The Integrity of Heme Is Essential for Reproducible Detection of Metronidazole-Resistant Clostridioides difficile by Agar Dilution Susceptibility Tests.

Metronidazole resistance in clinical Clostridioides difficile is often described as unstable, since resistant strains reportedly appear susceptible following freezer storage or brief passage. This has presented a conundrum for adopting susceptibility testing to accurately evaluate the connection between metronidazole resistance and decreased clinical efficacy of metronidazole in patients with C. difficile infections (CDIs). We discovered that supplementation of microbiological media with the metalloporphyrin heme is crucial for detection of metronidazole-resistant C. difficile using the agar dilution susceptibility testing method. Known metronidazole-resistant strains appeared susceptible to metronidazole in media lacking heme. Similarly, these resistant strains exhibited increased susceptibility to metronidazole when tested on heme-containing agars that were exposed to room light for more than 1 day, likely due to heme photodecomposition. In parallel experiments, resistance was reproducibly detected when heme-containing agars were either prepared and used on the same day or protected from light and then used on subsequent days. Notably, heme did not influence the susceptibilities of drug-susceptible strains that were of the same ribotype as the resistant strains. These findings firmly show that the consistent detection of metronidazole-resistant C. difficile is dependent upon heme and its protection from light. Studies are warranted to determine the extent to which this heme-associated metronidazole-resistant phenotype affects the clinical efficacy of metronidazole in CDI and the underlying genetic and biochemical mechanisms.

Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates.

OBJECTIVES: To describe, for the first time (to the best of our knowledge), the genetic mechanisms of vancomycin resistance in clinical isolates of Clostridioides difficile ribotype 027. METHODS: Clinical isolates and laboratory mutants were analysed: genomically to identify resistance mutations; by transcriptional analysis of vanGCd, the vancomycin resistance operon encoding lipid II d-alanine-d-serine that is less bound by vancomycin than native lipid II d-alanine-d-alanine; by imaging of vancomycin binding to cell walls; and for changes in vancomycin bactericidal activity and autolysis. RESULTS: Vancomycin-resistant laboratory mutants and clinical isolates acquired mutations to the vanSR two-component system that regulates vanGCd. The substitutions impaired VanSR's function, resulting in constitutive transcription of vanGCd. Resistance was reversed by silencing vanG, encoding d-alanine-d-serine ligase in the vanGCd operon. In resistant cells, vancomycin was less bound to the cell wall septum, the site where vancomycin interacts with lipid II. Vancomycin's bactericidal activity was reduced against clinical isolates and laboratory mutants (64 and ≥1024 mg/L, respectively) compared with WT strains (4 mg/L). Truncation of the potassium transporter TrkA occurred in laboratory mutants, which were refractory to autolysis, accounting for their survival in high drug concentrations. CONCLUSIONS: Ribotype 027 evolved first-step resistance to vancomycin by constitutively expressing vanGCd, which is otherwise silent. Experimental evolutions and bactericidal assays show that ribotype 027 can acquire mutations to drastically enhance its tolerance to vancomycin. Thus, further epidemiological studies are warranted to examine the extent to which vancomycin resistance impacts clinical outcomes and the potential for these strains to evolve higher-level resistance, which would be devastating.

A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses.

