IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

ISL1 regulates lung branching morphogenesis via Shh signaling pathway.

Lung branching morphogenesis relies on a complex coordination of multiple signaling pathways and transcription factors. Here, we found that ablation of the LIM homeodomain transcription factor Islet1 (Isl1) in lung epithelium resulted in defective branching morphogenesis and incomplete formation of five lobes. A reduction in mesenchymal cell proliferation was observed in Isl1ShhCre lungs. There was no difference in apoptosis between the wild-type (ShhCre) and Isl1ShhCre embryos. RNA-Seq and in situ hybridization analysis showed that Shh, Ptch1, Sox9, Irx1, Irx2, Tbx2, and Tbx3 were downregulated in the lungs of Isl1ShhCre embryos. ChIP assay implied the Shh gene served as a direct target of ISL1, since the transcription factor ISL1 could bind to the Shh epithelial enhancer sequence (MACS1). Also, activation of the Hedgehog pathway via ectopic gene expression rescued the defects caused by Isl1 ablation, confirming the genetic integration of Hedgehog signaling. In conclusion, our works suggest that epithelial Isl1 regulates lung branching morphogenesis through administrating the Shh signaling mediated epithelial-mesenchymal communications.

Biopsy and blood-based molecular biomarker of inflammation in IBD.

OBJECTIVE: IBD therapies and treatments are evolving to deeper levels of remission. Molecular measures of disease may augment current endpoints including the potential for less invasive assessments. DESIGN: Transcriptome analysis on 712 endoscopically defined inflamed (Inf) and 1778 non-inflamed (Non-Inf) intestinal biopsies (n=498 Crohn's disease, n=421 UC and 243 controls) in the Mount Sinai Crohn's and Colitis Registry were used to identify genes differentially expressed between Inf and Non-Inf biopsies and to generate a molecular inflammation score (bMIS) via gene set variance analysis. A circulating MIS (cirMIS) score, reflecting intestinal molecular inflammation, was generated using blood transcriptome data. bMIS/cirMIS was validated as indicators of intestinal inflammation in four independent IBD cohorts. RESULTS: bMIS/cirMIS was strongly associated with clinical, endoscopic and histological disease activity indices. Patients with the same histologic score of inflammation had variable bMIS scores, indicating that bMIS describes a deeper range of inflammation. In available clinical trial data sets, both scores were responsive to IBD treatment. Despite similar baseline endoscopic and histologic activity, UC patients with lower baseline bMIS levels were more likely treatment responders compared with those with higher levels. Finally, among patients with UC in endoscopic and histologic remission, those with lower bMIS levels were less likely to have a disease flare over time. CONCLUSION: Transcriptionally based scores provide an alternative objective and deeper quantification of intestinal inflammation, which could augment current clinical assessments used for disease monitoring and have potential for predicting therapeutic response and patients at higher risk of disease flares.

A Collaborative Classroom Investigation of the Evolution of SABATH Methyltransferase Substrate Preference Shifts over 120 My of Flowering Plant History.

Next-generation sequencing has resulted in an explosion of available data, much of which remains unstudied in terms of biochemical function; yet, experimental characterization of these sequences has the potential to provide unprecedented insight into the evolution of enzyme activity. One way to make inroads into the experimental study of the voluminous data available is to engage students by integrating teaching and research in a college classroom such that eventually hundreds or thousands of enzymes may be characterized. In this study, we capitalize on this potential to focus on SABATH methyltransferase enzymes that have been shown to methylate the important plant hormone, salicylic acid (SA), to form methyl salicylate. We analyze data from 76 enzymes of flowering plant species in 23 orders and 41 families to investigate how widely conserved substrate preference is for SA methyltransferase orthologs. We find a high degree of conservation of substrate preference for SA over the structurally similar metabolite, benzoic acid, with recent switches that appear to be associated with gene duplication and at least three cases of functional compensation by paralogous enzymes. The presence of Met in active site position 150 is a useful predictor of SA methylation preference in SABATH methyltransferases but enzymes with other residues in the homologous position show the same substrate preference. Although our dense and systematic sampling of SABATH enzymes across angiosperms has revealed novel insights, this is merely the "tip of the iceberg" since thousands of sequences remain uncharacterized in this enzyme family alone.

