Safe plant Hsp90 adjuvants elicit an effective immune response against SARS-CoV2-derived RBD antigen.

To better understand the role of pHsp90 adjuvant in immune response modulation, we proposed the use of the Receptor Binding Domain (RBD) of the Spike protein of SARS-CoV2, the principal candidate in the design of subunit vaccines. We evaluated the humoral and cellular immune responses against RBD through the strategy "protein mixture" (Adjuvant + Antigen). The rRBD adjuvanted with rAtHsp81.2 group showed a higher increase of the anti-rRBD IgG1, while the rRBD adjuvanted with rNbHsp90.3 group showed a significant increase in anti-rRBD IgG2b/2a. These results were consistent with the cellular immune response analysis. Spleen cell cultures from rRBD + rNbHsp90.3-immunized mice showed significantly increased IFN-γ production. In contrast, spleen cell cultures from rRBD + rAtHsp81.2-immunized mice showed significantly increased IL-4 levels. Finally, vaccines adjuvanted with rNbHsp90.3 induced higher neutralizing antibody responses compared to those adjuvanted with rAtHsp81.2. To know whether both chaperones must form complexes to generate an effective immune response, we performed co-immunoprecipitation (co-IP) assays. The results indicated that the greater neutralizing capacity observed in the rRBD adjuvanted with rNbHsp90.3 group would be given by the rRBD-rNbHsp90.3 interaction rather than by the quality of the immune response triggered by the adjuvants. These results, together with our previous results, provide a comparative benchmark of these two novel and safe vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV2 subunit vaccines. Furthermore, these results revealed differences in the ability to modulate the immune response between these two pHsp90s, highlighting the importance of adjuvant selection for future rational vaccine and adjuvant design.

Nowcast flood predictions in the Amazon watershed based on the remotely sensed rainfall product PDIRnow and artificial neural networks.

The aim of this study was to develop artificial neural network (ANN) models to predict floods in the Branco River, Amazon basin. The input data for the models included the river levels and the average rainfall within the drainage area of the basin, which was estimated from the remotely sensed rainfall product PDIRnow. The hourly water level data used in the study were recorded by fluviometric telemetric stations belonging to the National Agency of Water. The multilayer perceptron was used as the neural framework of the ANNs, and the number of neurons in each layer of the model was determined via optimization with the SCE-UA algorithm. Most of the fitted ANN models showed Nash-Sutcliffe efficiency index values greater than 0.9. It is possible to conclude that the ANNs are effective for predicting the flood levels of the Branco River, with horizons of 6, 12 and 24 h; thus, constituting a viable option for use in river-flood warning systems in the Amazon basin. For the forecast with a 24-h horizon, it is essential to include the average rainfall of the basin that accumulated over the last 48 h as input data into the ANNs, along with the levels measured by the streamflow stations. The indirect rainfall estimates provided by PDIRnow are an excellent alternative as input data for ANN models used to predict floods and constitute a viable solution for regions where the density of rain gauge stations is low, as is the case in the Amazon basin.

Dysfunction, oxidative stress markers, and cytokine expression in the placentae of mice experimentally infected with Neospora caninum.

Neosporosis is the major cause of abortion and reproductive failures in cattle, leading to significant economic losses. In this study, we evaluated the impact of Neospora caninum infection on oxidative stress (OS) markers and local cytokine mRNA expression at the placenta, as well as its effect on the progesterone (P4) serum levels and systemic cytokine profile in a pregnant mouse model. Infected pregnant mice (NC-1 group) showed increased percentages of fetal losses and IFN-γ serum levels, decreased serum progesterone, increased placental mRNA expression levels of both Th1-type (IFN-γ and TNF-α) and Th2-type (IL-4) cytokines, and inhibited expression of TGF-ß1 (Treg) compare to control dams (CONTROL group). In addition, lipid peroxidation and ROS were increased, whereas the antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) activities were modified in the placentae of infected mice compared to control mice. These findings demonstrate that multiple factors, including placental OS, are involved in fetal losses associated with N. caninum infection in mice, thus OS contribution to the placental physiopathology of neosporosis in other hosts must not be ruled out.