The generation of reactive oxygen species (ROS) in a live-cell system is routinely measured using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF). However, it is difficult to simultaneously monitor cellular oxidative responses and ROS generation in cells, and analyses of cellular oxidative responses are typically performed after ROS generation has been evaluated. In this study, we developed a modified fixed staining method that allows the simultaneous analysis of ROS generation and oxidative responses using standard immunostaining techniques. A microplate reader-based assay showed that of the fixatives tested, only methanol did not alter the hydrogen peroxide (H(2)O(2))-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), a chloromethyl derivative of H(2)DCFDA, or the fluorescence of oxidized DCF in vitro. Further in vivo assays using flow cytometry showed that both methanol and acetic acid maintained the fluorescence of oxidized DCF in H(2)O(2)-, antimycin A-, and serum starvation-treated human lung adenocarcinoma A549 cells and human microvascular endothelial HMEC-1 cells. Following acetic acid-based fixation, the ROS generation in starved HMEC-1 cells could be evaluated by flow cytometric analysis while simultaneously monitoring the phosphorylation status of p38 mitogen-activated protein kinase. Immunostaining also revealed the synchronization of ROS generation and the H(2)O(2)-induced phosphorylation of Src homology-2 domain-containing phosphatase2. This study describes a modified method that may be used in future biomedical investigations to simultaneously measure intracellular ROS production and cellular oxidative responses.

The natural tannins oligomeric proanthocyanidins and punicalagin are potent inhibitors of infection by SARS-CoV-2.

The Coronavirus Disease 2019 (COVID-19) pandemic continues to infect people worldwide. While the vaccinated population has been increasing, the rising breakthrough infection persists in the vaccinated population. For living with the virus, the dietary guidelines to prevent virus infection are worthy of and timely to develop further. Tannic acid has been demonstrated to be an effective inhibitor of coronavirus and is under clinical trial. Here we found that two other members of the tannins family, oligomeric proanthocyanidins (OPCs) and punicalagin, are also potent inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with different mechanisms. OPCs and punicalagin showed inhibitory activity against omicron variants of SARS-CoV-2 infection. The water extractant of the grape seed was rich in OPCs and also exhibited the strongest inhibitory activities for viral entry of wild-type and other variants in vitro. Moreover, we evaluated the inhibitory activity of grape seed extractants (GSE) supplementation against SARS-CoV-2 viral entry in vivo and observed that serum samples from the healthy human subjects had suppressive activity against different variants of SARS-CoV-2 Vpp infection after taking GSE capsules. Our results suggest that natural tannins acted as potent inhibitors against SARS-CoV-2 infection, and GSE supplementation could serve as healthy food for infection prevention.

Since it first surfaced in late 2019, the COVID-19 pandemic has had a significant impact on people's lives. While several vaccines have been created, infections have not disappeared. This is largely due to new variants of the virus responsible for the disease (SARS-CoV-2) emerging, which current vaccines do not work as well against. Indeed, several reports suggest that protection from the omicron variant wanes as shortly as four to six months after vaccination. Therefore, other strategies are needed to reduce the risk of SARS-CoV-2 infections. In 2022, researchers discovered that tannic acid blocked two proteins that SARS-CoV-2 needs to enter and replicate inside human cells. Tannic acid is part of the tannin family, which includes natural molecules found in plant-based meals and beverages. Here, Chen et al. ­ including some of the researchers involved in the 2022 studies ­ set out to find whether two other tannins found in nature (OPCs and punicalagin) could also inhibit SARS-CoV-2. Chen et al. administered tannic acid, OPCs and punicalagin to human cells cultured in a laboratory that had been infected with SARS-CoV-2. This revealed that all three tannins suppress the activity of the same proteins required for viral entry and replication, but to varying degrees suggesting that they block SARS-CoV-2 infections via different mechanisms. The compounds were also able to inhibit different variants of the virus, including omicron, from infecting the lab-grown cells. Further experiments revealed that water extracted from seeded grapes, which contains high levels of OPCs, could also block SARS-CoV-2 entry in the cell culture system. To test this further, Chen et al. gave 18 healthy individuals capsules containing different concentrations of grape seed extract and collected samples of their serum. The serum samples suppressed entry of different variants of SARS-CoV-2 in the cell culture system, with serums from subjects that received the higher dose having the greatest effect. These findings suggest that naturally occurring tannins can suppress multiple variants of SARS-CoV-2 from entering and replicating in cells. Consuming supplements of grape seed extract could potentially reduce the risk of SARS-CoV-2 infections. However, further experiments, including clinical trials, are needed to test this possibility.

