Methods for measuring gut microbiota biochemical activities in vivo are needed to characterize its functional states in health and disease. To illustrate one approach, an arabinan-containing polysaccharide was isolated from pea fiber, its structure defined, and forward genetic and proteomic analyses used to compare its effects, versus unfractionated pea fiber and sugar beet arabinan, on a human gut bacterial strain consortium in gnotobiotic mice. We produced ‘Microbiota Functional Activity Biosensors’ (MFABs) consisting of glycans covalently linked to the surface of fluorescent paramagnetic microscopic glass beads. Three MFABs, each containing a unique glycan/fluorophore combination, were simultaneously orally gavaged into gnotobiotic mice, recovered from their intestines, and analyzed to directly quantify bacterial metabolism of structurally distinct arabinans in different human diet contexts. Colocalizing pea-fiber arabinan and another polysaccharide (glucomannan) on the bead surface enhanced in vivo degradation of glucomannan. MFABs represent a potentially versatile platform for developing new prebiotics and more nutritious foods. Tens of trillions of microbes living in the gut help humans and other animals digest their food. In the process, the microbes provide necessary nutrients for themselves and the animal. Learning more about the interaction of food components and gut bacteria could help scientists to better understand how different diets affect human health. Currently, studying these complex interactions is challenging, but new technologies that measure microbial nutrient processing in the gut could help.Now, Wesener et al. show that swallowable microscopic biosensors can measure how gut bacteria break down nutrients from food. To make the biosensors, Wesener et al. attached complex carbohydrates extracted from peas and fluorescent tags to microscopic beads. In the experiments, mice colonized with human gut microbes were fed the beads along with a traditional low fiber, Western diet. Some of the animals also received fiber supplements.The microscopic beads were then recovered from the intestines after digestion and the remaining carbohydrates on the beads were measured. The genetic makeup of the gut microbiome and the expression of microbial genes was also examined. The experiments revealed which pea carbohydrates the gut microbes consumed and showed that pairing certain carbohydrates together on the microbead surface increased their digestion in mice that received fiber supplements.If future studies prove that the microbead biosensors created by Wesener et al. are safe for humans to ingest, they could be used to help diagnose how well a person’s gut microbiota can process different foods. Studies using the microbead sensors may also help scientists develop more nutritious foods or supplements that promote the growth of microbes important for health. Increasing effort is being directed to determining how components of diets consumed by various human populations impact the composition and expressed functional features of their gut microbial communities e.g., . A hopedPlant fibers epitomize these challenges. Plant fibers are complex mixtures of biomolecules whose composition varies depending upon their source, method of initial recovery, and the food processing techniques used to incorporate them into food products that have satisfactory organoleptic properties  . Plant fPisum sativum, produced a significant increase in the abundance of Bacteroides thetaiotaomicron . The mole percent representation of arabinose in the end product was 71%    . Glycosyabinose)  attachedabinose) . Xylose O-branched as revealed by glycosyl-linkage analysis . B. vulgatus ATCC 8482 also reached exponential growth more quickly than when d-glucose was present in this medium  is primarily 3-analysis . PFABN aanalysis . In vitranalysis  with Bacs medium .B. thetaiotaomicron, three additional Bacteroides species  plus six other types of bacteria  were each represented by previously described libraries of tens of thousands of transposon (Tn) insertion mutants  abundance of 0.1% under any of the diet conditions tested.Two days post-gavage (dpg 2) of the defined bacterial consortium, mice were switched from the unsupplemented HiSF-LoFV diet to a HiSF-LoFV diet supplemented with either unfractionated pea fiber, isolated PFABN, or isolated SBABN. Animals were fed these diets ad libitum for 10 days and then euthanized. A control group was maintained on unsupplemented HiSF-LoFV chow . Of the B. thetaiotaomicron VPI-5482 responded to all three supplements; (2) B. ovatus ATCC 8483 increased after exposure to both intact pea fiber and PFABN but not to SBABN; (3) B. vulgatus ATCC 8482 exhibited significant increases with SBABN; and (4) B. cellulosilyticus WH2 and Ruminococcaceae sp. TSDC 17.2 increased significantly when exposed to intact pea fiber but not to either of the isolated arabinan preparations  arations   involved in polysaccharide depolymerization, as well as transcriptional regulators that allow the locus to be induced in the presence of glycans it can recognize/utilize ; a shared feature of PULs is an adjacent pair of /utilize . Regulat/utilize .B. thetaiotaomicron VPI-5482 (BT), B. ovatus ATCC 8483 (Bovatus), B. cellulosilyticus WH2 (BcellWH2), and B. vulgatus ATCC 8482 (BVU), respectively .Supplemental results, B. thetaiotaomicron VPI-5482, 135 genes in B. ovatus ATCC 8483, 346 genes in B. cellulosilyticus WH2, and 82 genes in B. vulgatus ATCC 8482 were significantly decreased during diet supplementation with either pea fiber, PFABN, or SBABN  reactive amines per bead versus 1.20 × 109 ± 1.92 × 108 reactive amines after surface acetylation (8 ± 1.13 × 108 molecules of biotin per bead). Second, we attached unique fluorogenic tags directly to the bead surface so that multiple bead types with different immobilized polysaccharides could be analyzed simultaneously within a given gnotobiotic animal. To do so, surface-modified beads were reacted with an N-hydroxysuccinimide (NHS) ester-activated fluorophore  to generate an electrophilic cyanate-ester intermediate  (nM ~1300 Da) was activated with CDAP (0.2 mg/mg oligosaccharide) prior to attachment to the bead surface. Conjugation resulted in 4.43 ± 1.03 ng of immobilized glucose per 1000 beads or 2.05 × 109 molecules of maltodextrin per bead  . Immobilonation) . Conjugaonation) . A low aonation) , likely per bead ; this rel-arabinofuranosidase enzymes removed 88 ± 1.2% (mean ± s.d.) of the immobilized PFABN after 30 min while incubation with each enzyme individually removed greater than 80% of the glycan after 30 min and more than 90% after 20 hr. In contrast, an endo-inulinase with specificity toward β(1-2)-linked fructose resides failed to degrade PFABN , we first performed several in vitro experiments. We initially incubated PFABN-coated beads with glycosyl hydrolyses and the fraction of arabinan remaining on the bead surface was measured by gas chromatography–mass spectrometry (GC–MS). Incubation with a combination of endo-arabinanase and α-de PFABN . These rB. thetaiotaomicron VPI-5482 or B. cellulosilyticus WH2 grown to mid-log phase in defined minimal medium  was more efficiently processed by the microbiota in all three supplemented diet contexts . In contAs noted in Introduction, plant fibers have complex physical–chemical properties manifest in part by their mixtures of different glycan structures and by their varying shapes and surface properties. Plant fiber particles are impacted by methods, such as extrusion, that are commonly used to incorporate plant fibers into food products so that these products have acceptable organoleptic properties , and by d-mannose and d-glucose. We found that among the pea fiber-responsive Bacteroides identified above, only B. ovatus ATCC 8483 and B. cellulosilyticus WH2 were able to grow in minimal medium containing glucomannan as the sole carbon source ; each of these PULs encodes at least one GH26 enzyme with β-mannanase activity .To explore this notion, we turned to glucomannan, a hemicellulosic linear β(1-4) polysaccharide composed of n source . Both oractivity . MultiplFV diets . Only twus_PUL80 . Neitherentation   isolated from two distinct plant sources. Robust isolation procedures and analytical characterization are required to isolate grams of polysaccharide and test their biological effects in gnotobiotic mice. The approach we employed allowed us to isolate 50 g of material to ~85% purity with the remaining material comprised of starch and xylan. Starch can be degraded by host enzymes. The xylan present in our PFABN preparation could, in principle, influence the microbiota response. However, there is arabinoxylan already present in the base HiSF-LoFV diet, and we found that expression of known xylan-responsive PULs in entation . The speentation , emphasientation . Althoug7 beads contained 100 µg of bead-immobilized polysaccharide, 1000 times more polysaccharide was consumed daily in the diet by mice in the polysaccharide-supplemented arms of our experiments.The bead-based MFABs described in this report represent a technology for measuring biochemical activities expressed by a microbial community of interest in vivo or ex vivo. Future studies where MFABs are used in conjunction with genetic tools that enable rapid deletion of genes or entire multi-kilobase loci could provide new insights about the mechanisms by which community members acquire/degrade nutrients (polysaccharides). We emphasize that in this study, we use MFABs to quantify community degradative activity and not to analyze community consumption of MFAB-bound polysaccharides and its relationship to bacterial growth. Determining the latter is challenging in our animal model; a typical gavage of 10Installing specific functional groups on the surfaces of microscopic paramagnetic glass beads using commercially available organosilane reagents allows ‘modular’ incorporation of different biomolecules. This approach represents an alternative to a procedure we described recently, where bifunctional biotinylated ligands are generated prior to immobilization on glass beads coated with streptavidin . Each ofB. cellulosilyticus WH2 and Ruminococcaceae sp. TSDC 17.2 increase significantly when animals were consuming the crude pea fiber-supplemented diet, but not during supplementation with either of the isolated arabinans. The chemistry for covalent polysaccharide attachment to MFABs not only allows for dense ligand presentation, but also enables multiple ligands to be simultaneously immobilized to create ‘hybrid’ beads to model the effects of physical colocalization of different fiber components on microbial degradation. In principle, a wide range of different glycan combinations with varying stoichiometries can be explored owing to the fact that different hybrid bead types, each with its own fluorophore, can be created and tested simultaneously in vitro and in vivo to investigate the mechanism, generality, and biological significance of colocalization of glycans on their degradation by gut microbes.A unifying question for food science, microbiome science, and nutrition research is how to decipher the effects of a nutrient and the matrix in which it resides within a food on modulation of microbiota functions . Crude pThe selection of gut bacterial taxa for in vivo tests of fiber effects in gnotobiotic animal models is critical for yielding ecologically and physiologically relevant results. Characterizing defined communities comprised of organisms beyond those represented in the 14-member consortium will undoubtedly be informative. For example, members of Lactobacillus, Bifidobacterium, and Faecalibacterium are more tolerant of acidic conditions after carbohydrate fermentation and can outcompete acid intolerant bacteria such as Bacteroides; their inclusion would likely change the response of community members to the presence of different plant fiber preparations and the dynamics of plant fiber degradation documented by various collections of MFABs.The approach we describe for ligand immobilization does not require the synthesis of bifunctional ligands (or fluorophores); instead, custom functional groups can be incorporated into the probe through modification of the organosilane donor molecule. As such, the MFAB platform provides an opportunity to develop chemistries for nondestructively releasing ligands for analysis . For exag, 5 min), and the supernatant was removed. Neat ethanol was added to the pelleted fiber, and the solution was mixed for 2 min. Fiber was centrifuged , and the supernatant was removed. Neat acetone was added to the pelleted fiber, the solution was mixed for 2 min, centrifuged , and the supernatant was removed. The resulting ‘defatted’ pea fiber was dried in a chemical hood overnight. Defatted pea fiber was subsequently resuspended in 200 mL of 50 mM ammonium oxalate (pH = 5.7) and stirred at 23°C for 20 hr. The suspension was centrifuged , the supernatant was collected, concentrated , and then dialyzed extensively against water . The precipitate from the dialysis was recovered by centrifugation . The precipitate and soluble material from the dialysis, representing fractions one and two, respectively, were dried with lyophilization.Raw pea fiber (Rattenmaier) was fractionated using serial extractions with aqueous buffers of increasing harshness . Pea fibg, 15 min), and the supernatant was discarded. The pellet was resuspended in 200 mL of 50 mM sodium carbonate (pH 10) containing 0.5% (wt:wt) sodium borohydride and stirred at 23°C for 20 hr. The suspension was centrifuged , and the supernatant was collected. Borohydride was quenched by slowly adding glacial acetic acid. A stringy precipitate began to form as the pH decreased. The suspension was concentrated (as above); the insoluble and soluble portions of the resulting concentrated sodium carbonate suspension were separated with centrifugation , yielding fractions three and four, respectively. Fractions were dialyzed and dried with lyophilization.The pellet from the ammonium oxalate extraction was washed with 200 mL of water, centrifuged , and the supernatant was removed. Five drops of 1-octanol were added to prevent foaming during borohydride quenching. A light precipitate began to form in the solution as the pH decreased. The suspension was concentrated; the insoluble and soluble portions of the concentrated 1 M potassium hydroxide extract were separated by centrifugation , yielding fractions five and six, respectively. The fractions were dialyzed and dried with lyophilization.The pellet from the sodium carbonate extraction was washed with water before resuspension in 200 mL of 1 M potassium hydroxide containing 1% wt:wt sodium borohydride and stirring for 20 hr at 23°C. The suspension was centrifuged , and the supernatant was removed. 1-Octanol was added to prevent foaming during borohydride quenching; during this process, a precipitate formed, dissolved, and then reformed as the pH was lowered to 6.0. The resulting suspension was concentrated; the insoluble and soluble portions of the concentrated 4 M potassium hydroxide extract were separated by centrifugation , yielding fractions seven and eight, respectively. These fractions were dialyzed and dried with lyophilization. Note that after each extraction, sodium azide was added to a final concentration of 0.05% prior to concentration and dialysis.The pellet from the 1 M potassium hydroxide extraction was washed with water before resuspension in 200 mL of 4 M potassium hydroxide containing 1% wt:wt sodium borohydride. The mixture was stirred at 23°C for 20 hr. The suspension was then centrifuged . The soluble material was assayed for protein content (bicinchoninic acid assay) using bovine serum albumin as a standard, DNA content  and total carbohydrate content  was used as an internal standard. Twofold dilutions of free monosaccharide standards  were simultaneously derivatized and used to quantify the absolute abundance of each monosaccharide in each fraction. GC–MS peaks were quantified using metaMS (Each of the eight fractions was resuspended in water (1 mg/mL) by heating to 90°C and sonication (Branson Sonifer). Insoluble material was removed by centrifugation , and the supernatant was discarded. The pellet from the 1 M potassium hydroxide extraction was resuspended in 4 M potassium hydroxide containing 0.5% (wt:wt) sodium borohydride (50 mg/mL) and stirred at room temperature for 24 hr. The suspension was centrifuged, and the supernatant was collected and neutralized with 4 M acetic acid. Neat ethanol was added (7.5:1 [vol:vol]), and polysaccharide was precipitated at −20°C. Precipitated polysaccharide was isolated by centrifugation  and rinsed with 250 mL of 80% ethanol (4°C) three times. The pellet was dried overnight under a dry nitrogen stream. The entire procedure was repeated five times to isolate 51 g of PFABN . Isolated PFABN was pulverized , and total carbohydrate content was defined (phenol-sulfuric acid assay).The isolation procedure described above was slightly modified to recover gram quantities of PFABN. Raw pea fiber was resuspended at 50 mg/mL in 1 M potassium hydroxide containing 0.5% (wt:wt) sodium borohydride and stirred at room temperature for 24 hr. The suspension was centrifuged . PFABN was hydrolyzed for 2 hr at 95°C. Samples were then centrifuged , the supernatant was transferred to a new glass vial, and the material was dried under reduced pressure. Samples were subsequently oximated by adding 20 μL of methoxyamine (15 mg/mL in pyridine) and incubating the solution overnight at 37°C. Twenty microliters of MSTFA -acetamide, chlorotrimethylsilane]) (Thermo Scientific) was added, and the solution was incubated at 70°C for 1 hr. The material was subsequently diluted with 20 μL heptane before analysis using an Agilent 7890A gas chromatography system coupled with an Agilent 5975C mass spectrometer detector. Employing l-arabinose, d-galactose, d-glucose, d-mannose, d-rhamnose, d-xylose standards, peaks were identified and quantified using metaMS . One hundred and seventy-five microliters of 2 M trifluoroacetic acid containing 15 ng of D6-g metaMS ; peak arPFABN was enzymatically destarched using amyloglucosidase and α-amylase (Megazyme). To do so, PFABN was first resuspended by heating at 95°C in a solution containing 50 mM sodium malate (pH = 6) and 2 mM calcium chloride (5 mg/mL). Based on the manufacturer’s measurement of the specific activities of these two enzymes, we added an amount sufficient to degrade all starch within the PFABN fraction within one minute; nonetheless, we allowed degradation to proceed for 4 hr at 37°C before terminating the reaction by incubation at 95°C for 20 min. Polysaccharide was dialyzed extensively against water and dried by lyophilization. Complete digestion of starch was confirmed with GC–MS analysis of neutral monosaccharides.Glycosyl-linkage analysis was performed on the destarched PFABN at the Complex Carbohydrate Research Center (University of Georgia) using previously described methods . Briefly2, and 77% N2). Single colonies were picked and grown overnight on a defined Bacteroides minimal medium (BMM) (d-glucose. Bacteria were then diluted 1:500 (vol:vol) into BMM supplemented with a carbon source at a final concentration of 0.5% (wt:wt) and distributed into the wells of a 96-well half-area plate . Plates were sealed with an optically clear membrane  and growth at 37°C was monitored by measuring optical density at 600 nm every 15 min (Biotek Eon instrument with a BioStack 4). Carbon sources tested include d-glucose, PFABN, SBABN, and glucomannan (Megazyme). All conditions were tested in quadruplicate. Readings obtained from control wells inoculated with bacteria but lacking a carbon source were averaged and subtracted from data obtained from carbon-supplemented cultures to generate background subtracted OD600 growth curves.Bacterial stocks, previously stored at −80°C, were struck onto Brain-heart infusion  agar plates supplemented with 10% (vol:vol) horse blood. Plates were incubated in an anaerobic growth chamber   containiAll experiments involving mice were carried out in accordance with protocols approved by the Animal Studies Committee of Washington University in Saint Louis.6 CFUs per strain; equal volumes of each INSeq library) and introduced into mice using a plastic tipped oral gavage needle. Animals receiving communities with Tn mutant libraries were individually housed in cages containing cardboard shelters .Germ-free male C57BL/6J mice were maintained within flexible plastic isolators under a strict 12 hr light cycle (lights on a 0600) and fed an autoclavable mouse chow (Envigo). Animals were colonized with a 14-member microbial community of cultured, sequenced bacterial strains composed of a mixture of type strains or their Tn mutant library equivalent  and straFive days prior to colonization, mice were switched to a HiSF-LoFV diet. This diet was produced using human foods as described in a previous publication , freeze-6 reads per sample. Reads were demultiplexed and mapped to community member bacterial genomes, 2 ‘spiked-in’ bacterial genomes for absolute abundance calculation (see below), and two ‘distractor’ genomes , 210 μL 20% (wt:wt) sodium dodecyl sulfate, and 500 μL of phenol:chloroform:amyl alcohol  for 4 min. Four hundred and twenty microliters of the aqueous phase was removed; DNA was purified  according to the manufacture’s protocol and eluted into 10 mM Tris–HCl (pH 8.5). Sequencing libraries were prepared from purified DNA by tagmentation with the Nextera DNA Library Prep Kit (Illumina) and custom barcoded primers . Librari8 cells/mL suspension of Alicyclobacillus acidiphilus DSM 14558 [GenBank assembly accession: GCA_001544355.1] and 30 μL of a 9.93 × 108 cells/mL suspension of Agrobacterium radiobacter DSM 30147 [GenBank assembly accession: GCA_000421945.1]; i, in sample j, the absolute abundance in genome equivalents per gram of feces was calculated using the normalized relative abundance and the A. acidiphilus spike-in (A. a):To calculate bacterial absolute abundance, an aliquot containing a known number of two bacteria strains not encountered in mammalian gut communities or in the diet was added to each fecal sample prior to DNA extraction  . Loess-normalized protein abundance data were then used to build linear models (limma package) to identify diet-supplement-responsive proteins (relative to levels in control mice receiving the unsupplemented HiSF-LoFV diet) at dpg 6. p-values from the linear models were corrected for multiple hypotheses.Only data from peptides that uniquely map to a single protein were considered for analysis. Summed peptide abundance data for each protein was log6/mL, and the organosilane reagents were included at 1000-fold excess of what would be required to coat the bead surface  were incubated at 23°C overnight in a solution of 20 mM HEPES (pH 7.4) and 100 mM NaCl. Equal molar amounts of (3-aminopropyl)triethoxysilane  and 3-(trihydroxysilyl)propylmethylphosphonate  were subsequently added to a suspension of hydrolyzed NHS ester-activated beads in deionized water . Beads w6 beads /mL). Pyridine  was then added as a base followed by acetic anhydride . The reaction was allowed to proceed for 3 hr at 22°C and then terminated by repeated washing in water. Beads were stored in 20 mM HEPES (pH 7.2) and 100 mM NaCl at 4°C.Beads were washed repeatedly with multiple solvents with the goal of resuspending the beads in anhydrous methanol; to do so, beads were washed in water, then methanol, and then anhydrous methanol  ester-activated fluorophores: (1) Alexa Fluor 488 NHS ester (Fisher Scientific), (2) Promofluor 415 NHS ester (PromoKine); (3) Promofluor 633P NHS ester (PromoKine), and (4) Promofluor 510-LSS NHS ester (PromoKine). NHS-activated fluorophores were dissolved in dimethyl sulfoxide (DMSO) at 1 mM. The stock solution of each fluorophore was diluted in DMSO to 10 μM. The fluorophore was conjugated to amine plus phosphonate beads in 20 mM HEPES (pH 7.2) and 100 mM NaCl . The reaction was allowed to proceed for 50 min at 22°C and then terminated by repeated washing with water. Beads were stored in 20 mM HEPES (pH 7.2) and 100 mM NaCl at 4°C.Beads were labeled with the following Polysaccharides were resuspended at a concentration of 5 mg/mL in 50 mM HEPES (pH 7.8) using heat and sonication with the exception of maltodextrin , which was resuspended at 50 mg/mL. Triethylamine  and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate  were added to the polysaccharide solution. The optimal concentration of CDAP for polysaccharide activation, without overactivation and aggregation, was found to be 0.2 mg CDAP/mg of polysaccharide (or roughly 7 moles of hexose per mole of CDAP for a hexose polysaccharide). The polysaccharide/TEA/CDAP solution was mixed for 2 min at 22°C to allow for polysaccharide activation. Fluorophore-labeled amine plus phosphonate beads resuspended in 50 mM HEPES (pH 7.8) were added to the activated polysaccharide solution, and the reaction was allowed to proceed for 15 hr at 22°C . Any aggregated beads were disrupted by gentle sonication. Polysaccharide-conjugated beads were reduced by adding 2-picoline borane  dissolved in DMSO (10% wt:wt) and incubating the mixture for 40 min at 40°C. The reaction was terminated with repeated washing with water. Beads were stored in 20 mM HEPES (pH 7.2) and 100 mM NaCl at 4°C.Beads were counted using flow cytometry. Typically, 5 μL of a polysaccharide-coated bead solution were added to 200 μL of HNTB (20 mM 4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid [HEPES] [pH 7.4], 100 mM NaCl, 0.01% bovine serum albumin [wt:wt], and 0.01% Tween-20 [wt:wt]) containing CountBright Absolute Counting Beads (Thermo Scientific). Beads were analyzed using flow cytometry on a FACSAriaIII instrument (BD Biosciences).5/mL in 10 mM HEPES (pH 7.2) passed through a 0.22 μm filter (Millipore) and analyzed in triplicate. Measurements were obtained with the default settings of the instrument, using the refractive index of SiO2 as the material and water as the dispersant.Bead zeta potential was measured to characterize the extent of modification of the bead surface; zeta potential was determined for beads reacted with organosilane reagents with or without surface amine acetylation. Zeta potential measurements were made on a Malvern ZEN3600 instrument using disposable zeta potential cuvettes . Beads were resuspended to a concentration of 5 × 10g, 5 min) to remove the beads, and the absorbance at 570 nm was measured from the resulting supernatant (Biotek Eon). One million amine plus phosphonate beads were analyzed, while three million acetylated amine plus phosphonate beads were required for sufficient signal. Surface amines were calculated from linear regression of the octylamine standard curve.Bead surface amines were quantified using a ninhydrin-based assay per the manufacturer’s instructions (Anaspec) with slight modification. Octylamine (Sigma–Aldrich) in 60% (vol:vol) ethanol:water was used to generate a standard curve. Octylamine or beads resuspended in 60% ethanol were added to glass crimp top vials . One volume equivalent of each of three solutions used for detection of amines was added to the vials. Vials were then sealed and incubated at 95°C for 3 min. The reaction mixture was centrifuged (5000 x 6 beads were incubated for 1 hr at 22°C in 0.25 mL of 0.2 mM biotin dissolved in PBS (7.4). The concentration of biotin remaining was compared to the initial biotin solution to quantify the mols of biotin captured by streptavidin-coated beads.Biotin binding sites on streptavidin-coated beads (Millipore Sigma) were quantified using an avidin and 4′-hydroxyazobenzene-2-carbocylic acid assay according to the manufacturer’s instructions (Fisher Scientific). Briefly, 5 × 104 beads/well) and washed three times in water using a magnet. Beads were resuspended in 175 μL of 2M trifluoroacetic acid containing 15 ng of D6-myo-inositol as an internal standard and then transferred into 8 mm crimp top glass vials. An aliquot was removed from the vial, and flow cytometry was used to quantify the number of beads that had been transferred to that vial. The quantity of monosaccharide released from a bead was determined from the linear fit of standards divided by the number of beads transferred into the hydrolysis vial. For quantifying relative polysaccharide degradation, the absolute amount of monosaccharide released from the bead surface was divided by the mass of that monosaccharide quantified on input beads (with results expressed as a percentage). Samples were omitted when a insufficient number of beads was recovered for analysis or where an instrument malfunction occurred . Biological replicates represent beads from individual animals, bacterial cultures, or input beads collected from FACS. Technical replicates represent independent GC–MS derivatizations and analyses from beads recovered from the same biological sample.Polysaccharide degradation from beads was quantified by GC–MS as described in the section ‘GC-MS of neutral monosaccharide composition’. Briefly, polysaccharide-coated beads were counted using flow cytometry. Beads for hydrolysis were transferred to a 96-well skirted PCR plate . All reactions contained a total of 1 unit of enzyme (per the manufacturer’s documentation). Beads were incubated with rotation at 37°C and aliquots were removed after 30 min and 20 hr. Beads were then washed ≥3 times with 20 mM HEPES (7.2) and 50 mM NaCl on a magnetic tube stand, incubated at 80°C for 10 min to inactivate any residual enzyme, and stored in HNTB. The absolute mass of PFABN remaining on the bead surface was determined by using GC–MS as described above.Individual glycosyl hydrolases, or combinations of enzymes, were added to PFABN-coated beads (10d-glucose. Bacteria were diluted 1:250 (vol:vol) into BMM supplemented with either d-glucose or destarched PFABN to a final concentration of 0.5% (wt:wt). Bacterial growth was monitored by measuring optical density at 600 nm every 15 min in a 96-well plate (Biotek Eon). Mid-log phase cells were harvested by centrifugation  and washed once with BMM without a carbon source. Pelleted cells were resuspended in BMM, without or without added destarched PFABN, and aliquoted into sterile 5 mL snap cap tubes . PFABN-coated MFABs were sterilized by washing in 70% ethanol (vol:vol) twice on a magnetic tube stand before resuspension in BMM for at least 1 hr within a Coy chamber . The concentration of MFABs in the final incubation was 1 × 106 beads/mL. The bacteria/MFAB mixture, with or without added destarched PFABN, was incubated at 37°C with rotation; aliquots (200 μL) were withdrawn at various time points and the number of colony forming units was determined by 10-fold serial dilution in BMM and plating on LYBHI agar plates. Beads were washed ≥3 times with HNTB on a magnetic tube stand. The absolute mass of PFABN remaining on the bead surface was determined by using GC–MS as described above.Single bacterial colonies were picked and grown overnight in BMM  containi6 beads (2.5–3.75 × 106 per bead type) in 400 μL of HNTB was prepared for each mouse; a 350 μL aliquot of the pool was introduced by oral gavage; the remaining 50 μL was analyzed as the input beads (see above). Beads were isolated from the cecums of mice 4 hr after gavage or from all fecal pellets that had been collected from a given animal during the 3–6 hr period following gavage. Beads were typically used in mice within 30 days after polysaccharide conjugation.Each bead type was individually sterilized by washing in 70% ethanol (vol:vol) twice on a magnetic tube stand before resuspension in HNTB. A pool of 10–15 × 10g, 5 min) through Percoll Plus  in a 50 mL conical tube. Beads were recovered from the bottom of the tube; recovered beads from each animal were distributed into four 1.5 mL sterile tubes and washed at least three times with HNTB on a magnetic tube stand until macroscopic particulate debris from intestinal contents were no longer observed. The material from four tubes were subsequently recombined, and beads were stored in HNTB containing 0.01% (wt:wt) sodium azide at 4°C. The procedure described above typically recovers 20–50% of the input beads from the cecum 4 hr post-gavage.Recovered beads were resuspended in 10 mL of HNTB by pipetting and subsequently by vortexing. The resulting slurry was passed through a 100 μm nylon filter . Beads were isolated from the suspension by centrifugation , the supernatant was aspirated, and beads were transferred into a 0.2 mL 96-well skirted PCR plate. Beads were washed with HNTB using a magnetic plate holder and stored at 4°C in HNTB plus 0.01% (wt:wt) sodium azide until analysis. Beads were subjected to acid hydrolysis of the bound polysaccharide, and the amount of liberated neutral monosaccharides was determined by GC–MS. All samples of a given bead type were analyzed in the same GC–MS run; however, the order of analysis of a given bead type recovered from animals representing different treatment groups was randomized. If sufficient beads were available, each bead type from each animal was analyzed up to three times.Bead types were purified by fluorescence-activated sorting . Aliquots of input beads were sorted throughout the procedure to quantify and monitor sort yield and purity. Bead purity typically exceeded 98%. Sorted beads were centrifuged  under study accession: PRJEB38095. Proteomic data are available in the MassIVE database under project number: MSV000085341. COPRO-Seq analysis software can be accessed at  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your fluorescent bead technology enables the characterization of specific metabolic features within the gut microbiome. The new methodology has allowed you to begin to explore biochemical activities of gut microbiota in situ. We view this as an important step forward in the area of microbiome research.Decision letter after peer review:eLife. Your article has been evaluated by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wendy Garrett as the Senior Editor.Thank you for sending your article entitled "Microbiota functional activity biosensors for characterizing nutrient utilization in vivo" for peer review at eLife. The reviewers express a desire to add some minor additional supporting data for some of your experiments . We are providing reviewer comments but to clarify are not asking for extensive new experimentation.There was enthusiasm for acceptance of your work but some concern about focus. The reviewers are quite interested about the fluorogenic bead technology and its application for microbiome studies. They have made recommendations about how you might strengthen the connection between the results describing how Bacteroides degrade these fibers and the technology. Consensus of the reviewers is that this is a manuscript about a powerful tool and its application rather than a deep dive into the underlying biology. If you can recast the work in light of the reviewers' comments and concerns and modify the text so that it is more conservative it its claims, we are of the opinion that it would be quite appropriate for publication in Reviewer #1I agree with the conclusions the authors draw from the enumeration, INSeq, and proteomic measurements presented in Figure 2 (and its supplements) and Figure 5 - in short, that specific PULs harbored by strains in the synthetic community determine the degree of glycan degradation observed on the beads recovered from mice. It might be nice to run a confirmatory experiment with, e.g., a community in which wild-type BT is replaced by a δ-PUL7 or δ-PUL75 mutant, but the data are already convincing in their current form.Likely outside the scope of this manuscript, but are the authors able to isolate bacterial cells or genetic material from beads recovered from a mouse? One could imagine an anchor organism that consumes the fiber or simply uses it as a physical niche. Might be especially interesting if bead-associated communities had structure and could be imaged using FISH.Reviewer #2:Wesener and colleagues present a method for characterizing biochemical activities of gut microbes in vivo. First, the authors purified and extensively characterized a polysaccharide fraction rich in arabinan (PFABN) and compared the biological activity of PFABN to that of unfractionated pea fiber and arabinan purified from sugar beet (SBABN) using in vitro growth assays/substrate degradation using monocultures of several Bacteroides species, establishing the impact of these on bacterial metabolism. They also characterized responses to these fibers in vivo, in mice colonized with a synthetic community composed of 14 species, consuming diet with/without these fibers. Authors used community-wide proteomic measurements and transposon mutant analyses (TnSeq) for five Bacteroides species to investigate genes involved in the utilization of these fibers and genes important for fitness in these fibers for the 5 Bacteroides species. In the second half of the paper the authors describe methods for generating “Microbiota Functional Activity Biosensors” (MFABs), which consist of glycans covalently-linked to the surface of fluorescent paramagnetic microscopic glass beads. They characterize MFABs and perform validation studies  in vitro and in gnotobiotic mice. Using unique fluorogenic tags authors analyze simultaneously multiple bead types with different immobilized polysaccharides. Presented results suggest that colocalizing different polysaccharides on the bead surface can have a synergistic effect on their degradation modulated by diet. These newly developed Microbiota