Mechanistic and functional aspects of the Ruminococcin C sactipeptide isoforms.

In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1-C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms.

TREML4 polymorphisms increase the mRNA in blood leukocytes in the progression of atherosclerosis.

TREML4 and other members of the triggering receptor expressed in the myeloid cell family are associated with a risk of atherosclerosis and progression in coronary artery disease, acute coronary syndrome, and coronary artery calcification. Herein, the relationship between TREML4 expression and its polymorphisms (rs2803495 and rs280396) was evaluated in patients with subclinical atherosclerosis (n = 340) and heart failure post-acute myocardial infarction (MI) (n = 68) for the first time. TREML4 variants rs2803495 (A > G) and rs2803496 (T > C) and leukocyte mRNA expression was analyzed by qRT-PCR. The rs2803495 G allele was associated with TREML4 expression (OR 8.01, CI 3.78-16.99, p < 0.001). Patients carrying the rs2803496 C minor allele (TC/CC genotypes) were more likely to express TREML4 than those without the C allele (OR 10.42, CI 4.76-22.78, p < 0.001), as well as having higher levels of TREML4 expression (OR 4.88, CI 2.35-10.12, p < 0.001). Thus, we report for the first time that TREML4 is not associated with the early stages of atherosclerotic plaque formation and later stages after MI. In conclusion, TREML4 mRNA expression in blood leukocytes is influenced by minor alleles (G and C) and may regulate differently during the atherosclerosis progression stages, but not in asymptomatic atherosclerosis disease and post-MI.

Effects of noise on the vestibular system of normal-hearing workers.

BACKGROUND: Studies in noise-exposed animals have shown changes in vestibular structures. Likewise, studies in humans have been suggesting that noise can damage the vestibular system, even with normal assessment results. OBJECTIVE: To assess the vestibular system of workers exposed to noise and to compare with individuals not exposed. METHODS: Twenty normal-hearing male adults were divided in the study group (SG), exposed to occupational noise, and control group (CG). We conducted the following procedures: medical history, Dizziness Handicap Inventory (DHI), Dix-Hallpike maneuver, and electronystagmography (eye and caloric tests). RESULTS: The DHI score did not differ between groups. The Dix-Hallpike maneuver was normal for both groups. All individuals had normal responses in the eye tests. 50% of the SG had hyperreflexia in the caloric tests, with a significant difference between the groups. There was a trend towards a statistical significance in the absolute values of angular speed of the slow component in the cold-air test, which were higher in the SG. There was a significant difference between the groups in the relative values of labyrinthine preponderance, which were higher in the SG. CONCLUSION: Our findings showed that 70% of the workers exposed to occupational noise had vestibular alterations identified with electronystagmography, whereas 100% of the individuals in the CG had normal results in the vestibular assessment. Moreover, only 20% of the sample in both groups had vestibular complaints, indicating the presence of subclinical vestibular changes in 50% of the individuals exposed to occupational noise.

TREML4 polymorphisms increase the mRNA in blood leukocytes in the progression of atherosclerosis

ABSTRACT: TREML4 and other members of the triggering receptor expressed in the myeloid cell family are associated with a risk of atherosclerosis and progression in coronary artery disease, acute coronary syndrome, and coronary artery calcification. Herein, the relationship between TREML4 expression and its polymorphisms (rs2803495 and rs280396) was evaluated in patients with subclinical atherosclerosis (n = 340) and heart failure post-acute myocardial infarction (MI) (n = 68) for the first time. TREML4 variants rs2803495 (A > G) and rs2803496 (T > C) and leukocyte mRNA expression was analyzed by qRT-PCR. The rs2803495 G allele was associated with TREML4 expression (OR 8.01, CI 3.78-16.99, p < 0.001). Patients carrying the rs2803496 C minor allele (TC/CC genotypes) were more likely to express TREML4 than those without the C allele (OR 10.42, CI 4.76-22.78, p < 0.001), as well as having higher levels of TREML4 expression (OR 4.88, CI 2.35-10.12, p < 0.001). Thus, we report for the first time that TREML4 is not associated with the early stages of atherosclerotic plaque formation and later stages after MI. In conclusion, TREML4 mRNA expression in blood leukocytes is influenced by minor alleles (G and C) and may regulate differently during the atherosclerosis progression stages, but not in asymptomatic atherosclerosis disease and post-MI.