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile.

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests using molecularly intact heme. Here, we describe the mechanism underlying this trait. We find that most metronidazole-resistant C. difficile strains carry a T-to-G mutation (which we term PnimBG) in the promoter of gene nimB, resulting in constitutive transcription. Silencing or deleting nimB eliminates metronidazole resistance. NimB is related to Nim proteins that are known to confer resistance to nitroimidazoles. We show that NimB is a heme-dependent flavin enzyme that degrades nitroimidazoles to amines lacking antimicrobial activity. Furthermore, occurrence of the PnimBG mutation is associated with a Thr82Ile substitution in DNA gyrase that confers fluoroquinolone resistance in epidemic strains. Our findings suggest that the pandemic of fluoroquinolone-resistant C. difficile occurring over the past few decades has also been characterized by widespread resistance to metronidazole.

Inhibition of Galectin-9 sensitizes tumors to anthracycline treatment via inducing antitumor immunity.

Anthracyclines are a class of conventionally and routinely used first-line chemotherapy drugs for cancer treatment. In addition to the direct cytotoxic effects, increasing evidence indicates that the efficacy of the drugs also depends on immunomodulatory effects with unknown mechanisms. Galectin-9 (Gal-9), a member of the ß-galactoside-binding protein family, has been demonstrated to induce T-cell death and promote immunosuppression in the tumor microenvironment. Here, we asked whether anthracycline-mediated immunomodulatory activity might be related to Gal-9. We found that combining doxorubicin with anti-Gal-9 therapy significantly inhibited tumor growth and prolonged overall survival in immune-competent syngeneic mouse models. Moreover, Gal-9 expression was increased in response to doxorubicin in various human and murine cancer cell lines. Mechanistically, doxorubicin induced tumoral Gal-9 by activating the STING/interferon ß pathway. Clinically, Gal-9 and p-STING levels were elevated in the tumor tissues of breast cancer patients treated with anthracyclines. Our study demonstrates Gal-9 upregulation in response to anthracyclines as a novel mechanism mediating immune escape and suggests targeting Gal-9 in combination with anthracyclines as a promising therapeutic strategy for cancer treatment.

Albumin prevents reactive oxygen species-induced mitochondrial damage, autophagy, and apoptosis during serum starvation.

Aberrant levels of reactive oxygen species (ROS) rapidly generated from NADPH oxidase (NOX) activation can be cytotoxic due to activating pro-apoptotic signals. However, ROS also induce pro-survival autophagy through the engulfment of damaged mitochondria. This study is aimed at investigating the cytoprotective role of albumin against NOX/ROS-induced autophagy and apoptosis under serum starvation. Serum starvation induced apoptosis following a myeloid cell leukemia sequence 1 (Mcl-1)/Bax imbalance, loss of the mitochondrial transmembrane potential, and caspase activation accompanied by pro-survival autophagy following canonical inhibition of mammalian target of rapamycin complex 1 (mTORC1). Aberrant ROS generation, initially occurring through NOX, facilitated mitochondrial damage, autophagy, and apoptosis. Autophagy additionally regulated the accumulation of ROS-generating mitochondria. NOX/ROS permitted p38 mitogen-activated protein kinase (p38 MAPK)-regulated mitochondrial apoptosis, accompanied by non-canonical induction of autophagy. In addition, activation of glycogen synthase kinase (GSK)-3ß by NOX/ROS-inactivated Akt facilitated a decrease in Mcl-1, followed by mitochondrial apoptosis as well as autophagy. Restoring albumin conferred an anti-oxidative effect against serum starvation-deregulated NOX, p38 MAPK, and Akt/GSK-3ß/Mcl-1/caspase-3 signaling. Albumin also prevented autophagy by sustaining mTORC1. These results indicate an anti-oxidative role for albumin via preventing NOX/ROS-mediated mitochondrial signaling to stimulate apoptosis as well as autophagy. Autophagy, initially induced by canonical inhibition of mTORC1 and enhanced by non-canonical mitochondrial damage, acts physically as a pro-survival mechanism.

