Human MicroRNAs Attenuate the Expression of Immediate Early Proteins and HCMV Replication during Lytic and Latent Infection in Connection with Enhancement of Phosphorylated RelA/p65 (Serine 536) That Binds to MIEP

Attenuating the expression of immediate early (IE) proteins is essential for controlling the lytic replication of human cytomegalovirus (HCMV). The human microRNAs (hsa-miRs), miR-200b-3p and miR-200c-3p, have been identified to bind the 3′-untranslated region (3′-UTR) of the mRNA encoding IE proteins. However, whether hsa-miRs can reduce IE72 expression and HCMV viral load or exhibit a crosstalk with the host cellular signaling machinery, most importantly the NF-κB cascade, has not been evaluated. In this study, argonaute-crosslinking and immunoprecipitation-seq revealed that miR-200b-3p and miR-200c-3p bind the 3′-UTR of UL123, which is a gene that encodes IE72. The binding of these miRNAs to the 3′-UTR of UL123 was verified in transfected cells stably expressing GFP. We used miR-200b-3p/miR-200c-3p mimics to counteract the downregulation of these miRNA after acute HCMV infection. This resulted in reduced IE72/IE86 expression and HCMV VL during lytic infection. We determined that IE72/IE86 alone can inhibit the phosphorylation of RelA/p65 at the Ser⁵³⁶ residue and that p-Ser⁵³⁶ RelA/p65 binds to the major IE promoter/enhancer (MIEP). The upregulation of miR-200b-3p and miR-200c-3p resulted in the phosphorylation of RelA/p65 at Ser⁵³⁶ through the downregulation of IE, and the binding of the resultant p-Ser⁵³⁶ RelA/p65 to MIEP resulted in a decreased production of pro-inflammatory cytokines. Overall, miR-200b-3p and miR-200c-3p—together with p-Ser⁵³⁶ RelA/p65—can prevent lytic HCMV replication during acute and latent infection     

Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B–Z Transition of DNA

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZα_ADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZα_ADAR1 provided the main contribution to the three-state conformational changes of a hZα_ADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, k_ex and k_ZB, were found to be 844 and 9.8 s⁻¹, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.     

Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H₂O₂. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.     

Single-Cell RNA Sequencing Analysis for Oncogenic Mechanisms Underlying Oral Squamous Cell Carcinoma Carcinogenesis with Candida albicans Infection

Oral squamous cell carcinoma (OSCC) carcinogenesis involves heterogeneous tumor cells, and the tumor microenvironment (TME) is highly complex with many different cell types. Cancer cell–TME interactions are crucial in OSCC progression. Candida albicans (C. albicans)—frequently pre-sent in the oral potentially malignant disorder (OPMD) lesions and OSCC tissues—promotes malignant transformation. The aim of the study is to verify the mechanisms underlying OSCC car-cinogenesis with C. albicans infection and identify the biomarker for the early detection of OSCC and as the treatment target. The single-cell RNA sequencing analysis (scRNA-seq) was performed to explore the cell subtypes in normal oral mucosa, OPMD, and OSCC tissues. The cell composi-tion changes and oncogenic mechanisms underlying OSCC carcinogenesis with C. albicans infec-tion were investigated. Gene Set Variation Analysis (GSVA) was used to survey the mechanisms underlying OSCC carcinogenesis with and without C. albicans infection. The results revealed spe-cific cell clusters contributing to OSCC carcinogenesis with and without C. albicans infection. The major mechanisms involved in OSCC carcinogenesis without C. albicans infection are the IL2/STAT5, TNFα/NFκB, and TGFβ signaling pathways, whereas those involved in OSCC carcinogenesis with C. albicans infection are the KRAS signaling pathway and E2F target down-stream genes. Finally, stratifin (SFN) was validated to be a specific biomarker of OSCC with C. albicans infection. Thus, the detailed mechanism underlying OSCC carcinogenesis with C. albicans infection was determined and identified the treatment biomarker with potential precision medicine applications.     

