Biology subjects

Zenobi, R.

Publications and source records attributed to Zenobi, R..

12 recordsLinked to original sources

bioRxiv2026

Comprehensive analysis of ammonia and biogenic amines in ecologically diverse systems with high-resolution mass spectrometry

The acquisition of sufficient nitrogen for growth, as well as the disposal of highly toxic nitrogen-containing degradation products, represent serious metabolic challenges in all multicellular organisms and microbial ecosystems. Understanding these processes requires a broadly applicable system to accurately quantify the concentration of a wide spectrum of biological amines and inorganic nitrogen-containing compounds. To date, this has been challenging due to limited specificity, selectivity, breadth and/or sensitivity of assays. Here we present a simple, optimized method based on liquid chromatography and high-resolution mass spectrometry, with an accompanying analysis pipeline, for the accurate quantification of more than 30 of these compounds, including ammonia. We demonstrate the effectiveness of this method in quantifying relevant nitrogen-containing compounds in mouse and human gut content, bacterial cultures, algal symbiosis in a cnidarian and freshwater ecosystem samples. As a proof-of-concept, we applied the method to study the influence of the gut microbiome on cecal nitrogen distribution in mice with different gnotobiotic microbiomes. This revealed a profound influence of gut microbiota composition on amino acid, ammonia, and amine concentrations in the upper large intestine. Overall, we provide a broadly applicable approach that can be used across microbial ecology fields to generate novel insights into the biology of nitrogen assimilation, exchange, and disposal.

microbiology↗

bioRxiv2025

Real-time volatilomics reveals microbiota and pathogen fingerprints in the honey bee

Understanding the complex relationship between gut microbiota and their hosts often relies on invasive sampling techniques. Honey bees provide a tractable model for host-microbe studies. Here we establish single-bee volatilomics using secondary electrospray ionization (SESI-HRMS) to examine volatile organic compounds released to the air around an individual live honey bee. Specifically, we focused on primary gut microbiota metabolites present in gnotobiotic bees. Our findings reveal distinct volatilome profiles in honey bees that depend on their gut bacterial colonization state. We cross-validated our findings using an established metabolomics technique, LC-HRMS, to compare and contrast the metabolites detectable with each mass spectrometry-based method. Finally, we assessed the ability of SESI-HRMS to detect colonization with the bee pathogen S. marcescens. By comparing the volatile signature of this bacterium grown in liquid culture with that of infected honey bee headspace, we identified overlapping compounds, including butane-2,3-diol, that were absent in uninfected bees. SESI-HRMS volatilomics, paired with LC-HRMS, therefore has potential to identify non-invasive biomarkers of bee microbiome composition and infection at the level of individual insects. These biomarkers represent practical targets for the development of simple, field-ready diagnostic tools for monitoring pollinator health.

microbiology↗

bioRxiv2025

LC-Inspector: a simple open-source viewer for targeted hyphenated mass spectrometry analysis

The ubiquitousness of hyphenated mass spectrometry techniques across life sciences has made researchers around the world aware of their analytical power. However, the analysis of high-complexity mass spectrometry data remains virtually impossible for non-specialists. LC-Inspector is a standalone graphical user interface application for straightforward analysis of targeted mass spectrometry data, distributed under the MIT license and available free of charge for Windows and MacOS. The user can upload and process the data entirely locally on their machine by specifying the desired mass-to-charge ratios of the targeted compound ions and simply clicking "Process". It allows for the processing of multiple files simultaneously, freely modifying and exporting graphs in real time, and calculating compound concentrations based on calibration standards. We show the versatility and applicability of LC-Inspector to different kinds of mass spectrometry data by analyzing a series of publicly available datasets from various samples recorded on instruments from multiple vendors and by different research teams.

bioinformatics↗

bioRxiv2022

Prototyping of a lateral flow assay based on monoclonal antibodies for detection of Bothrops venoms