Functional Activity Biosensors are a significant contribution to the field as options for measuring gut microbiota biochemical activities in vivo are currently very limited. Also, the study contributes valuable datasets on bacterial (proteomics/InSeq for 5 Bacteroides species) that will be useful to other scientists studying the microbiome. Specific comments:The paper comes across as having two major parts that could be better integrated. Testing whether any of the key PULs identified for degradation of PFABN/SBABN (results from INSeq/proteomics) impact degradation of these fibers in the beads in vitro (or vivo) would strengthen the connection between the results describing how Bacteroides degrade these fibers and the new development.Not clear how the statistical differences in growth were determined in Figure 1. i.e., what values were used? Growth rate during exponential phase? Should be indicated.Bead assays: What fraction of the beads orally-gavaged are recovered from the mice? Also, in the Materials and methods authors mention that beads were collected from cecum and/or from fecal pellets. Does collection site affect the extent to which glycans on the beads are metabolized?Beads assays ii. The measurement of glycans that remain bound to the beads informs what it is released from them. It is likely that in the context of a complex community most/all? of the released carbohydrates are also used by microbes. This may not always be true, particularly in monocultures or in experiments in mice with small communities. This should be discussed."The results disclosed that the fitness scores of 332, 195, and 75 genes were significantly altered during diet supplementation with pea fiber, PFABN, or SBABN, respectively (Supplementary file 4) " Not clear what species is being discussed here. Supplementary file 4 contains 12 tables (3 supplemented substrates/4 Bacteroides strains).Reviewer #3:This manuscript represents a technical tour de force, and describes an approach to trace and understand metabolic pathways generated by single compounds in a complex ecosystem such as the mouse gut. An outstanding research team has applied many state of the art methodologies to examine the effects of particular dietary substrates on bacterial growth and gene expression. This represents a technological advance but the extent of the advance is not clear, since the results often are marginal. Although a great deal of work has been performed, the overall significance of the work is not obvious.Several specific points should be addressed as well:The pea fiber arabinan is only 71% of the purified polysaccharide. What is the significance of the substantial carbohydrate contamination? Can it really be called purified with such extensive contamination? The results in Figure 4 indicate that the xylan component materially affects the results.B. ovatus with glucose as the substrate? That pattern is unlike all other conditions.What is is the significance of the long lag phase for Figure 2. Panels B and C seem to be contradictory. All of the indicated bacteria grew as well on PF as on the “purified” polysaccharides in panels B . The authors should account for the fitness variation in both directions, and not just cherry-pick the ones that support their hypothesis.Figure 3C. Why are the results shown for SBABN when he main thrust of the paper is PFABN? If the method is universal, then the PFABN results should be similar.Figure 4. Panel B is confusing-are the germ-free mice shown under the heading colonized? That is the only way to reconcile the text and figure. But then why are sugars metabolized in the GF animals? Or are the GF mice not shown? This point needs to be clarified since it affects the interpretation of the results.Figure 5 panel D. The y-axis in the two sub-panels that deal with the glucomannan coated beads are different. In the HISF/LoFV columns, the results of the 2 conditions differ, but the pea fiber results are the same across the panels-this difference is the source of the author's claims, but this seems in the realm of experimental variation, rather than biological meaning. There should be more confirmation of the points that the authors are making, since they end with a strong statement of impact without the necessary rigor.Discussion: In general, the Discussion overstates the significance of the actual findings. The broad statements should be toned down. The limitations of the purification methods should be acknowledged and how that affects the results achieved should be addressed. Reviewer #1I agree with the conclusions the authors draw from the enumeration, INSeq, and proteomic measurements presented in Figure 2 (and its supplements) and Figure 5 - in short, that specific PULs harbored by strains in the synthetic community determine the degree of glycan degradation observed on the beads recovered from mice. It might be nice to run a confirmatory experiment with, e.g., a community in which wild-type BT is replaced by a δ-PUL7 or δ-PUL75 mutant, but the data are already convincing in their current form.Bacteroides, but this remains a “work in progress”. Therefore, we feel that targeted deletion of these or other PULs is outside the scope of the current study which focuses on development of MFAB technology and its application for quantifying polysaccharide degradation by a gut microbial community.We agree with the reviewer that deletion of an entire multi-kilobase PUL would provide confirmatory evidence of the importance of that PUL for polysaccharide utilization and fitness. We are working to develop CRISPR-based tools for removing specified PULs from Likely outside the scope of this manuscript, but are the authors able to isolate bacterial cells or genetic material from beads recovered from a mouse? One could imagine an anchor organism that consumes the fiber or simply uses it as a physical niche. Might be especially interesting if bead-associated communities had structure and could be imaged using FISH.We very much appreciate the reviewer’s suggestion and vision. However, quantifying the distribution of each member of our 13-member community within and across different bead-types, within and across different regions of the gut as a function of diet using FISH or other culture-independent methods is technically challenging. We agree with the reviewer at two levels: such an effort is outside the scope of the present work but certainly a future application of the bead technology.Reviewer #2:Wesener and colleagues present a method for characterizing biochemical activities of gut microbes in vivo. First, the authors purified and extensively characterized a polysaccharide fraction rich in arabinan (PFABN) and compared the biological activity of PFABN to that of unfractionated pea fiber and arabinan purified from sugar beet (SBABN) using in vitro growth assays/substrate degradation using monocultures of several Bacteroides species, establishing the impact of these on bacterial metabolism. They also characterized responses to these fibers in vivo, in mice colonized with a synthetic community composed of 14 species, consuming diet with/without these fibers. Authors used community-wide proteomic measurements and transposon mutant analyses (TnSeq) for five Bacteroides species to investigate genes involved in the utilization of these fibers and genes important for fitness in these fibers for the 5 Bacteroides species. In the second half of the paper the authors describe methods for generating “Microbiota Functional Activity Biosensors” (MFABs), which consist of glycans covalently-linked to the surface of fluorescent paramagnetic microscopic glass beads. They characterize MFABs and perform validation studies  in vitro and in gnotobiotic mice. Using unique fluorogenic tags authors analyze simultaneously multiple bead types with different immobilized polysaccharides. Presented results suggest that colocalizing different polysaccharides on the bead surface can have a synergistic effect on their degradation modulated by diet. These newly developed Microbiota Functional Activity Biosensors are a significant contribution to the field as options for measuring gut microbiota biochemical activities in vivo are currently very limited. Also, the study contributes valuable datasets on bacterial (proteomics/InSeq for 5 Bacteroides species) that will be useful to other scientists studying the microbiome. Specific comments:The paper comes across as having two major parts that could be better integrated. Testing whether any of the key PULs identified for degradation of PFABN/SBABN (results from INSeq/proteomics) impact degradation of these fibers in the beads in vitro (or vivo) would strengthen the connection between the results describing how Bacteroides degrade these fibers and the new development.As noted in our response to reviewer 1, we are working to develop CRISPR-based tools for PUL deletions. In the revised Discussion, we have emphasized how application of genetic (e.g. CRISPR-based) tools for single gene or entire PUL deletion, combined with the use of MFABs as a functional readout, could provide new insights about the mechanisms by which community members acquire/degrade nutrients (polysaccharides).We have modified the text:“Future studies where MFABs are used in conjunction with genetic tools that enable rapid deletion of genes or entire multi-kilobase loci could provide new insights about the mechanisms by which community members acquire/degrade nutrients (polysaccharides).”Not clear how the statistical differences in growth were determined in Figure 1. i.e., what values were used? Growth rate during exponential phase? Should be indicated.We have modified the text to state:B. ovatus ATCC 8483, B. cellulosilyticus WH2 and B. thetaiotaomicron VPI-5482 grew on isolated PFABN and SBABN, although less rapidly during the exponential phase of growth and to a lower cell density than in medium containing an equivalent concentration of d-glucose .”“in vitro assays performed in a minimal defined medium  with Bacteroides type strains established that Note, we have provided data summarizing the growth curves in Supplementary file 1 (maximum change in OD600 per hour and maximum OD600 achieved).Bead assays: What fraction of the beads orally-gavaged are recovered from the mice? Also, in the Materials and methods authors mention that beads were collected from cecum and/or from fecal pellets. Does collection site affect the extent to which glycans on the beads are metabolized?Materials and methods section to read:We have modified the “The procedure described above typically recovers 20-50% of the input beads from the cecum four hours post gavage.”Beads from Experiment #1 were recovered from the cecum and beads from Experiment #2 were isolated from the feces. In general, the results obtained were similar; this was reported in the original manuscript:“.”Beads assays ii. The measurement of glycans that remain bound to the beads informs what it is released from them. It is likely that in the context of a complex community most/all? of the released carbohydrates are also used by microbes. This may not always be true, particularly in monocultures or in experiments in mice with small communities. This should be discussed.We agree and have modified the manuscript throughout to emphasize that MFABs report on polysaccharide degradation. For example, the Discussion has been modified to include:7 beads contained 100 micrograms of bead immobilized polysaccharide, 1,000 times more polysaccharide was consumed daily in the diet by mice in the polysaccharide-supplemented arms of our experiments.”“We emphasize that in this study, we use MFABs to quantify community degradative activity and not to analyze community consumption of MFAB-bound polysaccharides and its relationship to bacterial growth. Determining the latter is challenging in our animal model; a typical gavage of 10"The results disclosed that the fitness scores of 332, 195, and 75 genes were significantly altered during diet supplementation with pea fiber, PFABN, or SBABN, respectively (Supplementary file 4) " Not clear what species is being discussed here. Supplementary file 4 contains 12 tables (3 supplemented substrates/4 Bacteroides strains).Bacteroides species:We have modified the text to link the number of genes to specific B. thetaiotaomicron VPI-5482, 135 genes in B. ovatus ATCC 8483, 346 genes in B. cellulosilyticus WH2, and 82 genes in B. vulgatus ATCC 8482 were significantly decreased during diet supplementation with either pea fiber, PFABN, or SBABN (Supplementary file 4) .”