Integrative Analysis of the Inflammatory Bowel Disease Serum Metabolome Improves Our Understanding of Genetic Etiology and Points to Novel Putative Therapeutic Targets.

BACKGROUND & AIMS: Polygenic and environmental factors are underlying causes of inflammatory bowel disease (IBD). We hypothesized that integration of the genetic loci controlling a metabolite's abundance, with known IBD genetic susceptibility loci, may help resolve metabolic drivers of IBD. METHODS: We measured the levels of 1300 metabolites in the serum of 484 patients with ulcerative colitis (UC) and 464 patients with Crohn's disease (CD) and 365 controls. Differential metabolite abundance was determined for disease status, subtype, clinical and endoscopic disease activity, as well as IBD phenotype including disease behavior, location, and extent. To inform on the genetic basis underlying metabolic diversity, we integrated metabolite and genomic data. Genetic colocalization and Mendelian randomization analyses were performed using known IBD risk loci to explore whether any metabolite was causally associated with IBD. RESULTS: We found 173 genetically controlled metabolites (metabolite quantitative trait loci, 9 novel) within 63 non-overlapping loci (7 novel). Furthermore, several metabolites significantly associated with IBD disease status and activity as defined using clinical and endoscopic indexes. This constitutes a resource for biomarker discovery and IBD biology insights. Using this resource, we show that a novel metabolite quantitative trait locus for serum butyrate levels containing ACADS was not supported as causal for IBD; replicate the association of serum omega-6 containing lipids with the fatty acid desaturase 1/2 locus and identify these metabolites as causal for CD through Mendelian randomization; and validate a novel association of serum plasmalogen and TMEM229B, which was predicted as causal for CD. CONCLUSIONS: An exploratory analysis combining genetics and unbiased serum metabolome surveys can reveal novel biomarkers of disease activity and potential mediators of pathology in IBD.

Evolution of immunogenetic components encoding ultralong CDR H3.

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

A Novel Variable Selection Method Based on Binning-Normalized Mutual Information for Multivariate Calibration.

Variable (wavelength) selection is essential in the multivariate analysis of near-infrared spectra to improve model performance and provide a more straightforward interpretation. This paper proposed a new variable selection method named binning-normalized mutual information (B-NMI) based on information entropy theory. "Data binning" was applied to reduce the effects of minor measurement errors and increase the features of near-infrared spectra. "Normalized mutual information" was employed to calculate the correlation between each wavelength and the reference values. The performance of B-NMI was evaluated by two experimental datasets (ideal ternary solvent mixture dataset, fluidized bed granulation dataset) and two public datasets (gasoline octane dataset, corn protein dataset). Compared with classic methods of backward and interval PLS (BIPLS), variable importance projection (VIP), correlation coefficient (CC), uninformative variables elimination (UVE), and competitive adaptive reweighted sampling (CARS), B-NMI not only selected the most featured wavelengths from the spectra of complex real-world samples but also improved the stability and robustness of variable selection results.

Convergent Biochemical Pathways for Xanthine Alkaloid Production in Plants Evolved from Ancestral Enzymes with Different Catalytic Properties.

Convergent evolution is widespread but the extent to which common ancestral conditions are necessary to facilitate the independent acquisition of similar traits remains unclear. In order to better understand how ancestral biosynthetic catalytic capabilities might lead to convergent evolution of similar modern-day biochemical pathways, we resurrected ancient enzymes of the caffeine synthase (CS) methyltransferases that are responsible for theobromine and caffeine production in flowering plants. Ancestral CS enzymes of Theobroma, Paullinia, and Camellia exhibited similar substrate preferences but these resulted in the formation of different sets of products. From these ancestral enzymes, descendants with similar substrate preference and product formation independently evolved after gene duplication events in Theobroma and Paullinia. Thus, it appears that the convergent modern-day pathways likely originated from ancestral pathways with different inferred flux. Subsequently, the modern-day enzymes originated independently via gene duplication and their convergent catalytic characteristics evolved to partition the multiple ancestral activities by different mutations that occurred in homologous regions of the ancestral proteins. These results show that even when modern-day pathways and recruited genes are similar, the antecedent conditions may be distinctive such that different evolutionary steps are required to generate convergence.