BackgroundBrazil is home to a multitude of venomous snakes, perhaps the most medically relevant of which belong to the Bothrops genus. Bothrops spp. are responsible for roughly 70% of all snakebites in Brazil, and envenomings caused by their bites can be treated with three types of antivenom: bothropic antivenom, bothro-lachetic antivenom, and bothro-crotalic antivenom. The choice in antivenom that is administered depends not only on its availability and how certain the treating physician is that the patient was bitten by a bothropic snake. The diagnosis of a bothropic envenoming can be made based on expert identification of a photo of the snake or based on a syndromic approach wherein the clinician examines the patient for characteristic manifestations of envenoming. This approach can be very effective but requires staff that has been trained in clinical snakebite management, which, unfortunately, far from all relevant staff has. ResultsIn this paper, we describe a prototype of the first lateral flow assay (LFA) capable of detecting venoms from Brazilian Bothrops spp. The monoclonal antibodies for the assay were generated using hybridoma technology and screened in sandwich enzyme-linked immunosorbent assays (ELISAs) to identify Bothrops spp. specific antibody sandwich pairs. The sandwich pairs were used to develop a prototype LFA that was able to detect venom from several different Bothrops spp. The limit of detection (LoD) of the prototype was evaluated using Brazilian B. atrox whole venom and was determined to be 8.0 ng/mL in spiked serum samples and 9.5 ng/mL in spiked urine samples, when using a portable reader, and < 25 ng/mL in spiked buffer when reading by eye. SignificanceThe work presented here serves as a proof of concept of a genus-specific venom detection kit, which could support physicians in diagnosing Bothrops envenomings. Although further optimization and testing is needed before the LFA can find clinical use, such a device could aid in decentralizing antivenoms in the Brazilian Amazon and help ensure optimal snakebite management for even more victims of this highly neglected disease.

immunology↗

bioRxiv2022

Discovery of a broadly-neutralizing human antibody that can rescue mice challenged with neurotoxin-rich snake venoms

Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and improved neutralization capacity. However, as new antivenom products may need to be polyvalent, i.e., target multiple different snake species, without dramatically increasing dose or cost of manufacture, such monoclonal antibodies need to be broadly-neutralizing. Here, we report the establishment of a pipeline for the discovery of high affinity broadly-neutralizing human monoclonal antibodies. We further demonstrate its utility by discovering an antibody that can prevent lethality induced by N. kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin, and which also prolongs survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.

bioengineering↗

bioRxiv2022

Non-invasive monitoring of microbiota and host metabolism using Secondary electrospray ionization-Mass spectrometry

The metabolic "handshake" between the microbiota and its mammalian host is a complex, dynamic process with potentially major influences on health. Dissecting the interaction between microbial species/strains and metabolites found in host tissues has been a challenge due to the high diversity of a complete micro-biota and the requirement for invasive sampling, which precludes high-resolution longitudinal analysis. Here we demonstrate that secondary electrospray ionization mass spectrometry can be used to non-invasively monitor metabolic activity of the intestinal microbiome of a live, awake mouse. This was achieved via analysis of the headspace volatile and semi-volatile metabolome of individual gut microbiota bacterial species growing in pure culture, as well as from live gnotobiotic mice specifically colonized with these microbes (i.e. metabolites released to the atmosphere via breath, the skin and from the gut). The microbial origin of these compounds was confirmed by feeding of heavy-isotope labeled microbiota-accessible sugars. This reveals that the microbiota is a major contributor to the released metabolites of a whole live mouse, and that it is possible to capture the catabolism of sugars and cross-feeding within the gut microbiota of a living animal using volatile/semi-volatile metabolite monitoring.

systems biology↗

bioRxiv2021

Circadian patterns of energy metabolism are only partially reconstituted in gnotobiotic mice

The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiome to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat-mass than germ-free or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure and respiratory quotient using a novel isolator-housed metabolic cage system which allows long-term measurements without contamination risk. This demonstrated that microbiota- released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and germ-free mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well-integrated into appetite control. We also observed no significant difference in energy expenditure per gram lean mass between the different microbiota groups, suggesting that cumulative very small differences in energy balance, or altered energy storage must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: in germ-free mice the respiratory exchange ratio was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied germ-free mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a germ-free-like drop in liver glycogen storage during the light cycle and both liver and plasma metabolomes of OligoMM12 mice clustered closely with germ-free mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle, and which are absent in the OligoMM12 consortium.

microbiology↗

bioRxiv2020

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.

biophysics↗

bioRxiv2020

Minimizing ion competition boosts volatile metabolome analysis by secondary electrospray ionization orbitrap mass spectrometry

Secondary electrospray ionization high-resolution mass spectrometry (SESI-HR-MS) is an emerging technique for the detection of volatile metabolites. However, sensitivity and reproducibility of SESI-HRMS have limited its applications in untargeted metabolomics profiling. Ion suppression in the SESI source has been considered to be the main cause. Here, we show that besides ion suppression, ion competition in the C-trap of Orbitrap instruments is another important factor that influences sensitivity and reproducibility of SESI-MS. Instead of acquiring the full mass-to-charge ratio (m/z) range, acquisition of consecutive m/z windows to minimize the ion competition effect allows the detection of more features. m/z window ranges are optimized to fill the C-trap either with an equal number of features or an equal cumulative intensity per window. Considering a balance between maximizing scanning speed and minimizing ion competition, splitting the m/z = 50-500 range into 4 windows is selected for measuring human breath and bacterial culture samples on SESI-Orbitrap MS, corresponding to a duty cycle of 2.3 s at a resolution of 140000. In a small cohort of human subjects, the proposed splitting into 4 windows allows three times more features to be detected compared to the classical full m/z range method.

systems biology↗

bioRxiv2020

Screening for New Interaction Partners for a Group IIB Intron Ribozyme with Surface Plasmon Resonance Imaging Coupled to MALDI Mass Spectrometry

We coupled SPR imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) to identify new potential RNA binders. Here, we improve this powerful method, especially by optimizing the proteolytic digestion (type of reducing agent, its concentration, and incubation time), to work with complex mixtures, specifically a lysate of the rough mitochondrial fraction from yeast. The advantages of this hyphenated method compared to column-based or separate analyses are (i) rapid and direct visual readout from the SPRi array, (ii) possibility of high-throughput analysis of different interactions in parallel, (iii) high sensitivity, and (iv) no sample loss or contamination due to elution or micro-recovery procedures. The model system used is a catalytically active RNA (group IIB intron from Saccharomyces cerevisiae, Sc.ai5{gamma}) and its cofactor Mss116. The protein supports the RNA folding process and thereby the subsequent excision of the intronic RNA from the coding part. Using the novel approach of coupling SPR with MALDI MS, we report the identification of potential RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach. Our results show that proteins other than the well-known cofactor Mss116 interact with Sc.ai5{gamma} (Dbp8, Prp8, Mrp13, and Cullin-3), suggesting that the intron folding and splicing are regulated by more than one cofactor in vivo. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/082776v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@50a09borg.highwire.dtl.DTLVardef@1602253org.highwire.dtl.DTLVardef@6d9cc7org.highwire.dtl.DTLVardef@4ff0f0_HPS_FORMAT_FIGEXP M_FIG C_FIG Author contributionUlrike Anders and Maya Gulotti-Georgieva contributed equally to this work.

molecular biology↗

bioRxiv2019

Occurrence and stability of hetero-hexamer associations formed by β-carboxysome CcmK shell components

