroscopy and transmission electron microscopy. The CuGaS(2) crystals obtained are mainly hexagonal plates of around 200 to 300 nm diameter and 10 to 30 nm thickness, exhibiting an unusual metastable hexagonal crystal structure, related to wurtzite. Partially, the usual tetragonal chalcopyrite structure or its disordered cubic zinc-blende analogue is realized by stacking faults, resulting in an overall similarity to the zinc-blende-wurtzite polytypism in ZnS and related compounds.

Syntheses and NMR characterizations of epimeric 4-deoxy-4-fluoro carbohydrates.

The synthesis and NMR characterizations of 1,2,3,6-tetra-O-benzoyl-4-deoxy-4-fluoro-ß-d-galactopyranoside, the 4-deoxy-4-fluoro epimer of an intermediate in the synthesis of a drug substance, needed for use as a potential impurity standard and to confirm the stereoselectivity of a key fluorination step, are described.

Pectenotoxin-2 synthetic studies. 3. Assessment of the capacity for stereocontrolled cyclization to form the entire C1-C26 subunit based upon the double bond geometry across C15-C16.

Second-generation synthetic routes to enantiopure sulfone 21 and aldehyde 24 are described. The union of these two intermediates by means of a Julia-Kocienski coupling gave rise to a series of E-configured building blocks that did not prove amenable to transannular cyclization. Alternatively, when the C15-C16 double bond was introduced with Z-geometry by Wittig olefination, spontaneous closure to generate a tetrahydrofuran culminated an ensuing direct dihydroxylation step. The structural assignment to 35, undergirded by detailed 1H and 13C NMR studies, is consistent with proper transannular bonding so as to deliver the entire C1-C26 fragment of PTX2.

Oxidative cleavage of p-methoxybenzyl ethers with methyl(trifluoromethyl)dioxirane.

[reaction: see text] The ability of methyl(trifluoromethyl)dioxirane to cleave p-methoxylbenzyl ethers oxidatively in the presence of various additional functional groups has been investigated. These reactions, performed in aqueous acetonitrile, transform a reasonably robust aryl substituent into a dienyl aldehydo ester. The originally generated E,Z-isomer undergoes slow conversion to the more stable E,E-form at 20 degrees C.

CoMFA, synthesis, and pharmacological evaluation of (E)-3-(2-carboxy-2-arylvinyl)-4,6-dichloro-1H-indole-2-carboxylic acids: 3-[2-(3-aminophenyl)-2-carboxyvinyl]-4,6-dichloro-1H-indole-2-carboxylic acid, a potent selective glycine-site NMDA receptor antagonist.

(E)-3-(2-Carboxy-2-phenylvinyl)-4,6-dichloro-1H-indole-2-carboxylic acid, 1, is a potent and selective antagonist of the glycine site of the N-methyl-d-aspartate (NMDA) receptor. Using 3D comparative molecular field analysis (CoMFA) to guide the synthetic effort, a series of aryl diacid analogues of 1 were synthesized to optimize in vivo potency, duration of action, and binding activity. It was found that the incorporation of a substituted aromatic with an electron withdrawing group or a heterocyclic group at the 2-position of the 3-propenyl moiety of 1 gave compounds with better affinity and potency in the murine stroke model. Ultimately this led to the discovery of 3-[2-(3-aminophenyl)-2-carboxyvinyl]-4,6-dichloro-1H-indole-2-carboxylic acid, 19, as a new potent selective glycine-site NMDA receptor antagonist.

Structural and stereochemical reassessment of sclerophytin-type diterpenes.

A complete reassessment of the structural assignments of the sclerophytin diterpenes is provided.

Novel steroidal vinyl fluorides as inhibitors of steroid C17(20) lyase.

20-fluoro-17(20)-pregnenolone derivatives were designed as enol mimics of pregnenolone. All of the targeted, novel fluoroolefins were potent inhibitors of C17(20) lyase.

Utilization of the Phenylthio Substituent as a Multipurpose Synthetic Tool. Direct Application to the Enantioselective Construction of (-)-Salsolene Oxide.

The architecturally unprecedented sesquiterpene (-)-salsolene oxide (1) has been synthesized in enantioselective fashion from (R)-(-)-carvone. Generation of the phenylthio-substituted vinyl ketene 4 is followed by intramolecular cyclization to the functionalized cyclobutanone 9. Vinyllithium addition to this intermediate proceeds in that stereocontrolled fashion which enables oxy-Cope rearrangement to operate readily under conditions of kinetic control. After hydride reduction, the desulfurization of 16 proceeds with inversion of bridgehead olefin geometry to deliver 17. This access route to the thermodynamically more stable geometric arrangement permits direct entry to 1. Attention is called specifically to the critical 3-fold function played by a phenylthio group introduced at the outset.