Key Properties of a Bioactive Ag-SiO2/TiO2 Coating on NiTi Shape Memory Alloy as Necessary at the Development of a New Class of Biomedical Materials

Recent years have seen the dynamic development of methods for functionalizing the surface of implants using biomaterials that can mimic the physical and mechanical nature of native tissue, prevent the formation of bacterial biofilm, promote osteoconduction, and have the ability to sustain cell proliferation. One of the concepts for achieving this goal, which is presented in this work, is to functionalize the surface of NiTi shape memory alloy by an atypical glass-like nanocomposite that consists of SiO₂-TiO₂ with silver nanoparticles. However, determining the potential medical uses of bio(nano)coating prepared in this way requires an analysis of its surface roughness, tribology, or wettability, especially in the context of the commonly used reference coat-forming hydroxyapatite (HAp). According to our results, the surface roughness ranged between (112 ± 3) nm (Ag-SiO₂)—(141 ± 5) nm (HAp), the water contact angle was in the range (74.8 ± 1.6)° (Ag-SiO₂)—(70.6 ± 1.2)° (HAp), while the surface free energy was in the range of 45.4 mJ/m² (Ag-SiO₂)—46.8 mJ/m² (HAp). The adhesive force and friction coefficient were determined to be 1.04 (Ag-SiO₂)—1.14 (HAp) and 0.247 ± 0.012 (Ag-SiO₂) and 0.397 ± 0.034 (HAp), respectively. The chemical data showed that the release of the metal, mainly Ni from the covered NiTi substrate or Ag from Ag-SiO₂ coating had a negligible effect. It was revealed that the NiTi alloy that was coated with Ag-SiO₂ did not favor the formation of E. coli or S. aureus biofilm compared to the HAp-coated alloy. Moreover, both approaches to surface functionalization indicated good viability of the normal human dermal fibroblast and osteoblast cells and confirmed the high osteoconductive features of the biomaterial. The similarities of both types of coat-forming materials indicate an excellent potential of the silver-silica composite as a new material for the functionalization of the surface of a biomaterial and the development of a new type of functionalized implants.     

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)—an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a K_D value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure–activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.     

Asymmetric Solvation of the Zinc Dimer Cation Revealed by Infrared Multiple Photon Dissociation Spectroscopy of Zn2+(H2O)n (n = 1–20)

Investigating metal-ion solvation—in particular, the fundamental binding interactions—enhances the understanding of many processes, including hydrogen production via catalysis at metal centers and metal corrosion. Infrared spectra of the hydrated zinc dimer (Zn₂⁺(H₂O)_n; n = 1–20) were measured in the O–H stretching region, using infrared multiple photon dissociation (IRMPD) spectroscopy. These spectra were then compared with those calculated by using density functional theory. For all cluster sizes, calculated structures adopting asymmetric solvation to one Zn atom in the dimer were found to lie lower in energy than structures adopting symmetric solvation to both Zn atoms. Combining experiment and theory, the spectra show that water molecules preferentially bind to one Zn atom, adopting water binding motifs similar to the Zn⁺(H₂O)_n complexes studied previously. A lower coordination number of 2 was observed for Zn₂⁺(H₂O)₃, evident from the highly red-shifted band in the hydrogen bonding region. Photodissociation leading to loss of a neutral Zn atom was observed only for n = 3, attributed to a particularly low calculated Zn binding energy for this cluster size.     

Design,Synthesis, Antibacterial,Antifungal and Anticancer Evaluations of Novel β-Pinene Quaternary Ammonium Salts

β-pinene is a monoterpene isolated from turpentine oil and numerous other plants’ essential oils, which has a broad spectrum of biological activities. In the current work, six novel β-pinene quaternary ammonium (β-PQA) salts were synthesized and evaluated in vitro for their antifungal, antibacterial and anticancer activities. The in vitro assay results revealed that compounds 4a and 4b presented remarkable antimicrobial activity against the tested fungi and bacteria. In particular, compound 4a showed excellent activities against F. oxysporum f.sp. niveum, P. nicotianae var.nicotianae, R. solani, D. pinea and Fusicoccumaesculi, with EC₅₀ values of 4.50, 10.92, 9.45, 10.82 and 6.34 μg/mL, respectively. Moreover, compound 4a showed the best antibacterial action against E. coli, P. aeruginosa, S. aureus and B. subtilis, with MIC at 2.5, 0.625, 1.25 and 1.25 μg/mL, respectively. The anticancer activity results demonstrated that compounds 4a, 4b, 4c and 4f exhibited remarkable activity against HCT-116 and MCF-7 cell lines, with IC₅₀ values ranged from 1.10 to 25.54 μM. Notably, the compound 4c displayed the strongest cytotoxicity against HCT-116 and MCF-7 cell lines, with the IC₅₀ values of 1.10 and 2.46 μM, respectively. Furthermore, preliminary antimicrobial mechanistic studies revealed that compound 4a might cause mycelium abnormalities of microbial, cell membrane permeability changes and inhibition of the activity of ATP. Altogether, these findings open interesting perspectives to the application of β-PQA salts as a novel leading structure for the development of effective antimicrobial and anticancer agents.     