Molecular Characterization of Limited Ulcerative Colitis Reveals Novel Biology and Predictors of Disease Extension.

BACKGROUND AND AIMS: Disease extent varies in ulcerative colitis (UC) from proctitis to left-sided colitis to pancolitis and is a major prognostic factor. When the extent of UC is limited there is often a sharp demarcation between macroscopically involved and uninvolved areas and what defines this or subsequent extension is unknown. We characterized the demarcation site molecularly and determined genes associated with subsequent disease extension. METHODS: We performed RNA sequence analysis of biopsy specimens from UC patients with endoscopically and histologically confirmed limited disease, of which a subset later extended. Biopsy specimens were obtained from the endoscopically inflamed upper (proximal) limit of disease, immediately adjacent to the uninvolved colon, as well as at more proximal, endoscopically uninflamed colonic segments. RESULTS: Differentially expressed genes were identified in the endoscopically inflamed biopsy specimens taken at each patient's most proximal diseased site relative to healthy controls. Expression of these genes in the more proximal biopsy specimens transitioned back to control levels abruptly or gradually, the latter pattern supporting the concept that disease exists beyond the endoscopic disease demarcation site. The gradually transitioning genes were associated with inflammation, angiogenesis, glucuronidation, and homeodomain pathways. A subset of these genes in inflamed biopsy specimens was found to predict disease extension better than clinical features and were responsive to biologic therapies. Network analysis revealed critical roles for interferon signaling in UC inflammation and poly(ADP-ribose) polymerase 14 (PARP14) was a predicted key driver gene of extension. Higher PARP14 protein levels were found in inflamed biopsy specimens of patients with limited UC that subsequently extended. CONCLUSION: Molecular predictors of disease extension reveal novel strategies for disease prognostication and potential therapeutic targeting.

Intestinal Inflammation Modulates the Expression of ACE2 and TMPRSS2 and Potentially Overlaps With the Pathogenesis of SARS-CoV-2-related Disease.

BACKGROUND AND AIMS: The presence of gastrointestinal symptoms and high levels of viral RNA in the stool suggest active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within enterocytes. METHODS: Here, in multiple, large cohorts of patients with inflammatory bowel disease (IBD), we have studied the intersections between Coronavirus Disease 2019 (COVID-19), intestinal inflammation, and IBD treatment. RESULTS: A striking expression of ACE2 on the small bowel enterocyte brush border supports intestinal infectivity by SARS-CoV-2. Commonly used IBD medications, both biologic and nonbiologic, do not significantly impact ACE2 and TMPRSS2 receptor expression in the uninflamed intestines. In addition, we have defined molecular responses to COVID-19 infection that are also enriched in IBD, pointing to shared molecular networks between COVID-19 and IBD. CONCLUSIONS: These data generate a novel appreciation of the confluence of COVID-19- and IBD-associated inflammation and provide mechanistic insights supporting further investigation of specific IBD drugs in the treatment of COVID-19. Preprint doi: https://doi.org/10.1101/2020.05.21.109124.

The combination of gene hyperamplification and PD-L1 expression as a biomarker for the clinical benefit of tislelizumab in gastric/gastroesophageal junction adenocarcinoma.