The carboxysome is a bacterial micro-compartment (BMC) subtype that encapsulates enzymatic activities necessary for carbon fixation. Carboxysome shells are composed of a relatively complex cocktail of proteins, their precise number and identity being species dependent. Shell components can be classified in two structural families, the most abundant class associating as hexamers (BMC-H) that are supposed to be major players for regulating shell permeability. Up to recently, these proteins were proposed to associate as homo-oligomers. Genomic data, however, demonstrated the existence of paralogs coding for multiple shell subunits. Here, we studied cross-association compatibilities among BMC-H CcmK proteins of Synechocystis sp. PCC6803. Co-expression in Escherichia coli proved a consistent formation of hetero-hexamers combining CcmK1 and CcmK2 or, remarkably, CcmK3 and CcmK4 subunits. Unlike CcmK1/K2 hetero-hexamers, the stoichiometry of incorporation of CcmK3 in associations with CcmK4 was low. Cross-interactions implicating other combinations were weak, highlighting a structural segregation of the two groups that could relate to gene organization. Sequence analysis and structural models permitted to localize interactions that would favor formation of CcmK3/K4 hetero-hexamers. Attempts to crystallize these CcmK3/K4 associations conducted to the unambiguous elucidation of a CcmK4 homo-hexamer structure. Yet, subunit exchange could not be demonstrated in vitro. Biophysical measurements showed that hetero-hexamers are thermally less stable than homo-hexamers, and impeded in forming larger assemblies. These novel findings are discussed in frame with reported data to propose a functional scenario in which minor CcmK3/K4 incorporation in shells would introduce sufficient local disorder as to allow shell remodeling necessary to adapt rapidly to environmental changes.

biochemistry↗

bioRxiv2018

Thermal Denaturation of DNA G-Quadruplexes and their Complexes with Ligands: Thermodynamic Analysis of the Multiple States Revealed by Native Mass Spectrometry

As the idea that G-quadruplex nucleic acid structures are involved in cellular processes is gaining support, it becomes important to develop ligands that specifically target G-quadruplexes. However, ligand design is complicated because there are multiple G-quadruplex target sequences, some sequences are polymorphic, and very few ligand-quadruplex structures in solution were solved to date. Further, structure alone does not reveal the driving forces for ligand binding. To know why a ligand binds, the thermodynamics of binding must be characterized. Electrospray mass spectrometry makes it possible to detect and quantify each specific stoichiometry in terms of number of strands, number of specific cations, and number of ligands, and thus allows one to simultaneously determine the equilibrium constants for the formation of each complex. We designed and built a temperature-controlled nano-electrospray source to monitor thermal denaturation by mass spectrometry (\"MS-melting\"). We studied the thermal denaturation of G-quadruplexes, including the c-myc promoter and several telomeric sequence variants, and their complexes with popular ligands (Phen-DC3, TrisQ, TMPyP4, Cu-ttpy). From the temperature dependence of the equilibrium constants, we determined the enthalpic and entropic contributions to the formation of each stoichiometric state. In absence of ligand, we untangled the potassium-induced G-quadruplex folding thermodynamics, one potassium ion at a time. The formation of each quartet-K+-quartet units is strongly enthalpy driven, with entropy penalty. In contrast, the formation of quartet-K+-triplet units is entropically driven. For this reason, such misfolded structures can become more abundant as the temperature increases. In the presence of ligands, mass spectrometry also revealed new states at intermediate temperatures. For example, even in cases where only a 1:1 (ligand:quadruplex) is observed at room temperature, a 2:1 complex predominates at intermediate temperatures. Mass spectrometry also makes it easy to distinguish ligand bound to the 2-quartet structures (containing 1 K+), the 3-quartet structures (containing 2 K+) and to the unfolded strand (no specific K+). We confirm that TrisQ binds preferably, but not exclusively, to 3-quartet structures, Phen-DC3 binds to a 2-quartet structure, while the porphyrin ligand TMPyP4 is characterized as non-selective, because it binds to all forms including the unfolded one. The thermodynamics of ligand binding to each form, one ligand at a time, provides unprecedented detail on the interplay between ligand binding and changes in G-quadruplex topology.\n\nTOC Graphics\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC=\"FIGDIR/small/370254_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@e624adorg.highwire.dtl.DTLVardef@171fa1dorg.highwire.dtl.DTLVardef@1dbc32eorg.highwire.dtl.DTLVardef@eb301f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