Conserved and Distinct Elements of Phagocytosis in Human and C. elegans

Endocytosis provides the cellular nutrition and homeostasis of organisms, but pathogens often take advantage of this entry point to infect host cells. This is counteracted by phagocytosis that plays a key role in the protection against invading microbes both during the initial engulfment of pathogens and in the clearance of infected cells. Phagocytic cells balance two vital functions: preventing the accumulation of cell corpses to avoid pathological inflammation and autoimmunity, whilst maintaining host defence. In this review, we compare elements of phagocytosis in mammals and the nematode Caenorhabditis elegans. Initial recognition of infection requires different mechanisms. In mammals, pattern recognition receptors bind pathogens directly, whereas activation of the innate immune response in the nematode rather relies on the detection of cellular damage. In contrast, molecules involved in efferocytosis—the engulfment and elimination of dying cells and cell debris—are highly conserved between the two species. Therefore, C. elegans is a powerful model to research mechanisms of the phagocytic machinery. Finally, we show that both mammalian and worm studies help to understand how the two phagocytic functions are interconnected: emerging data suggest the activation of innate immunity as a consequence of defective apoptotic cell clearance.     

Size dependence of the magnetic properties of Ni nanoparticles prepared by thermal decomposition method

By means of thermal decomposition, we prepared single-phase spherical Ni nanoparticles (23 to 114 nm in diameter) that are face-centered cubic in structure. The magnetic properties of the Ni nanoparticles were experimentally as well as theoretically investigated as a function of particle size. By means of thermogravimetric/differential thermal analysis, the Curie temperature T_C of the 23-, 45-, 80-, and 114-nm Ni particles was found to be 335°C, 346°C, 351°C, and 354°C, respectively. Based on the size-and-shape dependence model of cohesive energy, a theoretical model is proposed to explain the size dependence of T_C. The measurement of magnetic hysteresis loop reveals that the saturation magnetization M_S and remanent magnetization increase and the coercivity decreases monotonously with increasing particle size, indicating a distinct size effect. By adopting a simplified theoretical model, we obtained M_S values that are in good agreement with the experimental ones. Furthermore, with increase of surface-to-volume ratio of Ni nanoparticles due to decrease of particle size, there is increase of the percentage of magnetically inactive layer.     

Chemical Compounds Released from Specific Osteoinductive Bioactive Materials Stimulate Human Bone Marrow Mesenchymal Stem Cell Migration

In this work, a poly(L-lactide-co-glycolide) (PLGA)-based composite was enriched with one of the following sol-gel bioactive glasses (SBG) at 50 wt.%: A1—40 mol% SiO₂, 60 mol% CaO, CaO/SiO₂ ratio of 1.50; S1—80 mol% SiO₂, 20 mol% CaO, CaO/SiO₂ ratio of 0.25; A2—40 mol% SiO₂, 54 mol% CaO, 6 mol% P₂O₅, CaO/SiO₂ ratio of 1.35; S2—80 mol% SiO₂,16 mol% CaO, 4 mol% P₂O₅, CaO/SiO₂ ratio of 0.20. The composites and PLGA control sheets were then soaked for 24 h in culture media, and the obtained condition media (CM) were used to treat human bone marrow stromal cells (hBMSCs) for 72 h. All CMs from the composites increased ERK 1/2 activity vs. the control PLGA CM. However, expressions of cell migration-related c-Fos, osteopontin, matrix metalloproteinase-2, C-X-C chemokine receptor type 4, vascular endothelial growth factor, and bone morphogenetic protein 2 were significantly increased only in cells treated with the CM from the A1/PLGA composite. This CM also significantly increased the rate of human BMSC migration but did not affect cell metabolic activity. These results indicate important biological markers that are upregulated by products released from the bioactive composites of a specific chemical composition, which may eventually prompt osteoprogenitor cells to colonize the bioactive material and accelerate the process of tissue regeneration.     

Quantitative Proteomics Analysis Reveals the Function of the Putative Ester Cyclase UvEC1 in the Pathogenicity of the Rice False Smut Fungus Ustilaginoidea virens

Rice false smut is a fungal disease distributed worldwide and caused by Ustilaginoidea virens. In this study, we identified a putative ester cyclase (named as UvEC1) as being significantly upregulated during U. virens infection. UvEC1 contained a SnoaL-like polyketide cyclase domain, but the functions of ketone cyclases such as SnoaL in plant fungal pathogens remain unclear. Deletion of UvEC1 caused defects in vegetative growth and conidiation. UvEC1 was also required for response to hyperosmotic and oxidative stresses and for maintenance of cell wall integrity. Importantly, ΔUvEC1 mutants exhibited reduced virulence. We performed a tandem mass tag (TMT)-based quantitative proteomic analysis to identify differentially accumulating proteins (DAPs) between the ΔUvEC1-1 mutant and the wild-type isolate HWD-2. Proteomics data revealed that UvEC1 has a variety of effects on metabolism, protein localization, catalytic activity, binding, toxin biosynthesis and the spliceosome. Taken together, our findings suggest that UvEC1 is critical for the development and virulence of U. virens.     