BACKGROUND: In solid tumor Phase 1/2 trials (NCT02407990; NCT04068519), tislelizumab demonstrated clinical benefit, including in advanced gastroesophageal adenocarcinoma (GEA). However, the majority of patients with GEA did not respond, highlighting the need to understand mechanisms of resistance and identify predictive biomarkers for response. METHODS: All tislelizumab-treated patients with GEA from the Phase 1/2 trials were included (N = 105). Programmed death-ligand 1 (PD-L1) expression (Tumor Area Positivity [TAP] ≥ 5%), interferon gamma (IFNγ)-related gene signature, gene expression profile, tumor mutational burden (TMB), and gene hyperamplification (HA) were analyzed for correlation with tislelizumab. RESULTS: A moderate association was observed between PD-L1 TAP ≥ 5%, IFNγ gene signature, TMB-high and efficacy. A potential correlation between hyperamplification (HA +) and worse outcomes with programmed cell death protein 1 (PD-1) inhibition was identified. Hyperamplified genes were mainly enriched in cancer progression pathways, including cell cycle and RTK-RAS-PI3K pathways. Joint PD-L1 TAP ≥ 5% and lack of hyperamplification showed the most favorable benefit with an objective response rate of 29.4%, and median progression-free survival and overall survival of 4.1 and 14.7 months, respectively. Tumors with TAP ≥ 5% and HA - had inflamed immune signatures with increased immune cell infiltration, enhanced anti-tumor cytotoxic activity and antigen presentation signatures. Findings were validated in two independent gastric and gastrointestinal cancer cohorts treated with immune checkpoint inhibitors. CONCLUSIONS: In GEA, PD-L1 positivity, IFNγ-related gene signature and TMB-high status were positively associated with tislelizumab clinical benefit, whereas HA was associated with worse clinical outcomes. Combining PD-L1 positivity and HA - may help identify patients more likely to benefit from PD-1 blockade.

Diversity of ACE2 and its interaction with SARS-CoV-2 receptor binding domain.

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing hundreds of millions of infections and over two million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species' ACE2 and human ACE2. Recombinant ACE2 from human, hamster, horseshoe bat, cat, ferret, and cow were evaluated for RBD binding. A gradient of binding affinities were seen where human and hamster ACE2 were similarly in the low nanomolar range, followed by cat and cow. Surprisingly, horseshoe bat (Rhinolophus sinicus) and ferret (Mustela putorius) ACE2s had poor binding activity compared with the other species' ACE2. The residue differences and binding properties between the species' variants provide a framework for understanding ACE2-RBD binding and virus tropism.

Implementation of Dynamic and Static Moisture Control in Fluidized Bed Granulation.

The application of process analysis and control is essential to enhance process understanding and ensure output material quality. The present study focuses on the stability of the feedback control system for a fluidized bed granulation process. Two strategies of dynamic moisture control (DMC) and static moisture control (SMC) were established based on the in-line moisture value obtained from the near-infrared sensor and control algorithm. The performance of these strategies on quality consistency control was examined using process moisture similarity analysis and principal component analysis. The stable moisture control performance and low batch-to-batch variability indicated that the DMC method was significantly better than other granulation methods. In addition, the investigation of robustness further showed that the implemented DMC method was able to produce predetermined target moisture values by varying process parameters. This study provides an advanced and simple control method for fluidized bed granulation quality assurance.

Dualmarker: a flexible toolset for exploratory analysis of combinatorial dual biomarkers for clinical efficacy.