Circadian rhythm-related factors of PER and CRY family genes function as novel therapeutic targets and prognostic biomarkers in lung adenocarcinoma.

The period (PER) and cryptochrome (CRY) families play critical roles in circadian rhythms. The imbalance of circadian factors may lead to the occurrence of cancer. Expressions of PER and CRY family members decrease in various cancers. Nevertheless, expression levels, genetic variations, and molecular mechanisms of PER and CRY family members in lung adenocarcinoma (LUAD) and their correlations with prognoses and immune infiltration in LUAD patients are still unclear. In this study, to identify their biological functions in LUAD development, comprehensive high-throughput techniques were applied to analyze the relationships of expressions of PER and CRY family members with genetic variations, molecular mechanisms, and immune infiltration. The present results showed that transcription levels of PER1 and CRY2 in LUAD were significantly downregulated. High expression levels of PER2, PER3, CRY1, and CRY2 indicated longer overall survival. Some cancer signaling pathways were related to PER and CRY family members, such as cell-cycle, histidine metabolism, and progesterone-mediated oocyte maturation pathways. Expressions of PER and CRY family members significantly affected the infiltration of different immune cells. In conclusion, our findings may help better understand the molecular basis of LUAD, and provide new perspectives of PER and CRY family members as novel biomarkers for LUAD.

Penetrating Exploration of Prognostic Correlations of the FKBP Gene Family with Lung Adenocarcinoma.

The complexity of lung adenocarcinoma (LUAD), the development of which involves many interacting biological processes, makes it difficult to find therapeutic biomarkers for treatment. FK506-binding proteins (FKBPs) are composed of 12 members classified as conservative intracellular immunophilin family proteins, which are often connected to cyclophilin structures by tetratricopeptide repeat domains and have peptidyl prolyl isomerase activity that catalyzes proline from residues and turns the trans form into the cis form. Since FKBPs belong to chaperone molecules and promote protein folding, previous studies demonstrated that FKBP family members significantly contribute to the degradation of damaged, misfolded, abnormal, and foreign proteins. However, transcript expressions of this gene family in LUAD still need to be more fully investigated. In this research, we adopted high-throughput bioinformatics technology to analyze FKBP family genes in LUAD to provide credible information to clinicians and promote the development of novel cancer target drugs in the future. The current data revealed that the messenger (m)RNA levels of FKBP2, FKBP3, FKBP4, FKBP10, FKBP11, and FKBP14 were overexpressed in LUAD, and FKBP10 had connections to poor prognoses among LUAD patients in an overall survival (OS) analysis. Based on the above results, we selected FKBP10 to further conduct a comprehensive analysis of the downstream pathway and network. Through a DAVID analysis, we found that FKBP10 was involved in mitochondrial electron transport, NADH to ubiquinone transport, mitochondrial respiratory chain complex I assembly, etc. The MetaCore pathway analysis also indicated that FKBP10 was involved in "Ubiquinone metabolism", "Translation_(L)-selenoaminoacid incorporation in proteins during translation", and "Transcription_Negative regulation of HIF1A function". Collectively, this study revealed that FKBP family members are both significant prognostic biomarkers for lung cancer progression and promising clinical therapeutic targets, thus providing new targets for treating LUAD patients.

Novel Potential Therapeutic Targets of PTPN Families for Lung Cancer.

Despite the treatment of lung adenocarcinoma (LUAD) having partially improved in recent years, LUAD patients still have poor prognosis rates. Therefore, it is especially important to explore effective biomarkers and exploit novel therapeutic developments. High-throughput technologies are widely used as systematic approaches to explore differences in expressions of thousands of genes for both biological and genomic systems. Recently, using big data analyses in biomedicine research by integrating several high-throughput databases and tools, including The Cancer Genome Atlas (TCGA), cBioportal, Oncomine, and Kaplan-Meier plotter, is an important strategy to identify novel biomarkers for cancer therapy. Here, we used two different comprehensive bioinformatics analysis and revealed protein tyrosine phosphatase non-receptor type (PTPN) family genes, especially PTPN1 and PTPN22, were downregulated in lung cancer tissue in comparison with normal samples. The survival curves indicated that LUAD patients with high transcription levels of PTPN5 were significantly associated with a good prognosis. Meanwhile, Gene Ontology (GO) and MetaCore analyses indicated that co-expression of the PTPN1, PTPN5, and PTPN21 genes was significantly enriched in cancer development-related pathways, including GTPase activity, regulation of small GTPase-mediated signal transduction, response to mechanical stimuli, vasculogenesis, organ morphogenesis, regulation of stress fiber assembly, mitogen-activated protein kinase (MAPK) cascade, cell migration, and angiogenesis. Collectively, this study revealed that PTPN family members are both significant prognostic biomarkers for lung cancer progression and promising clinical therapeutic targets, which provide new targets for treating LUAD patients.

Peimine inhibits variants of SARS-CoV-2 cell entry via blocking the interaction between viral spike protein and ACE2.

Coronavirus disease 2019 (COVID-19) is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several vaccines against SARS-CoV-2 have been approved; however, variants of concern (VOCs) can evade vaccine protection. Therefore, developing small compound drugs that directly block the interaction between the viral spike glycoprotein and ACE2 is urgently needed to provide a complementary or alternative treatment for COVID-19 patients. We developed a viral infection assay to screen a library of approximately 126 small molecules and showed that peimine inhibits VOCs viral infections. In addition, a fluorescence resonance energy transfer (FRET) assay showed that peimine suppresses the interaction of spike and ACE2. Molecular docking analysis revealed that peimine exhibits a higher binding affinity for variant spike proteins and is able to form hydrogen bonds with N501Y in the spike protein. These results suggest that peimine, a compound isolated from Fritillaria, may be a potent inhibitor of SARS-CoV-2 variant infection. PRACTICAL APPLICATIONS: In this study, we identified a naturally derived compound of peimine, a major bioactive alkaloid extracted from Fritillaria, that could inhibit SARS-CoV-2 variants of concern (VOCs) viral infection in 293T/ACE2 and Calu-3 lung cells. In addition, peimine blocks viral entry through interruption of spike and ACE2 interaction. Moreover, molecular docking analysis demonstrates that peimine has a higher binding affinity on N501Y in the spike protein. Furthermore, we found that Fritillaria significantly inhibits SARS-CoV-2 viral infection. These results suggested that peimine and Fritillaria could be a potential functional drug and food for COVID-19 patients.

Potential natural products that target the SARS-CoV-2 spike protein identified by structure-based virtual screening, isothermal titration calorimetry and lentivirus particles pseudotyped (Vpp) infection assay.

BACKGROUND AND AIM: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells through the binding of the viral spike protein with human angiotensin-converting enzyme 2 (ACE2), resulting in the development of coronavirus disease 2019 (COVID-19). To date, few antiviral drugs are available that can effectively block viral infection. This study aimed to identify potential natural products from Taiwan Database of Extracts and Compounds (TDEC) that may prevent the binding of viral spike proteins with human ACE2 proteins. METHODS: The structure-based virtual screening was performed using the AutoDock Vina program within PyRX software, the binding affinities of compounds were verified using isothermal titration calorimetry (ITC), the inhibitions of SARS-CoV-2 viral infection efficacy were examined by lentivirus particles pseudotyped (Vpp) infection assay, and the cell viability was tested by 293T cell in MTT assay. RESULTS AND CONCLUSION: We identified 39 natural products targeting the viral receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in silico. In ITC binding assay, dioscin, celastrol, saikosaponin C, epimedin C, torvoside K, and amentoflavone showed dissociation constant (K d) = 0.468 µM, 1.712 µM, 6.650 µM, 2.86 µM, 3.761 µM and 4.27 µM, respectively. In Vpp infection assay, the compounds have significantly and consistently inhibition with the 50-90% inhibition of viral infection efficacy. In cell viability, torvoside K, epimedin, amentoflavone, and saikosaponin C showed IC50 > 100 µM; dioscin and celastrol showed IC50 = 1.5625 µM and 0.9866 µM, respectively. These natural products may bind to the viral spike protein, preventing SARS-CoV-2 from entering cells. SECTION 1: Natural Products. TAXONOMY CLASSIFICATION BY EVISE: SARS-CoV-2, Structure-Based Virtual Screening, Isothermal Titration Calorimetry and Lentivirus Particles Pseudotyped (Vpp) Infection Assay, in silico and in vitro study.