Neosporosis in sheep: A systematic review and meta-analysis of global seroprevalence and related risk factors.

Neosporosis is recognized as the main cause of abortions in cattle worldwide and there is an increasing concern about its role in ovine reproductive losses; however, epidemiological studies regarding neosporosis in sheep are still limited. This meta-analysis aimed to estimate the global pooled seroprevalence and associated risk factors of ovine neosporosis. In the current report, a comprehensive strategy of search and data collection from 7 worldwide databases was performed. A final set of 73 studies (80 datasets) published from 2000 to 2021 were selected based on inclusion criteria, comprising data on 35,740 sheep (corresponding to 37,565 evaluated samples) from 30 countries worldwide. The global pooled seroprevalence of Neospora caninum infection in sheep estimated by the random-effects model was 13% (95% CI, 10-15) and showed high heterogeneity (Q = 5147.15, I2 = 98%, p< 0.001). Furthermore, by meta-analyses of subgroups it was demonstrated for the first time that seroprevalence significantly varied between continents (highest in Africa; 20%, 95% CI, 4-44), WHO regions (highest in African Region; 42%, 95% CI, 36-48), countries (highest in Colombia; 79%, 95% CI, 61-92%) and diagnostic methods (highest by IFAT; 17%, 95% CI, 12-23). Meta-regression indicated significant increasing trends in the prevalence of ovine neosporosis with decrease in geographical latitude (coefficient = -0.013; p<0.001), whereas longitude did not influence it (coefficient = -0.001; p=0.365). Regarding associated risk factors, older sheep were more likely to be infected with N. caninum than younger ones (OR 1.42; 95% CI 1.08-1.87), and sheep bred under intensive or semi-intensive systems resulted less susceptible to be seropositive than those bred under extensive system (OR 0.65; 95% CI 0.42-0.99 and OR 0.74; 95% CI 0.62-0.89, respectively). Conversely, no apparent association was found between seroprevalence and other variables, such as sex (OR 1.06; 95% CI 0.9-1.24), the presence of dogs on the farm (OR 1.15; 95% CI 0.63-2.12) or the presence of abortion (OR 1.80; 95% CI 0.87-3.74). In conclusion, the seroprevalence of ovine neosporosis is widely and heterogeneously distributed throughout the world, and it is negatively associated with increasing geographical latitude. In addition, age and extensive production system represent risk factors, which suggest that the horizontal transmission route is relevant for this host species. It is recommended to pay more attention to this disease and emphasize the global need for more indexed studies concerning the seroprevalence and risk factors of ovine neosporosis to better understand the epidemiology of this coccidian infection.

Oral Immunization With a Plant HSP90-SAG1 Fusion Protein Produced in Tobacco Elicits Strong Immune Responses and Reduces Cyst Number and Clinical Signs of Toxoplasmosis in Mice.

