Biological characteristics and pathogenicity of a Staphylococcus aureus strain with an incomplete hemolytic phenotype isolated from bovine milk.

Staphylococcus aureus is a common pathogen capable of infecting both humans and animals and causing various severe diseases. Here, we aimed to determine the biological features and pathogenicity of S. aureus strain Sa9, of the incomplete hemolysis phenotype, isolated from bovine milk. Sa9 was classified as ST97 by multilocus sequence typing, and it showed increased ß-hemolysin expression and lower Hla and Hld expression levels compared with that in the S. aureus USA300 strain LAC. RT-PCR and ELISA results showed that the expression levels of inflammatory cytokines were higher in Sa9-induced mouse primary peritoneal macrophages compared with those induced by the LAC strain. However, the Sa9 strain also mediated anti-inflammatory effects by upregulating IL-10 and IFN-ß in macrophages, which were not apparently induced by S. aureus culture supernatants. Phagocytosis and whole-blood survival assays were also performed to assess the in vitro survival of bacteria, and the virulence was evaluated in mice. Although the Sa9 strain showed lower ability of intracellular survival in macrophages than LAC, similar multiplication in human whole blood and pathogenicity toward mice were observed. Taken together, we report that the distinctive immune response induced by the S. aureus strain with an incomplete hemolysis phenotype occurs in cattle, and its potential pathogenicity and risk of transmission to humans require attention.

Copper-Mediated Oxidative Chloro- and Bromodifluoromethylation of Phenols.

The first oxidative chloro- and bromodifluoromethylation of phenols with (CH3)3SiCF2X and CuX (X = Cl or Br) in the presence of Selectfluor under mild reaction conditions was developed. This protocol provided a practical and efficient method for the synthesis of a diverse range of biologically valuable and synthetically challenging chloro- and bromodifluoromethyl aryl ethers. Preliminary mechanistic studies suggest that this reaction proceeded through a difluorocarbene-involved oxidative coupling process.

N-Trifluoromethoxyphthalimide: A Shelf-Stable Reagent for Nucleophilic Trifluoromethoxylation.

Due to the unique properties of the OCF3 group, trifluoromethyl ether compounds play an important role in pharmaceuticals and agrochemicals. Recently, considerable attention has been focused on the development of practical and convenient reagents for the direct incorporation of the OCF3 group into organic compounds. Herein, we reported a new trifluoromethoxylating reagent N-trifluoromethoxyphthalimide (Phth-OCF3). The reagent was a stable solid and released an OCF3 anion under mild reaction conditions. We demonstrated the application of Phth-OCF3 for the nucleophilic trifluoromethoxylation of various alkyl electrophiles.

Visible-light-induced nickel-catalyzed α-hydroxytrifluoroethylation of alkyl carboxylic acids: Access to trifluoromethyl alkyl acyloins.

A visible-light-induced nickel-catalyzed cross coupling of alkyl carboxylic acids with N-trifluoroethoxyphthalimide is described. Under purple light irradiation, an α-hydroxytrifluoroethyl radical generated from a photoactive electron donor-acceptor complex between Hantzsch ester and N-trifluoroethoxyphthalimide was subsequently engaged in a nickel-catalyzed coupling reaction with in situ-activated alkyl carboxylic acids. This convenient protocol does not require photocatalysts and metal reductants, providing a straightforward and efficient access to trifluoromethyl alkyl acyloins in good yields with broad substrate compatibility. The complex bioactive molecules were also compatible with this catalytic system to afford the corresponding products.

General Synthesis of N-Trifluoromethyl Compounds with N-Trifluoromethyl Hydroxylamine Reagents.

N-CF3 compounds constitute valuable targets in medicinal chemistry. Extensive studies have been reported for the preparation of N-CF3 compounds through fluorination and trifluoromethylation of N-containing compounds. The development of new synthetic methods from abundant and easily available substrates is highly desirable but still challenging. Herein, we report the design and synthesis of novel N-Cbz- and N-Boc-N-trifluoromethyl hydroxylamine reagents by silver-mediated oxidative trifluoromethylation. These reagents have been successfully applied to the direct incorporation of a NCF3 moiety into the commonly used unsaturated substrates under photoredox catalysis. This protocol enables the efficient and regioselective C-H trifluoromethylamination of various (hetero)arenes, including complex bioactive molecules. Furthermore, a variety of alkenes, dienes, and isonitriles undergo tandem trifluoromethylamination/functionalization delivering structurally diverse N-trifluoromethyl aliphatic and heteroaromatic amines. Notably, previously unknown cyclic N-CF3 compounds including N-CF3 oxazolidinones and oxazolones were conveniently prepared with N-Boc-N-trifluoromethyl hydroxylamine reagents. Furthermore, diversification of the resulting α-trifluoromethylamino ketones afforded the largely underexplored N-alkenyl- and N-alkynyl-N-CF3 compounds.

