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Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient–derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients.Significance:Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis.See related commentary by Jain and Dudeja, p. 1838.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Table from Human Colon Cancer–Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice

Supplementary Figure from Human Colon Cancer–Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice

Human gut microbial species found to associate with clinical responses to immune checkpoint inhibitors (ICIs) are often tested in mice using fecal microbiota transfer (FMT), wherein tumor responses in recipient mice may recapitulate human responses to ICI treatment. However, many FMT studies have reported only limited methodological description, details of murine cohorts, and statistical methods. To investigate the reproducibility and robustness of gut microbial species that impact ICI responses, we performed human to germ-free mouse FMT using fecal samples from patients with non-small cell lung cancer who had a pathological response or nonresponse after neoadjuvant ICI treatment. R-FMT mice yielded greater anti-tumor responses in combination with anti-PD-L1 treatment compared to NR-FMT, although the magnitude varied depending on mouse cell line, sex, and individual experiment. Detailed investigation of post-FMT mouse microbiota using 16S rRNA amplicon sequencing, with models to classify and correct for biological variables, revealed a shared presence of the most highly abundant taxa between the human inocula and mice, though low abundance human taxa colonized mice more variably after FMT. Multiple Clostridium species also correlated with tumor outcome in individual anti-PD-L1-treated R-FMT mice. RNAseq analysis revealed differential expression of T and NK cell-related pathways in responding tumors, irrespective of FMT source, with enrichment of these cell types confirmed by immunohistochemistry. This study identifies several human gut microbial species that may play a role in clinical responses to ICIs and suggests attention to biological variables is needed to improve reproducibility and limit variability across experimental murine cohorts.

Distant organ crosstalk during AKI, first studied mechanistically in the lung (1), is now known to play an important role in disease outcomes. The relationship between intestinal microbiota and AKI has also been explored, resulting in exciting findings that are promising for future human therapeutics. In search of ways to reduce kidney T cells by using “cleaner,” germfree (GF) mice, it was unexpectedly found that GF mice not only retained their kidney immune cells, but also had a higher number of natural killer T (NKT) cells and reduced IL-4 levels compared with normal wild-type (WT) mice. Moreover, GF mice subjected to kidney ischemia-reperfusion (IR) injury had increased CD8 T-cell trafficking and inflammatory cytokine mediators compared with normal WT mice (2). Induction of ischemic AKI in GF mice led to a worse course of AKI, in terms of both structure and function. Conventionalizing GF mice with normal mouse stool led to normalizing T-cell and NKT populations, and relative protection from AKI, compared with GF mice (2). It was subsequently observed that early exposure to gut microbiota during development was associated with long-term changes in NKT-cell function in nonrenal tissues, and NKT cells in GF mice had a less mature phenotype with diminished activation capacity upon antigen encounter (3,4). To elucidate whether the effects were bidirectional and AKI led to changes in the gut microbiome, gut microbiota was evaluated in C57BL/6 WT mice after ischemic or nephrotoxic (cisplatin; CP) injury. DNA was isolated from the fecal pellets ( n =4–5 per group) at baseline (D0) and at 3 days (D3) post-IR. The V3–V5 region of the 16S ribosomal RNA gene was amplified using the 357F/926R primer set and sequenced using the Roche/454 platform. IR and CP modified the relative abundance of specific bacterial species belonging to the phyla Actinobacteria, Bacteroidetes, Firmicutes, …

The main goal of this work is to study a numerical method for certain hybrid fuzzy differential equations with an application of a reproducing kernel Hilbert space technique for fuzzy differential equations. Meanwhile, we construct a system of orthogonal functions of the space W 2 2 [ a , b ] ⊕ W 2 2 [ a , b ] depending on a Gram-Schmidt orthogonalization process to get approximate-analytical solutions of a hybrid fuzzy differential equation. A proof of convergence of this method is also discussed in detail. The exact as well as the approximate solutions are displayed by a series in terms of their α-cut representation form in the Hilbert space W 2 2 [ a , b ] ⊕ W 2 2 [ a , b ] . To demonstrate behavior, efficiency, and appropriateness of the present technique, two different numerical experiments are solved numerically in this paper.