MCL1 participates in leptin-promoted mitochondrial fusion and contributes to drug resistance in gallbladder cancer.

Obesity is a risk factor for gallbladder cancer (GBC) development, and it correlates with shorter overall survival. Leptin, derived from adipocytes, has been suggested to contribute to the growth of cancer cells; however, the detailed mechanism of leptin in GBC drug resistance remains uninvestigated. In this study, our finding that patients with GBC with a higher BMI were associated with increased GBC risks, including shortened survival, is clinically relevant. Moreover, obese NOD/SCID mice exhibited a higher circulating concentration of leptin, which is associated with GBC growth and attenuated gemcitabine efficacy. We further revealed that leptin can inhibit gemcitabine-induced GBC cell death through myeloid cell leukemia 1 (MCL1) activation. The transcription factor C/EBP δ (CEBPD) is responsive to activated STAT3 (pSTAT3) and contributes to MCL1 transcriptional activation upon leptin treatment. In addition, MCL1 mediates leptin-induced mitochondrial fusion and is associated with GBC cell survival. The findings in this study suggest the involvement of the pSTAT3/CEBPD/MCL1 axis in leptin-induced mitochondrial fusion and survival and provide a potentially new therapeutic target to improve the efficacy of gemcitabine in patients with GBC.

Potential Prognostic Biomarkers of OSBPL Family Genes in Patients with Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy with poor survival outcomes. In addition, oxysterol-binding protein-like (OSBPL) family members are reported to be involved in lipid binding and transport and play critical roles in tumorigenesis. However, relationships between PDAC and OSBPL family members have not comprehensively been elucidated. In this study, we used the Oncomine and GEPIA 2 databases to analyze OSBPL transcription expressions in PDAC. The Kaplan-Meier plotter and TIMER 2.0 were used to assess the relationships between overall survival (OS) and immune-infiltration with OSBPL family members. Co-expression data from cBioPortal were downloaded to assess the correlated pathways with OSBPL gene family members using DAVID. The expressions of OSBPL3, OSBPL8, OSBPL10, and OSBPL11 were found to be highly upregulated in PDAC. Low expressions of OSBPL3, OSBPL8, and OSBPL10 indicated longer OS. The functions of OSBPL family members were mainly associated with several potential signaling pathways in cancer cells, including ATP binding, integrin binding, receptor binding, and the renin-angiotensin system (RAS) signaling pathway. The transcription levels of OSBPL gene family members were connected with several immune infiltrates. Collectively, OSBPL family members are influential biomarkers for the early diagnosis of PDAC and have prognostic value, with the promise of precise treatment of PDAC in the future.

Reduced Susceptibility to Metronidazole Is Associated With Initial Clinical Failure in Clostridioides difficile Infection.