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins

Interferon-β (IFN-β) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-β and S100P lowering IFN-β cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633–639). S100P is a member of large family of multifunctional Ca²⁺-binding proteins with cytokine-like activities. To probe selectivity of IFN-β—S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca²⁺-dependent binding to IFN-β with equilibrium dissociation constants, K_d, of 0.04–1.5 µM for their Ca²⁺-bound homodimeric forms. Calcium depletion abolishes the S100—IFN-β interactions. Monomerization of S100A1/A4/A6 decreases K_d values down to 0.11–1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-β-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-β activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-β.     

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury—Time-Dependent Changes in the Cortex,Striatum, Thalamus and Hippocampus in a Mouse Model

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein—GFAP), microglia/macrophages (allograft inflammatory factor 1—IBA-1), and microglia (transmembrane protein 119—TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.     

The Effect of the Aerial Part of Lindera akoensis on Lipopolysaccharides (LPS)-Induced Nitric Oxide Production in RAW264.7 Cells

Four new secondary metabolites, 3α-((E)-Dodec-1-enyl)-4β-hydroxy-5β-methyldihydrofuran-2-one (1), linderinol (6), 4′-O-methylkaempferol 3-O-α-l-(4″-E-p-coumaroyl)rhamnoside (11) and kaempferol 3-O-α-l-(4″-Z-p-coumaroyl) rhamnoside (12) with eleven known compounds—3-epilistenolide D₁ (2), 3-epilistenolide D₂ (3), (3Z,4α,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (4), (3E,4β,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (5), matairesinol (7), syringaresinol (8), (+)-pinoresinol (9), salicifoliol (10), 4″-p-coumaroylafzelin (13), catechin (14) and epicatechin (15)—were first isolated from the aerial part of Lindera akoensis. Their structures were determined by detailed analysis of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Lindera akoensis showed that in vitro anti-inflammatory activity decreases the LPS-stimulated production of nitric oxide (NO) in RAW 264.7 cell, with IC₅₀ values of 4.1–413.8 μM.     

Molecular Modeling and MM-PBSA Free Energy Analysis of Endo-1,4-β-Xylanase from Ruminococcus albus 8

Endo-1,4-β-xylanase (EC 3.2.1.8) is the enzyme from Ruminococcus albus 8 (R. albus 8) (Xyn10A), and catalyzes the degradation of arabinoxylan, which is a major cell wall non-starch polysaccharide of cereals. The crystallographic structure of Xyn10A is still unknown. For this reason, we report a computer-assisted homology study conducted to build its three-dimensional structure based on the known sequence of amino acids of this enzyme. In this study, the best similarity was found with the Clostridium thermocellum (C. thermocellum) N-terminal endo-1,4-β-d-xylanase 10 b. Following the 100 ns molecular dynamics (MD) simulation, a reliable model was obtained for further studies. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methods were used for the substrate xylotetraose having the reactive sugar, which was bound in the −1 subsite of Xyn10A in the ⁴C₁ (chair) and ²S_O (skew boat) ground state conformations. According to the simulations and free energy analysis, Xyn10A binds the substrate with the −1 sugar in the ²S_O conformation 39.27 kcal·mol⁻¹ tighter than the substrate with the sugar in the ⁴C₁ conformation. According to the Xyn10A-²S_O Xylotetraose (X4(sb) interaction energies, the most important subsite for the substrate binding is subsite −1. The results of this study indicate that the substrate is bound in a skew boat conformation with Xyn10A and the −1 sugar subsite proceeds from the ⁴C₁ conformation through ²S_O to the transition state. MM-PBSA free energy analysis indicates that Asn187 and Trp344 in subsite −1 may an important residue for substrate binding. Our findings provide fundamental knowledge that may contribute to further enhancement of enzyme performance through molecular engineering.     

Carvacrol and trans-Cinnamaldehyde Reduce Clostridium difficile Toxin Production and Cytotoxicity in Vitro

Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo.