Design and synthesis of 1-aroyl-2-ylidene hydrazines under conventional and microwave irradiation conditions and their cytotoxic activities

We report the design and synthesis of 1-aroyl-2-(alkenyl/aryl)idene hydrazines as hybrid molecules derived from mefenamic acid and substituted hydrazones. A number of compounds based on this new scaffold were prepared in good yields. The key intermediate N-acylhydrazine, prepared from mefenamic acid, was coupled with a variety of aldehydes under conventional as well as microwave irradiation conditions. The second approach, that requires short reaction time, can be carried out under a solvent free condition and does not require the use of an acid catalyst or solid support. Some of the compounds synthesized showed cytotoxic activities in vitro

One-Step, Effective, and Cascade Syntheses of Highly Functionalized Cyclopentenes with High Diastereoselectivity

Tetrabutylammonium fluoride works as an effective organocatalyst for the cycloaddition between phenacylmalononitriles and electron-deficient olefins (having substituent groups of NO₂, CHO, and COR), providing a facile synthetic route to versatile multifunctionalized cyclopentenes having an allylic quaternary carbon center bearing both cyano and carboxamide groups with high yields and high diastereoselectivity. Preliminary studies reveal that these functionalized cyclopentenes are convenient precursors for making α-cyano-functionalized cyclopentadienone oximes

Nanomaterial Delivery Systems for mRNA Vaccines

The recent success of mRNA vaccines in SARS-CoV-2 clinical trials is in part due to the development of lipid nanoparticle delivery systems that not only efficiently express the mRNA-encoded immunogen after intramuscular injection, but also play roles as adjuvants and in vaccine reactogenicity. We present an overview of mRNA delivery systems and then focus on the lipid nanoparticles used in the current SARS-CoV-2 vaccine clinical trials. The review concludes with an analysis of the determinants of the performance of lipid nanoparticles in mRNA vaccines

UDP-glucose dehydrogenase (UGDH) activity is suppressed by peroxide and promoted by PDGF in fibroblast-like synoviocytes: Evidence of a redox control mechanism

UDP-glucose dehydrogenase (UGDH) generates essential precursors of hyaluronic acid (HA) synthesis, however mechanisms regulating its activity are unclear. We used enzyme histostaining and quantitative image analysis to test whether cytokines that stimulate HA synthesis upregulate UGDH activity. Fibroblast-like synoviocytes (FLS, from N = 6 human donors with knee pain) were cultured, freeze-thawed, and incubated for 1 hour with UDP-glucose, NAD+ and nitroblue tetrazolium (NBT) which allows UGDH to generate NADH, and NADH to reduce NBT to a blue stain. Compared to serum-free medium, FLS treated with PDGF showed 3-fold higher UGDH activity and 6-fold higher HA release, but IL-1beta/TGF-beta1 induced 27-fold higher HA release without enhancing UGDH activity. In selected proliferating cells, UGDH activity was lost in the cytosol, but preserved in the nucleus. Cell-free assays led us to discover that diaphorase, a cytosolic enzyme, or glutathione reductase, a nuclear enzyme, was necessary and sufficient for NADH to reduce NBT to a blue formazan dye in a 1-hour timeframe. Primary synovial fibroblasts and transformed A549 fibroblasts showed constitutive diaphorase/GR staining activity that varied according to supplied NADH levels, with relatively stronger UGDH and diaphorase activity in A549 cells. Unilateral knee injury in New Zealand White rabbits (N = 3) stimulated a coordinated increase in synovial membrane UGDH and diaphorase activity, but higher synovial fluid HA in only 2 out of 3 injured joints. UGDH activity (but not diaphorase) was abolished by N-ethyl maleimide, and inhibited by peroxide or UDP-xylose. Our results do not support the hypothesis that UGDH is a rate-liming enzyme for HA synthesis under catabolic inflammatory conditions that can oxidize and inactivate the UGDH active site cysteine. Our novel data suggest a model where UGDH activity is controlled by a redox switch, where intracellular peroxide inactivates, and high glutathione and diaphorase promote UGDH activity by maintaining the active site cysteine in a reduced state, and by recycling NAD+ from NADH

In situ enzyme staining reaction conditions.

In situ enzyme staining reaction conditions.</p

Nuclear UGDH enzyme histostaining was observed in selected FLS cells.

(A, B) UGDH enzyme histostaining was occasionally detected in the nuclear or perinuclear compartment in sub-confluent cells in the middle of the culture well, and (C, D) was mainly cytosolic in more confluent cells at the edge of the well. (A, C) bright field and (B, D) matching epifluorescence image of the same field showing Hoechst-stained nuclei of FLS stimulated here with 1 ng/mL each IL1β + TGF-β1 and stained for UGDH enzyme activity. Scale bars: 20 μm. (TIF)</p

Intense UGDH and diaphorase enzyme histostaining activity in transformed A549 cells.

By in situ enzyme staining, A549 cells showed relatively high (A) 99.8% diaphorase/GR staining with 0.1 mM NADH, (B) 93.1% UGDH enzyme staining and (C) 12.1% background NBT staining in the absence of UDP-Glc compared to primary human FLS. Scale bars: 100 μm.</p

UDP-xylose and peroxide inhibited UGDH but not diaphorase staining activity in cultured monolayer FLS.

Cells were from a 23-year old male donor (passage 4). FLS stained for UGDH (A: bright field; B: phase contrast) or UGDH with UDP-xylose (C: bright field; D: phase contrast), diaphorase (E), or diaphorase with UDP-xylose (F). In panel (G), quantitative histomorphometry was used to measure the relative staining intensity in the absence or presence of UDP-xylose or peroxide. Symbols: *pvs UGDH; ** p (TIF)</p

TLC demonstration that NADH and GSH reduce NBT to the same formazan product with different kinetics.

Arrowheads show the origin and final solvent front; UV light was absorbed in the TLC plate by NADH and NBT at the origin, and by diformazan at the arrow. Reduced formazan had an Rf value ranging from 0.42 to 0.44 [38] depending on whether the lane was in the middle or edge of the TLC plate. In the absence of enzyme, GSH reduced NBT after 60 minutes but NADH only reduced NBT after 24 hours. Diaphorase or GR was necessary and sufficient for NADH to reduce NBT within 1 hour. See (S1 File) for original TLC plate images. (TIF)</p

Hypothetical model representing 2-enzyme UGDH activity staining mechanisms in cultured cells and unfixed cryosections.

Boxed molecules are enzyme staining components, encircled factors and dashed lines represent activities modulated in vivo by cytokines. Oxidative stress induced by inflammatory cytokines (TGF-β1+IL-1β) can lead to peroxide generation and reversible inactivation of UGDH unless GSH scavenges the free radicals to sustain UGDH activity. High endogenous GSH can produce background staining by directly reducing NBT and by-passing UGDH. Abbreviations: PDGF: platelet derived growth factor; GR: glutathione reductase; GSH: glutathione; H2O2: peroxide; NEM: N-ethyl maleimide (thiol alkylating agent). In the upper panel, Dark blue cells: UGDH-stained cell compartments where cytosolic diaphorase, or nuclear glutathione reductase activity is present; dashed lines: freeze-thaw permeabilized cell membranes.</p