Electrochemical Trifluoromethoxylation of (Hetero)aromatics with a Trifluoromethyl Source and Oxygen.

Trifluoromethoxylated aromatics (ArOCF3 ) are valuable structural motifs in the area of drug discovery due to the enhancement of their desired physicochemical properties upon the introduction of the trifluoromethoxy group (CF3 O). Although significant progress has been made recently in the introduction of CF3 O group into aromatics, current methods either require the use of expensive trifluoromethoxylation reagents or require harsh reaction conditions. We present a conceptually new and operationally simple protocol for the direct C-H trifluoromethoxylation of (hetero)aromatics by the combination of the readily available trifluoromethylating reagent and oxygen under electrochemical reaction conditions. This reaction proceeds through the initial generation of CF3 radical followed by conversion to CF3 O radical, addition to (hetero)aromatics and rearomatization. The utility of this electrochemical trifluoromethoxylation is illustrated by the direct incorporation of CF3 O group into a variety of (hetero)aromatics as well as bio-relevant molecules.

Nucleophilic and Radical Heptafluoroisopropoxylation with Redox-Active Reagents.

The heptafluoroisopropyl group (CF(CF3 )2 ) is prevalent in pharmaceuticals and agrichemicals. However, heptafluoroisopropoxylated (OCF(CF3 )2 ) compounds remain largely underexplored, presumably due to the lack of efficient access to these compounds. Herein, we disclose the practical and efficient heptafluoroisopropoxylation reactions through the invention of a series of redox-active N-OCF(CF3 )2 reagents. These reagents were readily prepared from the oxidative heptafluoroisopropylation of hydroxylamines with AgCF(CF3 )2 . The substitutions on the nitrogen atom significantly affected the properties and reactivities of N-OCF(CF3 )2 reagents. Accordingly, two types of N-OCF(CF3 )2 reagents including N-OCF(CF3 )2 phthalimide A and N-OCF(CF3 )2 benzotriazolium salt O' were used as OCF(CF3 )2 anion and radical precursors, respectively. This protocol enables the direct heptafluoroisopropoxylation of a range of substrates, delivering the corresponding products in moderate to excellent yields.

Chemoselective Hydro(Chloro)pentafluorosulfanylation of Diazo Compounds with Pentafluorosulfanyl Chloride.

Pentafluorosulfanyl chloride (SF5 Cl) is the most prevalent reagent for the incorporation of SF5 group into organic compounds. However, the preparation of SF5 Cl often relies on hazardous reagents and specialized apparatus. Herein, we described a safe and practical synthesis of a bench-stable and easy-to-handle solution of SF5 Cl in n-hexane under gas-reagent-free conditions. The synthetic application of SF5 Cl was demonstrated through the unprecedented reaction with diazo compounds. The chemoselective hydro- and chloropentafluorosulfanylations of α-diazo carbonyl compounds were developed in the presence of K3 PO4 or copper catalyst, respectively. These reactions provide a direct and efficient access to various α-pentafluorosulfanyl carbonyl compounds of high value for potential applications.

Nickel-Mediated Trifluoromethylation of Phenol Derivatives by Aryl C-O Bond Activation.

The increasing pharmaceutical importance of trifluoromethylarenes has stimulated the development of more efficient trifluoromethylation reactions. Tremendous efforts have focused on copper- and palladium-mediated/catalyzed trifluoromethylation of aryl halides. In contrast, no general method exists for the conversion of widely available inert electrophiles, such as phenol derivatives, into the corresponding trifluoromethylated arenes. Reported herein is a practical nickel-mediated trifluoromethylation of phenol derivatives with readily available trimethyl(trifluoromethyl)silane (TMSCF3 ). The strategy relies on PMe3 -promoted oxidative addition and transmetalation, and CCl3 CN-induced reductive elimination. The broad utility of this transformation has been demonstrated through the direct incorporation of trifluoromethyl into aromatic and heteroaromatic systems, including biorelevant compounds.

Cascade trifluoromethylthiolation and cyclization of N-[(3-aryl)propioloyl]indoles.