BACKGROUND: An increasing number of clinical trials require biomarker-driven patient stratification, especially for revolutionary immune checkpoint blockade therapy. Due to the complicated interaction between a tumor and its microenvironment, single biomarkers, such as PDL1 protein level, tumor mutational burden (TMB), single gene mutation and expression, are far from satisfactory for response prediction or patient stratification. Recently, combinatorial biomarkers were reported to be more precise and powerful for predicting therapy response and identifying potential target populations with superior survival. However, there is a lack of dedicated tools for such combinatorial biomarker analysis. RESULTS: Here, we present dualmarker, an R package designed to facilitate the data exploration for dual biomarker combinations. Given two biomarkers, dualmarker comprehensively visualizes their association with drug response and patient survival through 14 types of plots, such as boxplots, scatterplots, ROCs, and Kaplan-Meier plots. Using logistic regression and Cox regression models, dualmarker evaluated the superiority of dual markers over single markers by comparing the data fitness of dual-marker versus single-marker models, which was utilized for de novo searching for new biomarker pairs. We demonstrated this straightforward workflow and comprehensive capability by using public biomarker data from one bladder cancer patient cohort (IMvigor210 study); we confirmed the previously reported biomarker pair TMB/TGF-beta signature and CXCL13 expression/ARID1A mutation for response and survival analyses, respectively. In addition, dualmarker de novo identified new biomarker partners, for example, in overall survival modelling, the model with combination of HMGB1 expression and ARID1A mutation had statistically better goodness-of-fit than the model with either HMGB1 or ARID1A as single marker. CONCLUSIONS: The dualmarker package is an open-source tool for the visualization and identification of combinatorial dual biomarkers. It streamlines the dual marker analysis flow into user-friendly functions and can be used for data exploration and hypothesis generation. Its code is freely available at GitHub at  https://github.com/maxiaopeng/dualmarker under MIT license.

Effect of Concomitant Therapy With Steroids and Tumor Necrosis Factor Antagonists for Induction of Remission in Patients With Crohn's Disease: A Systematic Review and Pooled Meta-analysis.

BACKGROUND & AIMS: It is not clear whether concomitant therapy with corticosteroids and anti-tumor necrosis factor (TNF) agents is more effective at inducing remission in patients with Crohn's disease (CD) than anti-TNF monotherapy. We aimed to determine whether patients with active CD receiving corticosteroids during induction therapy with anti-TNF agents had higher rates of clinical improvement than patients not receiving corticosteroids during induction therapy. METHODS: We systematically searched the MEDLINE, Embase, and CENTRAL databases, through January 20, 2016, for randomized trials of anti-TNF agents approved for treatment of CD and identified 14 trials (5 of adalimumab, 5 of certolizumab, and 4 of infliximab). We conducted a pooled meta-analysis of individual patient and aggregated data from these trials. We compared data from participants who continued oral corticosteroids during induction with anti-TNF therapy to those treated with anti-TNF agents alone. The endpoints were clinical remission (CD activity index [CDAI] scores <150) and clinical response (a decrease in CDAI of 100 points) at the end of induction (weeks 4-14 of treatment). RESULTS: We included 4354 patients who received induction therapy with anti-TNF agents, including 1653 [38.0%] who were receiving corticosteroids. The combination of corticosteroids and an anti-TNF agent induced clinical remission in 32.0% of patients, whereas anti-TNF monotherapy induced clinical remission in 35.5% of patients (odds ratio [OR], 0.93; 95% CI, 0.74-1.17). The combination of corticosteroids and an anti-TNF agent induced a clinical response in 42.7% of patients, whereas anti-TNF monotherapy induced a clinical response in 46.8% (OR 0.84; 95% CI, 0.73-0.96). These findings did not change with adjustment for baseline CDAI scores and concurrent use of immunomodulators. CONCLUSIONS: Based on a meta-analysis of data from randomized trials of anti-TNF therapies in patients with active CD, patients receiving corticosteroids during induction therapy with anti-TNF agents did not have higher rates of clinical improvement compared with patients not receiving corticosteroids during induction therapy. Given these findings and the risks of corticosteroid use, clinicians should consider early weaning of corticosteroids during induction therapy with anti-TNF agents for patients with corticosteroid-refractory CD.

Induction of right open reading frame kinase 3 (RIOK3) during ovulation and luteinisation in rat ovary.