This work brings a one-dimensional logistic harvesting model with Allee effect to the time-varying framework. This new framework is more sober than the autonomous version of the system because it; the framework, permits all environment-dependent coefficients to depend on time. Based on these coefficients, we derive sets of conditions that drive population to “mathematical” extinction. More precisely, we investigate various local and global stability notions including uniform stability, attractivity, asymptotic stability and the (uniform) exponential stability.

BackgroundHuman gut microbial species found to associate with clinical responses to immune checkpoint inhibitors (ICIs) are often tested in murine models using fecal microbiota transfer (FMT), wherein tumor responses in recipient mice may recapitulate human responses to ICI treatment. However, many FMT studies have reported only limited methodological description, including identification of colonizing species associated with murine outcomes, details of murine cohorts, and statistical methods. Thus, the reproducibility and robustness of ICI murine models remain uncertain.MethodsTo investigate gut microbial species that impact ICI responses, we performed human to germ-free (GF) mouse FMT using pre-treatment stools from a pathologic lung cancer responder (R) and a pathologic lung cancer non-responder (NR) after neoadjuvant anti-PD-1 and anti-CTLA4 treatment, followed by implantation of the mice with syngeneic tumors and anti-PD-L1 treatment. Cohorts of GF mice varied by sex, age and syngeneic cell line implanted. To identify relevant microbes, murine tumor progressors (MT-P) and non-progressors (MT-NP) to anti-PD-L1 were classified based on tumor growth curves, 16S rRNA sequencing of human and mouse stools was performed, and data was statistically corrected for mouse characteristics using a generalized linear model. RNA sequencing was performed to assess transcriptional changes in murine tumors.ResultsR-FMT mice yielded a greater anti-tumor response in combination with anti-PD-L1 treatment compared to NR-FMT, although the magnitude varied depending on the mouse cell line, sex, and individual experiment. Microbiota analysis revealed a shared presence of the most highly abundant taxa between the human inocula and mice, however low abundance human taxa colonized mice more variably after FMT. Multiple Clostridium species correlated with tumor outcome in individual anti-PD-L1-treated R-FMT mice. RNAseq analysis revealed differential expression of T cell and NK cell-related pathways in responding tumors, irrespective of FMT source, and enrichment of these cell types were confirmed by immunohistochemistry.ConclusionsThis study identifies several human intestinal microbial species that may play a role in clinical responses to ICIs and suggests attention to biological variables is needed to improve reproducibility and limit variability across experimental murine models.Ethics ApprovalAll studies in this abstract have been approved by Johns Hopkins University Animal Care and Use and Johns Hopkins Medicine Institutional Review Board.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient–derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. Significance: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825

In this paper, we introduce the local fractional semilinear and the local fractional Dahl models and investigate their consistency with hysteresis. The notion of consistency was initially introduced in Ikhouane (Math Control Signals Syst 25:294–310) to analyze the hysteretic behavior of causal systems and to describe analytically the so-called “hysteresis loops”. The validity of our results is confirmed by numerical simulations.

In this work, we consider the problem of estimating the parameters and predicting the unobserved or removed ordered data for the progressive type II censored flexible Weibull sample. Frequentist and Bayesian analyses are adopted for conducting the estimation and prediction problems. The likelihood method as well as the Bayesian sampling techniques is applied for the inference problems. The point predictors and credible intervals of unobserved data based on an informative set of data are computed. Markov Chain Monte Carlo samples are performed to compare the so-obtained methods, and one real data set is analyzed for illustrative purposes.