BACKGROUND: Clinical studies have demonstrated inferior cure rates when metronidazole (MTZ) is used to treat Clostridioides difficile infection (CDI). We hypothesized that a newly identified, heme-inducible form of reduced MTZ susceptibility in C. difficile leads to higher odds of initial clinical failure in patients with CDI treated with MTZ. METHODS: This multicenter cohort study included adults diagnosed with CDI between 2017 and 2018. C. difficile isolated from stool samples underwent agar dilution MTZ susceptibility testing with incorporation of fresh heme. Blinded investigators reviewed medical records for initial clinical failure and other relevant clinical variables. Classification and regression tree (CART) analysis was used to identify the MTZ minimum inhibitory concentration (MIC) breakpoint that was predictive of initial clinical failure. Results were confirmed using univariate and multivariable logistic regression analyses to account for potential confounders. RESULTS: Of the 356 patients included, 72% received MTZ-based therapy and 27% experienced initial clinical failure. CART analysis identified an MTZ MIC ≥1 µg/mL above which patients had a higher rate of initial clinical failure. MTZ MICs ranged from 0.25 to 8 µg/mL (MIC50/90 = 0.25/2 µg/mL), and approximately 18% of isolates had MTZ MICs ≥1 µg/mL. In multivariable analysis, an MTZ MIC ≥1 µg/mL was an independent predictor of initial clinical failure in patients receiving an MTZ-based treatment regimen (odds ratio, 2.27 [95% confidence interval, 1.18-4.34]). CONCLUSIONS: Using a reproducible method to determine C. difficile MICs to MTZ, a breakpoint of ≥1 µg/mL identified patients at higher risk of initial clinical failure.

Microbiota regulate innate immune signaling and protective immunity against cancer.

Microbiota play critical roles in regulating colitis and colorectal cancer (CRC). However, it is unclear how the microbiota generate protective immunity against these disease states. Here, we find that loss of the innate and adaptive immune signaling molecule, TAK1, in myeloid cells (Tak1ΔM/ΔM) yields complete resistance to chemical-induced colitis and CRC through microbiome alterations that drive protective immunity. Tak1ΔM/ΔM mice exhibit altered microbiota that are critical for resistance, with antibiotic-mediated disruption ablating protection and Tak1ΔM/ΔM microbiota transfer conferring protection against colitis or CRC. The altered microbiota of Tak1ΔM/ΔM mice promote IL-1ß and IL-6 signaling pathways, which are required for induction of protective intestinal Th17 cells and resistance. Specifically, Odoribacter splanchnicus is abundant in Tak1ΔM/ΔM mice and sufficient to induce intestinal Th17 cell development and confer resistance against colitis and CRC in wild-type mice. These findings identify specific microbiota strains and immune mechanisms that protect against colitis and CRC.

Pharmacophore Modeling, Synthesis, and Antibacterial Evaluation of Chalcones and Derivatives.

A series of novel chalcone and thiol-Michael addition analogues was synthesized and tested against Mycobacterium tuberculosis and other clinically significant bacterial pathogens. Previously reported chalcone-like antibacterials (1a-c and 2) were used as a training set to generate a pharmacophore model. The chalcone derivative hit compound 3 was subsequently identified through a pharmacophore-based virtual screen of the Specs library of >200 000 compounds. Among the newly synthesized chalcones and thiol-Michael addition analogues, chalcones 6r and 6s were active (minimum inhibitory concentrations (MICs) = 1.56-6.25 µg/mL) against Gram-positive pathogens Bacillus anthracis and Staphylococcus aureus [methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA)]. The chalcone thiol-Michael addition derivatives 7j-m showed good to excellent antibacterial activities (MICs = 0.78-6.25 µg/mL) against Enterococcus faecalis, B. anthracis, and S. aureus. Interestingly, the amine-Michael addition analogue 12a showed promising anti-MRSA activity (MIC = 1.56 µg/mL) with a selectivity index of 14 toward mammalian Vero cells. In addition, evaluation of selected compounds against biofilm and planktonic S. aureus (MSSA and MRSA) revealed that 12a exhibited bactericidal activities in these assays, which was overall superior to vancomycin. These properties may result from the compounds dissipating the proton motive force of bacterial membranes.