Atypical protein serine kinase RIOK3 is involved in cellular invasion and survival. The spatiotemporal expression pattern and regulatory mechanisms controlling expression of Riok3 were investigated in the rat ovary during the periovulatory period. Immature female rats (22-23 days old) were treated with pregnant mare's serum gonadotropin (PMSG) to stimulate follicular development, followed 48h later by injection with human chorionic gonadotrophin (hCG). Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after hCG administration. Both real-time polymerase chain reaction (PCR) and in situ hybridisation analysis revealed that Riok3 was highly induced in both granulosa cells and theca-interstitial cells by hCG. Riok3 expression was induced in theca-interstitial cells at 4h after hCG. However, the expression of Riok3 mRNA was stimulated in granulosa cells at 8h. Both protein kinase C inhibitor (GF109203) and the protein kinase A inhibitor (H89) could block the stimulation of Riok3 mRNA by hCG. Furthermore, Riok3 induction is dependent on new protein synthesis. Inhibition of prostaglandin synthesis or progesterone action did not alter Riok3 mRNA expression, whereas inhibition of the epidermal growth factor (EGF) pathway downregulated Riok3 expression. In conclusion, our findings suggest that the induction of the RIOK3 may be important for ovulation and luteinisation.

Infants born to mothers with IBD present with altered gut microbiome that transfers abnormalities of the adaptive immune system to germ-free mice.

BACKGROUND AND AIMS: Prenatal and early life bacterial colonisation is thought to play a major role in shaping the immune system. Furthermore, accumulating evidence links early life exposures to the risk of developing IBD later in life. We aimed to assess the effect of maternal IBD on the composition of the microbiome during pregnancy and on the offspring's microbiome. METHODS: We prospectively examined the diversity and taxonomy of the microbiome of pregnant women with and without IBD and their babies at multiple time points. We evaluated the role of maternal IBD diagnosis, the mode of delivery, antibiotic use and feeding behaviour on the microbiome composition during early life. To assess the effects of IBD-associated maternal and infant microbiota on the enteric immune system, we inoculated germ-free mice (GFM) with the respective stool and profiled adaptive and innate immune cell populations in the murine intestines. RESULTS: Pregnant women with IBD and their offspring presented with lower bacterial diversity and altered bacterial composition compared with control women and their babies. Maternal IBD was the main predictor of the microbiota diversity in the infant gut at 7, 14, 30, 60 and 90 days of life. Babies born to mothers with IBD demonstrated enrichment in Gammaproteobacteria and depletion in Bifidobacteria. Finally, GFM inoculated with third trimester IBD mother and 90-day infant stools showed significantly reduced microbial diversity and fewer class-switched memory B cells and regulatory T cells in the colon. CONCLUSION: Aberrant gut microbiota composition persists during pregnancy with IBD and alters the bacterial diversity and abundance in the infant stool. The dysbiotic microbiota triggered abnormal imprinting of the intestinal immune system in GFM.

Activation of CB2R with AM1241 ameliorates neurodegeneration via the Xist/miR-133b-3p/Pitx3 axis.

Activation of cannabinoid receptor type II (CB2R) by AM1241 has been demonstrated to protect dopaminergic neurons in Parkinson's disease (PD) animals. However, the specific mechanisms of the action of the CB2R agonist AM1241 for PD treatment have not been characterized. Wild-type (WT), CB1R knockout (CB1-KO), and CB2R knockout (CB2-KO) mice were exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 1 week to obtain a PD mouse model. The therapeutic effects of AM1241 were evaluated in each group. Behavioral tests, analysis of neurotransmitters, and immunofluorescence results demonstrated that AM1241 ameliorated PD in WT animals and CB1-KO animals. However, AM1241 did not ameliorate PD symptoms in CB2-KO mice. RNA-seq analysis identified the lncRNA Xist as an important regulator of the protective actions of AM1241. Specifically, AM1241 allowed WT and CB1-KO animals treated with MPTP to maintain normal expression of Xist, which affected the expression of miR-133b-3p and Pitx3. In vitro, overexpression of Xist or AM1241 protected neuronal cells from death induced by 6-hydroxydopamine and increased Pitx3 expression. The CB2 receptor agonist AM1241 alleviated PD via regulation of the Xist/miR-133b-3p/Pitx3 axis, and revealed a new approach for PD treatment.

Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes.

Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study.