Background Long-term effects of chronic and/or recurrent C. difficile infections (CDI) are not well understood, and any potential role of CDI in colorectal cancer (CRC) risk is presently unknown. While pursuing efforts to identify novel procarcinogenic microbes, we identified two mucosal slurries from CRC patients (3728T and 3752T) that were tumorigenic in germ-free (GF) ApcMin/+ mice. Surprisingly, both of these CRC patient slurries were positive for C. difficile by 16S rRNA amplicon sequencing. Given the ability of other chronic infections to promote tumorigenesis (e.g., H. pylori), we hypothesized that chronic colonization with C. difficile could promote tumorigenesis in the colon. Methods A consortium of 30 bacterial isolates including a toxigenic tcdA+ tcdB+ C. difficile strain (CIm_3728T) was cultured from GF ApcMin/+ mice gavaged with the 3728T slurry. This consortium was gavaged into additional GF ApcMin/+ mice with or without C. difficile strains CIm_3728T, CIm_3752T (isolated from mice gavaged with the 3752T slurry), or isogenic tcdA/tcdB mutants of the M7404 R027 strain. Single cell RNA sequencing (scRNAseq), high dimensional (HD) flow cytometry, and fluorescence in situ hybridization (FISH) with EUB338 and Cd198 probes were performed on distal colons from mice gavaged with either complex CRC slurries or the 3728T isolates with CIm_3728T. Results C. difficile strains drove tumorigenesis of the 3728T isolate mixture (Fig. 1A,B). Tumorigenesis was associated with early procarcinogenic signaling and spatial changes including induction of Wnt signaling in colonic epithelial progenitor cells by scRNAseq, IL-17 induction in immune cells by HD flow cytometry, and bacterial biofilm invasion deep into epithelial crypts by FISH. Tumorigenesis correlated with chronic colonization with toxigenic strains of C. difficile and was toxin-dependent, as toxin mutant strains (M7404 tcdA-tcdB-) did not induce tumors. Figure 1. C. difficile strains from CRC patients induce distal colonic tumorigenesis in germ-free (GF) ApcMin/+ mice. A consortium of 30 bacteria, including C. difficile, were isolated from mice gavaged with the 3728T human CRC mucosal slurry. These isolates were then gavaged into additional GF ApcMin/+ mice, with or without C. difficile isolates from mice gavaged with the 3728T slurry or 3752T slurry. (A) Colonic tumor numbers in GF ApcMin/+ mice at 10 wk p.i. demonstrate that C. difficile (Cd) drives the tumorigenesis of this 30-member bacterial consortium. (B) Gross tumors can be observed in the colon of a representative mouse gavaged with the 3728T isolates with the CIm_3728T (top) or CIm_3752T (middle) strain of C. difficile but not in a mouse gavaged with the isolates lacking C. difficile (bottom). Conclusion Toxigenic C. difficile strains isolated from human CRC mucosal slurries were pro-carcinogenic in mice, suggesting that C. difficile is a potential driver of CRC. Given the public health burden of C. difficile, further studies are warranted to determine whether C. difficile infections (initial, recurrent, and chronic asymptomatic) increase CRC risk in patients. Disclosures Jada Domingue, PhD, AstraZeneca (Employee) James White, PhD, Personal Genome Diagnostics (Consultant) Patricia J. Simner, PhD, Accelerate Diagnostics (Grant/Research Support)Affinity Biosensors (Grant/Research Support)BD Diagnostics (Consultant, Grant/Research Support)GeneCapture (Consultant)OpGen, Inc (Consultant, Grant/Research Support)Shionogi, Inc (Consultant) Karen C. Carroll, MD, MeMed (Scientific Research Study Investigator)Meridian Diagnostics, Inc. (Grant/Research Support)Pattern Diagnostics (Advisor or Review Panel member)Scanogen, Inc. (Advisor or Review Panel member) Karen C. Carroll, MD, Pattern Diagnostics, Inc. (Individual(s) Involved: Self): Grant/Research Support; Scanogen, Inc. (Individual(s) Involved: Self): Consultant Cynthia L. Sears, MD, Bristol Myers Squibb (Grant/Research Support)Ferring (Advisor or Review Panel member)Janssen (Grant/Research Support)

N1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m1G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here, we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m1G37 deficiency on protein synthesis. Using E coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.