A cascade oxidative trifluoromethylthiolation and cyclization of N-[(3-aryl)propioloyl]indoles with AgSCF3 is described. This protocol allows for the synthesis of novel bis(trifluoromethylthiolated) or trifluoromethylthiolated pyrrolo[1,2-a]indol-3-ones in moderate to good yields. Mechanistic investigations indicated that radical processes were probably involved in these transformations.

Influence of transcription regulator SAUSA300_1968 on the virulence protein secretion and immune evasion by Staphylococcus aureus.

Staphylococcus aureus employs an arsenal of secreted virulence factors to evade host immune responses, such as inducing host cell death pathways or hiding within phagocytes. A previous study indicated that the transcription regulator SA1804 is a novel virulence repressor of S. aureus, and is regulated by SaeRS. However, the function of this regulator in S. aureus pathogenesis remains unclear. In this study, we created a SA1804 homologue, SAUSA300_1968, deletion mutant in the S. aureus USA300 strain LAC, and found that culture supernatant proteins of the mutant exhibit higher cytotoxicity and greater ability to induce the expression of inflammatory cytokines in mouse primary peritoneal macrophages as compared with that of its parent strain. Comparative proteome analysis of secreted protein expression profiles between the two strains was determined through tandem mass tags (TMT). Among the 18 well-known extracellular virulence-related factors that showed significantly different expressions, 17 proteins were up-regulated in the SAUSA300_1968 deletion mutant, including various pore-forming toxins and extracellular proteases. Accordingly, we also found that inactivation of SAUSA300_1968 enhanced S. aureus survival in mouse macrophages. Our data provide novel insights into the role of the SAUSA300_1968 regulator in host immune evasion and S. aureus pathogenesis.

Argentination of Fluoroform: Preparation of a Stable AgCF3 Solution with Diverse Reactivities.

The transformation of a large-volume industrial by-product and stable greenhouse gas fluoroform (HCF3 ) to useful products has recently received significant attention. Now, a simple and scalable preparation of AgCF3 by treatment of HCF3 with t-BuOK and AgOAc is disclosed. The reactivity of the HCF3 -derived AgCF3 has been demonstrated by hydrotrifluoromethylation of alkenes and C-H trifluoromethylation of (hetero)arenes. This work not only provides a new avenue for the utilization of HCF3 , but also presents a reliable and easy-to-execute synthesis of the relatively stable AgCF3 solution.

Hydrotrifluoromethylthiolation of Unactivated Alkenes and Alkynes with Trifluoromethanesulfonic Anhydride through Deoxygenative Reduction and Photoredox Radical Processes.

An ongoing challenge in trifluoromethylthiolation reactions is the use of less expensive and easily available trifluoromethylthio sources. Herein, we disclose an unprecedented usage of trifluoromethanesulfonic anhydride (Tf2 O) as a radical trifluoromethylthiolating reagent. Hydrotrifluoromethylthiolation of unactivated alkenes and alkynes with Tf2 O in the presence of PMePh2 and H2 O under visible-light photoredox catalysis gave the addition products. The trifluoromethylthio radical (. SCF3 ) was first formed from Tf2 O through a photoredox radical processes and deoxygenative reduction of PMePh2 , and H2 O serves as the H-atom donor for the hydrotrifluoromethylthiolation reaction. This reaction provides a new strategy for radical trifluoromethylthiolation.

Direct and Regioselective C-H Oxidative Difluoromethylation of Heteroarenes.

The difluoromethyl group (CF2H) is of great interest in the area of medicinal chemistry. However, the investigation of molecular scaffolds containing this group has been hampered by the limitation of synthetic methods for the introduction of CF2H into heteroarenes. Herein we disclose a new strategy for the direct introduction of a difluoromethyl group into heteroarenes via the copper-mediated C-H oxidative difluoromethylation of heteroarenes with TMSCF2H. This mild and regioselective method enables the convenient synthesis of a range of difluoromethylated heteroarenes in high yields. The usage of 9,10-phenanthrenequinone (PQ) as an oxidant is critical to the success of this new difluoromethylation reaction.

Application of ex-vivo spheroid model system for the analysis of senescence and senolytic phenotypes in uterine leiomyoma.

Cellular senecence is an important biologic endpoint. Naturally occuring (aging) senescence is common in uterine leiomyoma (ULM). AKT is one of major pathways in promoting ULM growth and survival. Inactivation of AKT by MK2206 in ULM resulted in stress-induced senescence in vitro. Study of the senescent phenotypes and molecular changes in ULM may greatly facilitate the understanding of the tumor biology and potential clinical therapy for this common disease associated with high morbidity. To study senescence in a model system that closely resembles primary ULM in vivo, we applied an ex vivo model of three-dimensional (3D) spheroid culture system which maintained the molecular and cellular characteristics of primary ULM and matched myometrium as seen in vivo. Gene expression profiling done on ULM induced to undergo replication (passaging) or stress-induced (MK2206) senescence revealed that ROS and hypoxic-related genes were upregulated in the two types of senescences. Overexpression of two selected genes, WIPI1 and SLITKR4, induced cellular senescence in ULM spheroids. Additionally, administration of ABT263 (a BH3 mimetic) effectively reduced the senescent cells induced in ULM spheroids. This study identified novel genes associated with senescence in ULM and demonstrated a BH3 mimetic to act as a senolytic to remove senescent cells.

Copper-Catalyzed Sulfenylation, Sulfonylation, and Selenylation of 2,3-Allenoic Acids with Disulfides or Diselenides.

The efficient copper-catalyzed sulfenylation and selenylation of 2,3-allenoic acids with disulfides or diselenides were developed, respectively. These reactions proceeded through tandem radical addition/intramolecular cyclization processes, affording a series of 4-sulfenylated and 4-selenylated butenolides in moderate to excellent yields. Moreover, 4-sulfonylated butenolides could also be obtained by sulfenylation of 2,3-allenoic acids and subsequent oxidation. Further transformation of the sulfur- and selenium-containing butenolides afforded the corresponding furan derivatives in good yields.

Synthesis of CMe2CF3-Containing Heteroarenes via Tandem 1,1-Dimethyltrifluoroethylation and Cyclization of Isonitriles.

A tandem 1,1-dimethyltrifluoroethylation and cyclization of isonitriles with 3,3,3-trifluoro-2,2-dimethylpropanoic acid was developed. This protocol provides the efficient synthesis of a series of previously unknown CMe2CF3-containing heteroarenes, which are potentially useful in the drug discovery process.

Fumarate Hydratase Mutations and Alterations in Leiomyoma With Bizarre Nuclei.

Leiomyoma with bizarre nuclei (LM-BN), is a variant of uterine smooth muscle tumor with atypical histologic features. Although some LM-BN share several significant genetic alterations with leiomyosarcoma, including p16 and p53, the underlying tumorigenesis of LM-BN remains largely unknown. As we previously reported, LM-BN can be divided into 2 subtypes, type I and type II, based on different nuclear features. Type I LM-BN have similar histologic features as uterine smooth muscle tumors with fumarate hydratase (FH) alterations. In this study, we examined FH expression and FH mutations in 77 LM-BN (40 type I cases and 37 type II cases). FH expression was examined by immunohistochemistry using S-(2-succino)-cysteine antibodies (2SC, a protein modification associated with FH inactivation and subsequent fumarate accumulation) and FH antibodies (FH gene products). Seventy-two LM-BN tumors underwent Sanger sequencing to detect FH mutations. We found that 51% (39/77) of LM-BN showed FH alterations detected by immunohistochemistry with both 2SC and FH. Mutational analysis showed that 21% (15/72) of LM-BN harbored FH gene mutations. Further analysis revealed that 85% (34/40) of those with FH alterations were type I LM-BN while 19% (7/37) were type II LM-BN. Our findings suggest that over half of histologically diagnosed LM-BN may be related to FH alterations or FH mutations and the majority of these have the characteristic histologic features of type I LM-BN.

Transition metal free decarboxylative fluoroalkylation of N-acrylamides with 3,3,3-trifluoro-2,2-dimethylpropanoic acid (TFDMPA).

A novel transition metal-free decarboxylative fluoroalkylation of activated alkenes and C-H functionalization cascade process has been developed. This approach provides an efficient way to construct valuable 1,1-dimethyl-2,2,2-trifluoroethyl substituted oxindoles.

Trifluoromethanesulfonic Anhydride as a Low-Cost and Versatile Trifluoromethylation Reagent.

A large number of reagents have been developed for the synthesis of trifluoromethylated compounds. However, an ongoing challenge in trifluoromethylation reaction is the use of less expensive and practical trifluoromethyl sources. We report herein the unprecedented direct trifluoromethylation of (hetero)arenes using trifluoromethanesulfonic anhydride as a radical trifluoromethylation reagent by merging photoredox catalysis and pyridine activation. Furthermore, introduction of both the CF3 and OTf groups of the trifluoromethanesulfonic anhydride into internal alkynes to access tetrasubstituted trifluoromethylated alkenes was achieved. Since trifluoromethanesulfonic anhydride is a low-cost and abundant chemical, this method provides a cost-efficient and practical route to trifluoromethylated compounds.