Plant 90kDa heat shock protein (HSP90) is a potent adjuvant that increases both humoral and cellular immune responses to diverse proteins and peptides. In this study, we explored whether Arabidopsis thaliana HSP90 (AtHsp81.2) can improve the immune effects of a Toxoplasma gondii surface antigen 1 (SAG1). We designed two constructs containing the sequence of mature antigen (SAG1m), from aa77 to aa322, and B- and T-cell antigenic epitope-containing SAG1HC, from aa221 to aa319 fused to AtHsp81.2 sequence. When comparing the transient expression in Nicotiana tabacum X-27-8 leaves, which overexpress the suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that in N. benthamiana leaves, co-agroinfiltrated with the suppressor p19, optimal conditions included 6-week-old N. benthamiana plants, 7-day time to harvest, Agrobacterium tumefaciens cultures with an OD600nm of 0.6 for binary vectors and LED lights. While AtHsp81.2-SAG1m fusion protein was undetectable by Western blot in any of the evaluated conditions, AtHsp81.2-SAG1HC was expressed as intact fusion protein, yielding up to 90µg/g of fresh weight. Besides, the AtHsp81.2-SAG1HC mRNA was strongly expressed compared to the endogenous Nicotiana tabacum elongation factor-alpha (NtEFα) gene, whereas the AtHsp81.2-SAG1m mRNA was almost undetectable. Finally, mice were orally immunized with AtHsp81.2-SAG1HC-infiltrated fresh leaves (plAtHsp81.2-SAG1HC group), recombinant AtHsp81.2-SAG1HC purified from infiltrated leaves (rAtHsp81.2-SAG1HC group), non-infiltrated fresh leaves (control group), or phosphate-buffered saline (PBS group). Serum samples from plAtHsp81.2-SAG1HC-immunized mice had significantly higher levels of IgGt, IgG2a, and IgG2b anti-SAG1HC antibodies than serum from rAtHsp81.2-SAG1HC, control, and PBS groups. The number of cysts per brain in the plAtHsp81.2-SAG1HC-immunized mice was significantly reduced, and the parasite load in brain tissue was also lower in this group compared with the remaining groups. In an immunoblot assay, plant-expressed AtHsp81.2-SAG1HC was shown to react with antibodies present in sera from T. gondii-infected people. Therefore, the plant expression of a T. gondii antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against T. gondii can improve the vaccine efficacy, and plant extract can be directly used for vaccination without the need to purify the protein, making this platform a suitable and powerful biotechnological system for immunogenic antigen expression against toxoplasmosis.

The Multifunctional Sactipeptide Ruminococcin C1 Displays Potent Antibacterial Activity In Vivo as Well as Other Beneficial Properties for Human Health.

The world is on the verge of a major antibiotic crisis as the emergence of resistant bacteria is increasing, and very few novel molecules have been discovered since the 1960s. In this context, scientists have been exploring alternatives to conventional antibiotics, such as ribosomally synthesized and post-translationally modified peptides (RiPPs). Interestingly, the highly potent in vitro antibacterial activity and safety of ruminococcin C1, a recently discovered RiPP belonging to the sactipeptide subclass, has been demonstrated. The present results show that ruminococcin C1 is efficient at curing infection and at protecting challenged mice from Clostridium perfringens with a lower dose than the conventional antibiotic vancomycin. Moreover, antimicrobial peptide (AMP) is also effective against this pathogen in the complex microbial community of the gut environment, with a selective impact on a few bacterial genera, while maintaining a global homeostasis of the microbiome. In addition, ruminococcin C1 exhibits other biological activities that could be beneficial for human health, as well as other fields of applications. Overall, this study, by using an in vivo infection approach, confirms the antimicrobial clinical potential and highlights the multiple functional properties of ruminococcin C1, thus extending its therapeutic interest.

Structural, biochemical and functional analyses of tRNA-monooxygenase enzyme MiaE from Pseudomonas putida provide insights into tRNA/MiaE interaction.

MiaE (2-methylthio-N6-isopentenyl-adenosine37-tRNA monooxygenase) is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide 2-methylthio-N6-isopentenyl-adenosine (ms2i6A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N6-4-hydroxyisopentenyl-adenosine (ms2io6A37). Here, we report the in vivo activity, biochemical, spectroscopic characterization and X-ray crystal structure of MiaE from Pseudomonas putida. The investigation demonstrates that the putative pp-2188 gene encodes a MiaE enzyme. The structure shows that Pp-MiaE consists of a catalytic diiron(III) domain with a four alpha-helix bundle fold. A docking model of Pp-MiaE in complex with tRNA, combined with site directed mutagenesis and in vivo activity shed light on the importance of an additional linker region for substrate tRNA recognition. Finally, krypton-pressurized Pp-MiaE experiments, revealed the presence of defined O2 site along a conserved hydrophobic tunnel leading to the diiron active center.