“The results disclosed that the fitness scores of a total of 39 genes in Reviewer #3:This manuscript represents a technical tour de force, and describes an approach to trace and understand metabolic pathways generated by single compounds in a complex ecosystem such as the mouse gut. An outstanding research team has applied many state of the art methodologies to examine the effects of particular dietary substrates on bacterial growth and gene expression. This represents a technological advance but the extent of the advance is not clear, since the results often are marginal. Although a great deal of work has been performed, the overall significance of the work is not obvious.Several specific points should be addressed as well:The pea fiber arabinan is only 71% of the purified polysaccharide. What is the significance of the substantial carbohydrate contamination? Can it really be called purified with such extensive contamination? The results in Figure 4 indicate that the xylan component materially affects the results.Arabinose represents 71 mole percent of the isolated pea fiber arabinan fraction. Similar to arabinan isolated from sugar beet, pea fiber arabinan contains galactose, likely as covalently attached galactan fragments, and glucose as starch .The reviewer is correct: xylose is present in the pea fiber arabinan fraction (12 mole percent). We had described the presence of xylan and starch in the text:“Xylose was present as a linear xylan (4-substituted xylose) and glucose in the form of residual starch.”.We have modified the text to include our rationale for naming the polysaccharide pea fiber arabinan and have replaced the word “purified” with “isolated”.“Xylose was present as a linear xylan polysaccharide (4-substituted xylose) and glucose in the form of residual starch. We named the isolated polysaccharide fraction “pea fiber arabinan” (PFABN) based on (i) these linkage results, (ii) the fact that arabinose comprises the majority of its monosaccharide content (71 mole percent) and (iii) our observation that ~90% of all non-starch carbohydrate is represented by what is likely a single species of polysaccharide, with the remaining being xylan.”What is the significance of the long lag phase for B. ovatus with glucose as the substrate? That pattern is unlike all other conditions.B. vulgatus. We do not understand why this organism exhibited a long lag phase when grown in defined minimal medium containing glucose as the sole carbon source, as opposed to either of the two isolated arabinan polysaccharides. All monocultures were seeded at the same density from a culture that had been grown overnight in minimal medium supplemented with glucose .We assume the reviewer is referring to Figure 2. Panels B and C seem to be contradictory. All of the indicated bacteria grew as well on PF as on the “purified” polysaccharides in panels B  .”Figure 2—figure supplement 2. Not surprisingly, results for PF and PFABN were similar, but they were not very different from SBABN. It is difficult to understand what actually was shown here. Although elegant, it is unclear what the fitness calculations add to the calculations of protein abundance. Almost all of the significant variance is along the axis of the protein abundances. A considerable number of the data points for the specified proteins are above the zero line for fitness. (mutant grew better than wild-type). The authors should account for the fitness variation in both directions, and not just cherry-pick the ones that support their hypothesis.Fitness benefits following gene deletion are observed in forward genetic screens, although the biological meaning of increased fitness can be obscure. We have paraphrased the point raised by the reviewer by noting in our discussion of the INSeq results that:“A substantial number of the genes in our forward genetic screen have a fitness score greater than zero, indicating that the mutant grew better than wild-type. The biological explanation for improved fitness in these cases remains to be determined.”Figure 3C. Why are the results shown for SBABN when he main thrust of the paper is PFABN? If the method is universal, then the PFABN results should be similar.Sugar beet arabinan was used when developing the MFAB conjugation reaction because it is commercially available in large quantities and is similar in composition to pea fiber arabinan.In our development of the CDAP immobilization chemistry for use with MFABs, we have not yet encountered a carbohydrate-based polymer that is not amenable to conjugation. We provide data using ligands with varying physical features . The results yielded similar ligand loading levels ; this includes our findings with a model oligosaccharide, maltodextrin .Figure 4. Panel B is confusing-are the germ-free mice shown under the heading colonized? That is the only way to reconcile the text and figure. But then why are sugars metabolized in the GF animals? Or are the GF mice not shown? This point needs to be clarified since it affects the interpretation of the results.We are sorry for the confusion. Our results from beads recovered from germ-free mice are not included in main text Figure 4. Rather, the results of quantifying six monosaccharides released from three bead types recovered from germ-free mice are presented, and compared to input beads, in Figure 4—figure supplement 3.We have modified the text to emphasize that only results from colonized animals are presented in Figure 4:“In contrast to germ-free controls , the mass of arabinose was significantly decreased when PFABN- or SBABN-coated beads were recovered from colonized mice fed the unsupplemented HiSF-LoFV diet .”We have also modified the title of Figure 4 to include “colonized”.“Figure 4. Quantifying microbial degradation of PFABN- and SBABN-coated beads in colonized gnotobiotic mice fed unsupplemented or supplemented HiSF-LoFV diets.”Figure 5 panel D. The y-axis in the two sub-panels that deal with the glucomannan coated beads are different. In the HISF/LoFV columns, the results of the 2 conditions differ, but the pea fiber results are the same across the panels-this difference is the source of the author's claims, but this seems in the realm of experimental variation, rather than biological meaning. There should be more confirmation of the points that the authors are making, since they end with a strong statement of impact without the necessary rigor.We have modified Figure 5D to make the y-axis equivalent across the subpanels and the text to state:“These results show that deliberate physical colocalization can result in quantitatively modest, albeit statistically significant synergistic degradation of polysaccharides during fiber supplementation .”The Discussion now contains the caveat:“In principle, a wide range of different glycan combinations with varying stoichiometries can be explored owing to the fact that different hybrid bead types, each with its own fluorophore, can be created and tested simultaneously in vitro and in vivo to investigate the mechanism, generality, and biological significance of this observation.”Discussion: In general, the Discussion overstates the significance of the actual findings. The broad statements should be toned down. The limitations of the purification methods should be acknowledged and how that affects the results achieved should be addressed.We appreciate the reviewer’s thoughtful comment and agree. We have modified the Discussion in the ways suggested. For example;We have emphasized the fact that MFABs were used to monitor degradation of polysaccharides not their consumption by members of the community.We now state in the text that:“The bead-based Microbiota Functional Activity Biosensors (MFABs) described in this report represent a technology for measuring biochemical activities expressed by a microbial community of interest in vivo or ex vivo. Future studies where MFABs are used in conjunction with genetic tools that enable rapid deletion of genes or entire multi-kilobase loci could provide new insights about the mechanisms by which community members acquire/degrade nutrients (polysaccharides). We emphasize that in this study, we use MFABs to quantify community degradative activity and not to analyze community consumption of MFAB-bound polysaccharides and its relationship to bacterial growth. Determining the latter is challenging in our animal model; a typical gavage of 107 beads contained 100 micrograms of bead immobilized polysaccharide, 1,000 times more polysaccharide was consumed daily in the diet by mice in the polysaccharide-supplemented arms of our experiments.”We have modified the text to tone down a general statement about how MFABs can help design more nutritious foods:“The selection of gut bacterial taxa for in vivo tests of fiber effects in gnotobiotic models is critical for yielding ecologically and physiologically relevant results.”We have mentioned caveats associated with the purity of our PFABN preparation in the Discussion:B. cellulosilyticus WH2 and B. ovatus ATCC 8483 was not induced by PFABN supplementation  .”“Robust isolation procedures and analytical characterization are required to isolate grams of polysaccharide and test their biological effects in gnotobiotic mice. The approach we employed allowed us to isolate 50 grams of material to ~85% purity with the remaining material comprised of starch and xylan. Starch can be degraded by host enzymes. The xylan present in our PFABN preparation could, in principle, influence the microbiota response. However, there is arabinoxylan already present the base HiSF-LoFV diet and we found that expression of known xylan-responsive PULs in


Diabrotica undecimpunctata virus 2 (DuV2), a putative positive-sense, single-stranded RNA virus identified from the southern corn rootworm transcriptome, comprises 5,313 nucleotides, including a short poly(A) tail. The two open reading frames encode a nonstructural polyprotein (p156) and a putative capsid protein (p25).The genome of  Diabrotica undecimpunctata virus 2 (DuV2), a putative positive-sense, single-stranded RNA virus identified from the southern corn rootworm transcriptome, comprises 5,313 nucleotides, including a short poly(A) tail. The two open reading frames encode a nonstructural polyprotein (p156) and a putative capsid protein (p25).The genome of  Diabrotica undecimpunctata howardi Barber, is one of three corn rootworm species that cause significant damage to agricultural and horticultural crops (Diabrotica virgifera virgifera (–The southern corn rootworm (SCR), al crops . Severalirgifera –4, but orgifera –. Here, wEggs, larvae , pupae, and adult  SCR were supplied by Crop Characteristics . The RNeasy minikit  was used for isolation of total RNA from three biological replicates of 100 eggs, 50 first-instar larvae, 5 each of second- and third-instar larvae, 1 pupa, 1 adult female, and 1 adult male. cDNA libraries were constructed using the NEBNext Ultra RNA library prep kit for Illumina (NEB) for each stage-specific sample with poly(A) selection and paired-end sequencing with 150-nucleotide (nt) reads conducted on a HiSeq 2500 platform . One or two replicate sequence data sets from each developmental stage were used for virus sequence discovery (Diabrotica undecimpunctata virus 2 (DuV2). Sequences derived from DuV2 were observed in transcriptome data sets from all developmental stages of SCR. The near-full-length sequences were assembled from first-instar larvae, suggesting increased abundance and active replication of DuV2 in larvae relative to other developmental stages. The 5′ coding sequence located between nt 156 and 4286 encodes a putative nonstructural protein of 1,376 amino acids (aa) , while the more 3′ sequence between nt 4366 and 5025 encodes a putative structural protein with 219 aa . A conserved protein domain search revealed that p156 contains three conserved domains, two overlapping viral helicase domains  (pfam00978). A Tomato mosaic virus coat protein domain (TMV_coat domain pfam00721) was identified in protein p25, supporting annotation as a capsid protein.The Trinity assembler v2.6.6 (Hubei beny-like virus 1 (GenBank accession no. KX883789.1), an RNA virus isolated from dipteran insects (Riboviria sp.) (Beihai charybdis crab virus 1 (GenBank accession no. YP_009333243.1), which has a genomic structure similar to that of DuV2.The highest BLASTp score for p156  was Huberia sp.) . BLASTp MN646771. The raw reads for SRA accession no. SRX8018653 to SRX8018656 are available at BioProject no. PRJNA615920.The genome sequence of DuV2 is available in GenBank under accession no.


This study aimed to comprehensively assess the diagnostic value of fibrinogen to prealbumin ratio (FPR) and gamma-glutamyl transpeptidase to platelet ratio (GPR) as single markers or in combination in patients with alpha-fetoprotein-negative (AFP-negative) hepatocellular carcinoma (HCC).A total of 199 healthy controls and 515 AFP-negative patients were enrolled in this study, including 180 HCC inpatients, 151 liver cirrhosis (LC) patients, and 184 chronic hepatitis (CH) cases. Mann–Whitney U or Kruskal–Wallis H test were used to analyze differences between groups in laboratory parameters and clinicopathological features. The diagnostic value of FPR and GPR, alone or in combination, in AFP-negative HCC (AFP-NHCC) patients was determined via a receiver operating characteristic (ROC) curve.The levels of FPR and GPR were gradually increased in the development of AFP-NHCC and positively correlated with the tumor size and Barcelona Clinic Liver Cancer (BCLC) stages. Moreover, GPR was associated with Edmondson–Steiner grades. After univariate logistic regression analysis, FPR and GPR remained independent predictors of adverse outcomes. The combination of FPR and GPR had a good ability to detect AFP-NHCC from the control group (area under curve [AUC] = 0.977), AFP-negative CH (AUC = 0.745), and AFP-negative LC (AUC = 0.666). FPR combined with GPR possessed a larger area  and sensitivity  than FPR or GPR alone for differentiating AFP-NHCC with tumor size < 3 cm or at the BCLC-A stage.The pretreatment levels of FPR and GPR played vital roles in the development of AFP-NHCC, especially in patients with early or small AFP-NHCC. Hepatocellular carcinoma (HCC) is the sixth-most commonly diagnosed cancer and the fourth leading cause of tumor-related death worldwide in 2018 . CurrentAn increasing amount of research has pinpointed systematic inflammation and abnormal metabolism involved in the diagnosis and progression of HCC, such as fibrinogen (Fib) , prealbuGamma-glutamyl transpeptidase (GGT) to platelet ratio (GPR) was proposed by Lemoine et al. as a novel predictor for liver disease . Ample eTo date, several studies have researched the value of FPR and GPR in the prognosis of HCC. However, there is a lack of data about the diagnostic value of FPR and GPR for the development of AFP-NHCC patients. Thus, this study evaluates whether the FPR and GPR could be used as predictive markers for patients with AFP-NHCC.180 HCC patients, 184 CH patients, and 151 LC patients who were all AFP-negative (< 20 ng/mL) were continuously recruited from the First Affiliated Hospital of Guangxi Medical University between Jan 2012 and Oct 2019. The inclusion criteria of AFP-negative patients were as follows: (1) newly diagnosed with HCC, and verified by surgical histopathology examination; LC was diagnosed via pathological examination and typical morphology upon ultrasonography or computed tomography (CT) imaging; or CH patients were confirmed to have been infected with hepatitis B virus (HBV)/hepatitis C virus (HCV) for at least 6 months; (2) no blood-system diseases or immunity-related diseases; (3) no presence of other types of cancers; (4) no infectious diseases other than hepatitis B or C; and (5) no organic disease outside of the liver. We also enrolled 199 healthy individuals with no history of cancer and no clinical evidence of liver disease as a control group. This research was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University, and informed consent was obtained from all the participants.A total of 180 patients (range 28–78 years) with AFP-NHCC were recruited in this study, including 151 patients with positive hepatitis B surface antigen, 21 patients with hepatitis C virus positive liver diseases, and 8 subjects with other types of liver cancer. And serum HBV-DNA levels ≥ 500 IU/mL was achieved in 69 of 151 AFP-NHCC patients who were hepatitis B surface antigen positive. According to the Barcelona Clinic Liver Cancer (BCLC) staging system, 108 patients (60.0%) had stage A, 53 (29.4%) had stage B, and 19 (10.6%) had stage C. Of the 151 cirrhosis patients, 130 were positive for hepatitis B surface antigen, 10 were positive for hepatitis C virus antibody, and 11 were other causes of cirrhosis, respectively. Subjects with hepatitis B surface antigen-positive were 159 in AFP-negative CH group, the rest were hepatitis C virus antibody-positive patients (25/184).All of the corresponding data for this study was extracted from the hospital’s electronic medical records before each qualified participant’s operation, including sex, age, white blood cells (WBC), platelets (PLT), hemoglobin (Hb), fibrinogen (Fib), prealbumin (PA), alpha-fetoprotein (AFP), alkaline phosphatase (ALP), total bilirubin (TBIL), alanine aminotransferase (ALT), aspartate amino transferase (AST), and gamma-glutamyl transpeptidase (GGT). Plasma fibrinogen concentrations were determined by the Clauss method using a Sysmex CA7000 automatic coagulation analyzer. Blood tests were performed with a Beckman-Coulter LH 780 hematology analyzer . The levels of PA, AST, ALT, TBIL, ALP, and GGT were tested by a Hitachi 7600 automatic biochemical analyzer  produced by Shanghai Zhicheng Bio-Technology Co., Ltd. Hepatitis B surface antigens and hepatitis C antibodies were analyzed using a chemiluminescent immunoassay . The values of FPR and GPR were calculated using the following formulas: FPR = fibrinogen level/prealbumin level; GPR = gamma-glutamyl transpeptidase level/platelet count.None of the data met the criteria for a normal distribution based on a Kolmogorov–Smirnov test. The median and interquartile ranges were applied for non-normally distributed data. The Mann–Whitney U test or Kruskal–Wallis H test were conducted to detect differences between groups in laboratory parameters and clinical characteristics. A receiver operating characteristic (ROC) curve and the area under the curve (AUC) were determined by MedCalc statistical software (version 18.1.1). The SPSS16.0 statistical software package was used for data processing and analysis, using a significant level of 0.05.p < 0.001) . Although 22 specimens were not clearly classified by Edmondson–Steiner grade, GPR showed a significant difference between the three subgroups of AFP-NHCC stages (p = 0.032).As shown in Table p < 0.05 were subjected to a multivariate analysis and further screened by enter method to evaluate their independent effect. Odd ratio (OR) and 95% confidence interval (CI) were also calculated for each other. After the univariate analysis, several significant parameters were chosen as potential independent predictors for further multivariate analysis, including TBIL , ALT , AST , ALP , FPR , and GPR . After adjusting for these six predictors, the results of the analysis demonstrated that FPR , GPR , and AST  were still important indicators closely related to the occurrence of AFP-NHCC. The optimal model for distinguishing AFP-NHCC patients from the control group was established through integration (logit P = 0.841 × FPR + 15.927 × GPR + 0.078 × ALT − 17.909). For this model, the AUC value was 0.981 (0.961 to 0.992), while the sensitivity and specificity were 92.22% and 97.99%, respectively.As shown in Table The results of the ROC curve analysis are shown in Table To predict AFP-NHCC, the optimal cut-offs of FPR were 16.70 and 20.72, for the diagnosis of AFP-negative CH , and AFP-negative LC , respectively. The optimal cut-offs of GPR were 0.23 and 0.20, for the diagnosis of AFP-negative CH , and AFP-negative LC , respectively. For the diagnosis of AFP-NHCC, the combined use of FPR and GPR resulted in a greater AUC (0.745 for AFP-negative CH and 0.666 for AFP-negative LC) than FPR or GPR alone.Early diagnosis of HCC is closely associated with its prognosis, which can substantially enhance the 5-year survival rate of patients . AFP, asResearch has proven that coagulation and nutritional status may affect the progression of HCC patients. Fibrinogen, as an important coagulation factor, synthesized by hepatocytes. Zhu et al.  demonstrAs a crucial enzyme in glutathione metabolism, Gamma-glutamyl transferase (GGT) was continually elevated in metabolic-induced hepatic injury , was obsThe present study first assessed the preoperatory FPR and GPR levels to evaluate their diagnostic efficacy in the development of AFP-NHCC. Both FRP and GPR significantly increased with the progression of AFP-NHCC as well as tumor size and cancer stage. Moreover, high FPR and GPR levels were also independent poor outcome predictors in multivariate logistic regression analyses adjusted for potential predictors. These suggested that FPR and GPR might be important markers in the progression of patients with AFP-NHCC. FPR and GPR had good AUCs and sensitivity in identifying AFP-NHCC patients from controls. Many papers indicated that a series of biomarkers could help diagnose AFP-NHCC, showing the AUC of GP73 , AFP-L3 Several limitations should be concerned. First, we failed to distinguish viral physical status and viral load from the stratified analyses due to the relatively small sample size. Second, AFP-negative cases were limited and all came from the same hospital, which may create bias in evaluating the predictive value of these markers. Third, personal information, including dietary habits and family histories, was not obtained, which may influence the final results. Hence, future prospective studies require multiple centers, a larger scale, and more detailed information to validate these results.The present study demonstrated that FPR and GPR were correlated with AFP-NHCC as well as tumor size and BCLC stage. The combination of FPR and GPR, as economic, simple, effective, and promising biomarkers, possessed a high diagnostic efficiency in the progression of patients with AFP-NHCC, especially in patients with early or small AFP-NHCC.


Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design. Cryo-electron microscopy (cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3 (picornaviruses) and T = 3 (flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development. Because crystallizing virus-antibody complexes can be challenging, morphological studies of virus-antibody interactions have long been carried out through transmission electron microscopy (TEM) with negative staining protocols , a Nobel-prize-winning technique, is now routinely used for studying virus-antibody complexes at high resolution  and their associated antibodies.There are two major 3D cryo-EM analysis strategies: single particle analysis (SPA) and cryo-electron tomography (cryo-ET)  has traditionally been achieved by the use of hybridoma technology where antibody-producing B cells derived from immunized mice are fused to an immortalized cell line   to the light (VL) chains with a flexible peptide linker  are present Fig. A, 1B, a et al.et al.et al.et al.H and VL domains have clearly defined densities  and variable domains (VH and VL). Because the antigen binding site is determined by six hypervariable loops, namely, the complementarity-determining regions (CDRs) on the VH and VL domains, these densities can provide detailed information about the virus-antibody interface. Based on high-resolution structural analysis, CDRs from five HAV antibodies  interact with a single conserved antigenic site, which has been shown as an attractive target for rational development of antiviral drugs  , West Nile virus (WNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV) B7 in total can bind to a DENV virion  and mature virions (infectious). Upon binding to cellular receptors, the native virions are converted into uncoated intermediates called A-particles   Intermediate complexes during ‘breathing’ motion. Although each cryo-EM reconstruction usually represents a static snapshot of a specific conformation, structural plasticity in immune complexes can also be visualized. Fab 1A1D-2 induces large conformation changes in the E protein and binds to normally partially hidden epitopes . Glycoprotein-specific antibody-inducing epitopes on the viral surface have been studied directly using cryo-ET. The open conformations of the HIV-1 Env spike induced by Fab b12 or CD4/Fab 17b have been characterized using cryo-ET analysis  is a mosquito-transmitted human pathogen with T = 4 icosahedral symmetry. On its surface, three E2 molecules form the major component of the viral spike and serve as the main target for antibodies. Because handling live infectious CHIKV requires biosafety level 3 facilities, cryo-EM studies are performed with the safe CHIKV vaccine strain (CHIKV 181/25)  it can investigate conformational epitopes with sequentially discontinuous residues on icosahedral virions; (2) it avoids tedious screening for diffractable crystals and can be incorporated into a standardized process for rapid and rational vaccine development; (3) it can help with analyzing intrinsic heterogeneous samples, like highly glycosylated viral glycoproteins; and (4) it can be exploited for developing and characterizing high-quality vaccine platforms. Finally, cryo-EM studies of antigen–antibody complexes are beginning to clarify the mechanisms of epitope–paratope recognition at atomic resolution, so we expect that high-resolution cryo-EM structures will play more important roles in future at guiding vaccine development.


We conducted a serologic survey for Crimean-Congo hemorrhagic fever virus antibodies in livestock  on Corsica (island of France) during 2014–2016. Overall, 9.1% of animals were seropositive, suggesting this virus circulates on Corsica. However, virus identification is needed to confirm these results. Nairoviridae family. Symptoms in humans vary from a nonspecific mild febrile syndrome to severe hemorrhagic disease that sometimes leads to death , the most widespread tickborne viral infection in humans, is a zoonotic disease caused by an orthonairovirus of the to death , panel AAs part of national surveillance for animal diseases, veterinarians collected cattle, goat, and sheep blood samples during 2014–2016. In total, 3,890 animals  were sampled from 269 farms, originating from 46% (137/298) of the municipalities with ruminant farming activities (https://www.id-vet.com) according to the manufacturer’s instructions  to be analyzed by the World Health Organization and World Organisation for Animal Health national reference center for CCHFV . We used the pseudo–plaque reduction neutralization test (PPRNT) ; estimated seroprevalence in cattle was 13.3% (95% CI 10.2%–17.3%), goats 3.1% (95% CI 1.4%–7.0%), and sheep 2.5% (95% CI 1.0%–5.9%). CCHFV antibodies were detected across the island; 35.8%  of the investigated municipalities had sitivity  panel A.sitivity , panel B is high , panel BOf 35 ELISA-positive serum samples tested, none showed neutralizing antibodies against Hazara and Dugbe viruses, and no ELISA-negative serum sample showed neutralizing antibodies against CCHFV, Hazara virus, or Dugbe virus  and PPRNT (23 positives) findings might result from their different target epitopes; the ELISA measures total immunoglobulin  and PPRNT just a subset  (As of February 2020, CCHFV has not been detected in ticks on Corsica (More information on Crimean-Congo hemorrhagic fever virus antibodies among livestock on Corsica, France, 2014–2016.


Variations in gene expression patterns represent a powerful source of evolutionary innovation. In a rodent living about 70 million years ago, a genomic accident led an immune formyl peptide receptor (FPR) gene to hijack a vomeronasal receptor regulatory sequence. This gene shuffling event forced an immune pathogen sensor to transition into an olfactory chemoreceptor, which thus moved from sensing the internal world to probing the outside world. We here discuss the evolution of the FPR gene family, the events that led to their neofunctionalization in the vomeronasal organ and the functions of immune and vomeronasal FPRs. The immune and olfactory systems, despite being respectively devoted to probe the internal and external worlds, face a very similar challenge: they must recognize millions of different molecules. An obvious answer to this task, which was the one selected during vertebrate evolution, is to build very large chemosensory toolboxes. The immune system generates this tool diversity using an original genomic recombination strategy, which, starting with a very limited number of genes, is able to produce millions of different transcripts. What evolution selected for the olfactory system is less parsimonious. It is in the numbers that diversity is achieved in the nose. Thus, mice, elephants, and most mammalian species benefit today from a vast repertoire of olfactory chemoreceptor genes, which in some species number over 5% of their coding genes. But the parallel between both systems does not end here. The present review points to such a parallel and will highlight a remarkable interaction between the immune and olfactory chemosensory toolboxes.N-formylmethionine, and the cleavage during the translocation process of the signal sequence containing this N-formylmethionine is a potential source of FPR ligands  are seven transmembrane domain G-protein coupled receptors belonging to the rhodopsin-like superfamily. They are expressed in myeloid cells, mainly in neutrophils and monocytes, but also in a number of tissues and cells including the lymphoid cells, spleen, platelets, bone marrow, hepatocytes, astrocytes, neurons, microglia, immature dendritic cells, and epithelial cells  . Phylogenetic analyses of immune mammalian FPR genes revealed that a first duplication led to the divergence of FPR1 and FPR2 and that a second duplication of FPR2 early in primate evolution led to FPR3  . However, Fpr-rs1 resulted from a duplication of Fpr-rs2, which is most closely related to the human FPR2. To avoid any confusion, we will here call this gene Fpr-rs1.Most mammalian genomes contain two immune FPR genes, namely in humans R3) Fig. . Of noteFpr-rs1, Fpr-rs3, Fpr-rs4, Fpr-rs6, and Fpr-rs7 were almost exclusively expressed in sensory neurons of the vomeronasal organ , downstream of a V1R promoter and first exon. This generated a splice trap that hijacked the endogenous V1R regulatory sequences, the pseudogenization of the endogenous V1R coding sequence, and the emergence of Fpr-rs3, the first vomeronasal FPR. The cluster of apically expressed FPRs later expanded through several duplications, leading to the birth of Fpr-rs4, Fpr-rs6, and Fpr-rs7. Consistent with this series of events, the promoter region of all apically expressed vomeronasal FPRs is highly similar to that of Fpr-rs3.How did immune receptors acquire vomeronasal tissue specificity, and how did they integrate into this sensory system? Phylogenetic analyses of FPR genes in mammalian species showed that the split between immune and vomeronasal FPRs occurred at the root of the Eumuroida group Fig. c Dietsc, which iFpr-rs1 are very different. Over 50 million years after the first FPR exon shuffling event, after the split between mice and rats, a second shuffling event took place . Possibly critical for this dynamic reshuffling are the long interspersed nuclear element (LINE) repeats that are enriched within V1R, V2R, and OR gene clusters . More specifically, the MKKFRW motif is highly conserved at the N-terminus of the virulence-regulating factor protein MgrB of these bacteria. Most relevant perhaps was the observation that this peptide triggers the activation of basal vomeronasal sensory neurons expressing Fpr-rs1. Moreover, the peptide induces avoidance behavior in male mice when added to female estrus urine, which otherwise is a highly attractive stimulus to males. Mice lacking the FPR-rs1 receptor or components of the vomeronasal signaling cascade do not display any avoidance of the MgrB formylated peptide, clearly suggesting a critical role for FPR-rs1 as a chemoreceptor involved in the behavioral response to bacterial cues (Fig. Much of our knowledge on FPR ligands originates from research on immune FPRs. Hence, after the discovery of FPR transcripts in mouse vomeronasal sensory neurons, the question was to know whether FPR-rs1, FPR-rs3, FPR-rs4, FPR-rs6, and FPR-rs7 share ligand affinities with immune FPRs. Initial in vitro and ex vivo screens for ligands showed that vomeronasal FPRs were responsive to several of the known immune-FPR ligands, including fMLF, CRAMP, lipoxin A4, and uPAR (Riviere et al. ues Fig.  (Bufe etues Fig. .Fpr-rs1 are still capable of discriminating between healthy and immune-challenged peers, thus excluding that FPR-rs1 alone is responsible for this behavior (Bufe et al. Mice have the ability to discriminate between sick and healthy conspecifics (Boillat et al. For several decades, FPRs were known for playing a critical role in the regulation of immune functions, responding to chemoattractants linked to pathogens and to inflammatory processes and triggering appropriate cellular responses. In the present review, we described the evolution of FPR genes and in particular the genomic events that led to their expansion and neofunctionalization in rodents, a lineage in which they appear to be involved in the innate behavioral response to pathogenic cues present in the environment. The acquisition of neuronal specificity by FPRs represents thus a remarkable example of genetic innovation, transitioning from pathogen sensors in the immune system to olfactory receptors in neurons.


Musa spp.) production as they affect the yield and limit the international movement of germplasm. Among all the viruses known to infect banana, the banana bunchy top virus and banana streak viruses are widespread and economically damaging. The use of virus-resistant bananas is the most cost-effective option to minimize the negative impacts of viral-diseases on banana production. CRISPR/Cas-based genome editing is emerging as the most powerful tool for developing virus-resistant crop varieties in several crops, including the banana. The availability of a vigorous genetic transformation and regeneration system and a well-annotated whole-genome sequence of banana makes it a compelling candidate for genome editing. A robust CRISPR/Cas9-based genome editing of the banana has recently been established, which can be applied in developing disease-resistant varieties. Recently, the CRISPR system was exploited to detect target gene sequences using Cas9, Cas12, Cas13, and Cas14 enzymes, thereby unveiling the use of this technology for virus diagnosis. This article presents a synopsis of recent advancements and perspectives on the application of CRISPR/Cas-based genome editing for diagnosing and developing resistance against banana viruses and challenges in genome-editing of banana.Viral diseases are significant biotic constraints for banana ( Babuvirus) and banana streak virus  are economically important viruses threatening banana production , is one of the chief staple food crops in 136 countries in tropics and sub-tropics, with an annual production of 155 million tons on 11 million hectares of land, and feeding millions of people , Musa balbisiana (BB genome), Musa schizocarpa (SS genome), and Australimusa species , or between M. acuminata and M. balbisiana  of dessert bananas for commercialization in local and international markets  as a marker gene , , banana bract mosaic virus  and cucumber mosaic virus  are the most significant  and infected planting materials. BBTD was first noticed in Fiji in the 1880s and is currently present in more than 36 countries in Africa, Asia, Oceania, and South Pacific , caused by BBTV, is the most important viral disease of banana responsible for the significant adverse economic impact on banana production. BBTV is transmitted by the banana aphid ( Pacific . In Afri Pacific . In the  Pacific . This in systems . The inf systems . SeverelBanana bunchy top virus is a single-stranded DNA (ssDNA) virus with a multipartite genome comprising of six circular components  with an Banana streak Obino l’Ewai virus, Banana streak Mysore virus, Banana streak Imové virus, and Banana streak Goldfinger virus, all of which are known to present commonly in banana plantations  virus with a monopartite genome of 7–8 Kb long with three ORFs . ORF 1 eBanana streak virus occurs in episomal and endogenous forms. The BSV genome sequence integrated into the host’s plant B genome is known as endogenous BSV eBSV; , whereasMusa hybrids as micropropagation and hybridization through conventional breeding triggered its activation , which are necessary for systemic infections in plants . RNA 1 aGenome editing technologies facilitated by various sequence/site-directed nucleases (SDN), such as zinc-finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas), have emerged as powerful tools for crop improvement and functional genomics. CRISPR/Cas has rapidly become the most popular genome-editing approach because of its simplicity, efficiency, versatility, specificity, and multiplexing .Streptococcus pyogenes that eliminates invasion of foreign plasmid or viral DNA. The CRISPR/Cas editing system consists of two main components: gRNA (guide RNA) and the Cas nuclease. The Cas protein exhibits nuclease activity, recognizes target DNA by gRNA-DNA pairing between the 5′ leading sequence of gRNA. It also recognizes the PAM (Protospacer adjacent motif) sequence and starts editing upstream of the sequence  or non-homologous end joining (NHEJ) to produce the desired mutation . The genThe types of genome-editing need to be distinguished due to potential discrepancies in the regulatory approaches. SDN1 and SDN2 are similar to mutations obtained through chemical mutagenesis, irradiation, or spontaneous natural mutations and do not lead to the insertion of foreign DNA . Any for1, advancement in bioinformatics tools, and robust genetic transformation protocols  machinery, as a visual marker to optimize the genome-editing protocol for banana. The CHAOS39 edited banana plants showed pale-green phenotypes with normal in vitro growth. Nevertheless, the researchers need to be careful in using cpSRP43/CHAOS39 as a visual marker as the pale green phenotype can be achieved with other factors such as nutrient deficiency and improper light.Genome-editing in banana was first demonstrated in the cultivar “Rasthali” (AAB genome), targeting the PDS as a marker gene . In thisM. acuminata gibberellin 20ox2 (MaGA20ox2) gene to develop semi-dwarf plants of the banana cultivar “Gros Michel” by disrupting gibberellin production  in Arabidopsis and Nicotiana benthamiana, targeting the replication-associated protein (Rep), coat protein (CP), and intergenic region  in N. benthamiana using CRISPR/Cas9 plasmid construct targeting the Rep gene delivered through Agrobacterium-mediated transformation was recorded , a monopartite begomovirus, by transiently delivering a CRISPR/Cas9 construct targeting the viral Rep, CP, and the conserved region of IR of TYLCV using the tobacco rattle virus (TRV) vector , the bipartite Merremia mosaic virus , and TYLCV. Later, N. benthamiana was engineered using a CRISPR/Cas9 system to interfere with the coding sequences of TYLCV, MeMV, and cotton leaf curl Kokhran virus  vector . The N. Begomovirus) was demonstrated in N. benthamiana using a CRISPR/Cas9 system by multiplexing gRNAs targeting Rep and IR sequences  failed to produce resistance against the virus in transgenic cassava  were generated by multiplexing four gRNAs targeting the overlapping region of MP and CP coding sequence, Rep/RepA coding region at the N-terminus of the proteins, long intergenic region (LIR) region, and the genomic region encoding the C-terminus of Rep , a dsDNA pararetrovirus, in Arabidopsis by creating targeted mutations in the coat protein  and a primary viral transcript encoding the viral microRNAs (miRNAs).CRISPR/Cas technology can also be applied to pararetroviruses such as BSV or retroviruses with dsDNA as part of their replication. Currently, the application of CRISPR/Cas9 to disrupt both episomal and integrated dsDNA viruses is reported for only one plant virus . HoweverAgrobacterium-mediated genetic transformation to inactivate the virus. The regenerated genome-edited plantain with targeted mutations in the viral genome prevented proper transcription or/and translation into infectious viral proteins. Phenotyping of the potted genome-edited plants under water stress conditions in the greenhouse confirmed the inactivation of the virus as 75% of the tested plants remained asymptomatic under stress conditions; in contrast, the wildtype control plants showed BSV disease symptoms. All the asymptomatic plants have mutations in all the three ORFs. The application of CRISPR/Cas9-mediated genome editing of banana for controlling viruses is only reported for BSV.Similarly, a multiplex CRISPR/Cas9 system targeting all three ORFs of BSV was used to inactivate the integrated dsDNA of eBSV from the banana genome . The CRIThere are reports of the emergence of new mutated virus variants in plants and human using the CRISPR-Cas9 with a single sgRNA targeting viral genes . The emeBanana is a polyploid heterozygous crop containing a high number of multigene families with paralogs . One of Agrobacterium transformation (Agrobacterium and microprojectile bombardment (Agrobacterium–derived DNA sequences through backcrossing is not feasible due to the sterility of the majority of the farmer-preferred cultivars, unlike seeded crops (Another major challenge is that genome editing in a banana crop is currently achieved by plasmid-based delivery through ormation . In othebardment . Howevered crops . To overed crops . Upon deed crops .In banana, preassembled RGENs-RNPs targeting viral genes or plant host factors could be coated to gold particles and delivered to banana cell suspension cultures by particle bombardment. Alternatively, the CRISPR/Cas9 constructs targeting the viral genes or plant host factors could transiently be delivered into banana cells through microprojectile bombardment. The complete plants can be regenerated from the bombarded banana cells. The edited plants with the targeted mutations and absence of foreign gene integration should be selected based on the molecular characterization. The virus-resistant genome-edited plants generated using preassembled RGENs-RNPs or transient delivery of CRISPR/Cas9 reagents will not have any foreign gene integration and might not require GMO regulatory approval .The major biosafety concerns with genome-edited crops are unwanted genetic changes in plants due to off-target mutations and transgene integration. The off-target effects can be minimized by improving strategies for designing the gRNA very specific to the target and RNP-based delivery as they are active for a short duration in the host cell and use of inducible CRISPR/Cas system to avoid the strong doses of CRISPR-Cas9 beyond the target cells/tissues and throughout the life span of the plant.Another pressing issue is the lack of high throughput screening methods to identify genome-edited events. Currently, the edited plants of banana are screened using PCR and target sequencing to detect the mutation. However, these methods are expensive and time-consuming. Screening the edited plants using the high-throughput phenotyping for the desired trait, followed by target sequencing of the selected events, will be more efficient and cost-efficient.The challenges with the differences among the countries regarding the regulation of genome-edited products cannot be ignored . The genClustered regularly interspaced short palindromic repeats/CRISPR-associated protein-based genome editing is fast revolutionizing its applications in crop improvement for desired traits such as disease resistance. BBTV and BSV are economically important viruses threatening banana production. The most sustainable way to reduce losses due to these viral diseases is the use of virus-resistant banana varieties. In the absence of any known resistant germplasm, it requires the development of durable virus-resistant varieties using modern biotechnological tools, complementing conventional breeding. Genome editing tools provide a new weapon in the arsenal against plant viruses. The availability of a robust genome-editing system and reference genome make the banana a potential candidate for developing virus-resistant varieties using CRISPR/Cas-based genome editing. So far, genome-editing is applied to banana only for control of BSV by inactivating the eBSV integrated into the host genome. CRISPR/Cas system targeting the essential genes of the virus or host plant genes involved in the susceptibility can be applied in banana to develop resistance against BBTV. Genome-edited virus-resistant banana varieties can be generated with no foreign gene integration, making them more acceptable for commercialization.LT conceived the idea. All authors contributed to writing, reviewing, and editing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.