Use of Veterinary Vaccines for Livestock as a Strategy to Control Foodborne Parasitic Diseases.

Foodborne diseases (FBDs) are a major concern worldwide since they are associated with high mortality and morbidity in the human population. Among the causative agents of FBDs, Taenia solium, Echinococcus granulosus, Toxoplasma gondii, Cryptosporidium spp., and Trichinella spiralis are listed in the top global risk ranking of foodborne parasites. One common feature between them is that they affect domestic livestock, encompassing an enormous risk to global food production and human health from farm to fork, infecting animals, and people either directly or indirectly. Several approaches have been employed to control FBDs caused by parasites, including veterinary vaccines for livestock. Veterinary vaccines against foodborne parasites not only improve the animal health by controlling animal infections but also contribute to increase public health by controlling an important source of FBDs. In the present review, we discuss the advances in the development of veterinary vaccines for domestic livestock as a strategy to control foodborne parasitic diseases.

The unusual structure of Ruminococcin C1 antimicrobial peptide confers clinical properties.

The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here we report its unique compact structure on the basis of four intramolecular thioether bridges with reversed stereochemistry introduced posttranslationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.

Heat Shock Proteins 90 kDa: Immunomodulators and Adjuvants in Vaccine Design Against Infectious Diseases.

Heat shock proteins 90 kDa (Hsp90s) were originally identified as stress-responsive proteins and described to participate in several homeostatic processes. Additionally, extracellular Hsp90s have the ability to bind to surface receptors and activate cellular functions related to immune response (cytokine secretion, cell maturation, and antigen presentation), making them very attractive to be studied as immunomodulators. In this context, Hsp90s are proposed as new adjuvants in the design of novel vaccine formulations that require the induction of a cell-mediated immune response to prevent infectious diseases. In this review, we summarized the adjuvant properties of Hsp90s when they are either alone, complexed, or fused to a peptide to add light to the knowledge of Hsp90s as carriers and adjuvants in the design of vaccines against infectious diseases. Besides, we also discuss the mechanisms by which Hsp90s activate and modulate professional antigen-presenting cells.

Ruminococcin C, a promising antibiotic produced by a human gut symbiont.

A major public health challenge today is the resurgence of microbial infections caused by multidrug-resistant strains. Consequently, novel antimicrobial molecules are actively sought for development. In this context, the human gut microbiome is an under-explored potential trove of valuable natural molecules, such as the ribosomally-synthesized and post-translationally modified peptides (RiPPs). The biological activity of the sactipeptide subclass of RiPPs remains under-characterized. Here, we characterize an antimicrobial sactipeptide, Ruminococcin C1, purified from the caecal contents of rats mono-associated with Ruminococcus gnavus E1, a human symbiont. Its heterologous expression and post-translational maturation involving a specific sactisynthase establish a thioether network, which creates a double-hairpin folding. This original structure confers activity against pathogenic Clostridia and multidrug-resistant strains but no toxicity towards eukaryotic cells. Therefore, the Ruminococcin C1 should be considered as a valuable candidate for drug development and its producer strain R. gnavus E1 as a relevant probiotic for gut health enhancement.

TREML4 mRNA Expression and Polymorphisms in Blood Leukocytes are Associated with Atherosclerotic Lesion Extension in Coronary Artery Disease.

Members of the triggering receptor expressed on myeloid cells (TREM) family are associated with atherosclerosis risk and progression. TREML4 is upregulated in the early phase of acute coronary syndrome. We investigated the relationship between the mRNA expression of 13 genes in blood leukocytes, TREML4 polymorphisms, and coronary artery lesion extension (Friesinger index) in patients with coronary artery disease (CAD) (n = 137). TREML4 rs2803495 (A > G) and rs2803496 (T > C) variants and leukocyte mRNA expression were analysed by qRT-PCR. TREML4 expression was higher in patients with major coronary artery lesions than in subjects without or with low and intermediate lesions (p < 0.05). However, TREML4 polymorphisms were not associated with coronary lesion extent. Presence of the rs2803495 G allele was not associated with increased TREML4 mRNA expression. Patients carrying the rs2803496 C allele (TC/CC genotypes) were more likely to express TREML4 mRNA than non-C allele carriers (allele C: OR 7.3, and 95% CI 1.9-27.5, p = 0.03). In conclusion, increased TREML4 mRNA expression in blood leukocytes is influenced by gene polymorphisms and is associated with more severe coronary artery lesions, suggesting its potential as a biomarker of the extent of coronary lesions in patients with CAD.

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming.

The serine protease inhibitors (SPIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of SPIs as protease enzymes is to regulate the proteolytic activity. In plants, most of the studies of SPIs have been focused on their physiological role. The initial studies carried out in plants showed that SPIs participate in the regulation of endogenous proteolytic processes, as the regulation of proteases in seeds. Besides, it was observed that SPIs also participate in the regulation of cell death during plant development and senescence. On the other hand, plant SPIs have an important role in plant defense against pests and phytopathogenic microorganisms. In the last 20 years, several transgenic plants over-expressing SPIs have been produced and tested in order to achieve the increase of the resistance against pathogenic insects. Finally, in molecular farming, SPIs have been employed to minimize the proteolysis of recombinant proteins expressed in plants. The present review discusses the potential biotechnological applications of plant SPIs in the agriculture field.

Relationship between circulating VCAM-1, ICAM-1, E-selectin and MMP9 and the extent of coronary lesions.

OBJECTIVES: Inflammatory molecules play a role in the development of atherosclerosis, which is the primary origin of cardiovascular disorders. However, to the best of our knowledge, no study has attempted to investigate the relationship between these circulating molecules and the prediction of cardiovascular risk. The present study aimed to investigate the relationships of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 serum concentrations with the extent of coronary lesions. METHODS: Seventy-four individuals who were undergoing coronary angiography for the first time for diagnostic purposes were enrolled in this study. The extent of the coronary lesion was assessed using the Friesinger Index, and subjects were classified into four groups: no lesions, minor lesions, intermediate lesions and major lesions. Serum biochemical parameters and serum concentrations of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 were analyzed. RESULTS: The vascular cell adhesion molecule-1 concentration was higher than 876 ng/mL in individuals with intermediate and major lesions (p<0.001 and p=0.020, respectively). Moreover, logistic regression analysis showed that these patients had an increased risk of having an intermediate lesion (p=0.007). Interestingly, all individuals with major lesions had vascular cell adhesion molecule-1 concentrations higher than 876 ng/mL. No association was found between the concentrations of the other proteins and the Friesinger Index. CONCLUSIONS: Serum vascular cell adhesion molecule-1 may be associated with the extent of coronary lesions. Moreover, it may represent an alternative to improve the cardiovascular risk classification in patients without acute coronary syndrome.

A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure.

Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC-MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high-resolution mass spectrometry. The structure of msms2 i6 A (2-methylthiomethylenethio-N6-isopentenyl-adenosine) was confirmed by methione-D3 feeding experiments and by synthesis of the nucleobase. The msms2 i6 A contains a thioacetal, shown in vitro to be biosynthetically derived from ms2 i6 A by the radical-SAM enzyme MiaB. This enzyme performs thiomethylation, forming ms2 i6 A from i6 A in a first turnover. The new thioacetal is formed by a second turnover. Along with the pool of 36 new modifications, this work describes a new layer of RNA modification chemistry.