Dual factors required for cytochrome-P450-mediated hydrocarbon ring contraction in bacterial gibberellin phytohormone biosynthesis.

Cytochromes P450 (CYPs) are heme-thiolate monooxygenases that prototypically catalyze the insertion of oxygen into unactivated C-H bonds but are capable of mediating more complex reactions. One of the most remarked-upon alternative reactions occurs during biosynthesis of the gibberellin A (GA) phytohormones, involving hydrocarbon ring contraction with coupled aldehyde extrusion of ent-kaurenoic acid to form the first gibberellin intermediate. While the unusual nature of this reaction has long been noted, its mechanistic basis has remained opaque. Building on identification of the relevant CYP114 from bacterial GA biosynthesis, detailed structure-function studies are reported here, including development of in vitro assays as well as crystallographic analyses both in the absence and presence of substrate. These structures provided insight into enzymatic catalysis of this unusual reaction, as exemplified by identification of a key role for the "missing" acid from an otherwise highly conserved acid-alcohol pair of residues. Notably, the results demonstrate that ring contraction requires dual factors, both the use of a dedicated ferredoxin and absence of the otherwise conserved acidic residue, with exclusion of either limiting turnover to just the initiating and more straightforward hydroxylation. The results provide detailed insight into the enzymatic structure-function relationships underlying this fascinating reaction and support the use of a semipinacol mechanism for the unusual ring contraction reaction.

GmSABP2-1 encodes methyl salicylate esterase and functions in soybean defense against soybean cyst nematode.

KEY MESSAGE: The soybean gene GmSABP2-1 encodes methyl salicylate esterase and its overexpression led to significant reduction in development of pathogenic soybean cyst nematode. Soybean cyst nematode (SCN, Heterodera glycines) is one of the most devastating pests of soybean (Glycine max L. Merr.). In searching for SCN-defense genes, a soybean gene of the methylesterase (MES) family was found to be upregulated in an SCN-resistant soybean line and downregulated in an SCN-susceptible line upon SCN infection. This gene was designated as GmSABP2-1. Here, we report on biochemical and overexpression studies of GmSABP2-1 to examine its possible function in SCN resistance. The protein encoded by GmSABP2-1 is closely related to known methyl salicylate esterases. To determine the biochemical function of GmSABP2-1, a full-length cDNA of GmSABP2-1 was cloned into a protein expression vector and expressed in Escherichia coli. The resulting recombinant GmSABP2-1 was demonstrated to catalyze the demethylation of methyl salicylate. The biochemical properties of GmSABP2-1 were determined. Its apparent Km value was 46.2 ± 2.2 µM for methyl salicylate, comparable to those of the known methyl salicylate esterases. To explore the biological significance of GmSABP2-1 in soybean defense against SCN, we first overexpressed GmSABP2-1 in transgenic hairy roots of an SCN-susceptible soybean line. When infected with SCN, GmSABP2-1-overexpressing hairy roots showed 84.5% reduction in the development of SCN beyond J2 stage. To provide further genetic evidence for the role of GmSABP2-1 in SCN resistance, stable transgenic soybean plants overexpressing GmSABP2-1 were produced. Analysis of the GmSABP2-1-overexpressing lines showed a significant reduction in SCN development compared to non-transgenic plants. In conclusion, we demonstrated that GmSABP2-1 encodes methyl salicylate esterase and functions as a resistance-related gene against SCN.

Emergency paediatric medicine consultation-a practical guide to a consultation with refugee and asylum-seeking children within the paediatric emergency department.

There are increasing numbers of refugee and asylum-seeking children entering the UK annually who face significant barriers to accessing healthcare services. Clinicians working in the emergency department should have an awareness of the journeys children may have taken and the barriers they face in accessing care and have a holistic approach to care provision. We conducted a narrative literature review and used experiential knowledge of paediatricians working in the Paediatric Emergency Department to formulate a step-by-step screening tool. We have formulated a step-by-step screening tool, CCHILDS (Communication, Communicable diseases, Health-physical and mental, Immunisation, Look after (safeguarding), Deficiencies, Sexual health) which can be used by healthcare professionals in the emergency department. CONCLUSION: Due to increasing numbers of refugee and asylum-seeking children, it is important that every point of contact with healthcare professionals is an impactful one on their health, well-being and development. Future work would include validation of our tool. WHAT IS KNOWN: •The number of refugees globally are rapidly increasing, leading to an increase in the number of presentations to the PED. These patients are often medically complex and may have unique and sometimes unexpected presentations that could be attributed to by their past. There are a multitude of resources available outlining guidance on the assessment and management of refugee children. WHAT IS NEW: •This review aims to succinctly summarise the guidance surrounding the assessment of refugee children presenting to the PED and ensure that healthcare professionals are aware of the pertinent information regarding this cohort. It introduces the CCHILDS assessment tool which has been formulated through a narrative review of the literature and acts as a mnemonic to aid professionals in their assessment of refugee children in the PED.

The Use of Ketamine Sedation for the Treatment of Nail Bed Repairs in the Pediatric Emergency Department.

AIM: This study aimed to determine if sedation with ketamine is safe and effective for the treatment of nail bed injuries in the pediatric emergency department (PED). METHOD: A retrospective cohort study was carried out during a 9-month period in children aged between 18 months and 15 years, presenting to PED requiring nail bed repair. We documented complications of sedation, clinical outcome of the repair both immediate and at follow-up, and parental satisfaction at 4 months. A cost analysis was also undertaken. RESULTS: Ten repairs were performed. There were no serious adverse events. The average satisfaction score was 9.4/10. All patients were discharged from follow-up by 3 months. There was a cost saving of approximately £1500 per case. CONCLUSIONS: We have demonstrated nail bed injury repair facilitated by sedation with ketamine to be safe, effective, and cost efficient in the PED. This management strategy, brought to the fore during the COVID-19 pandemic, should be adopted widely in PEDs.

Rational design and testing of abiotic stress-inducible synthetic promoters from poplar cis-regulatory elements.

Abiotic stress resistance traits may be especially crucial for sustainable production of bioenergy tree crops. Here, we show the performance of a set of rationally designed osmotic-related and salt stress-inducible synthetic promoters for use in hybrid poplar. De novo motif-detecting algorithms yielded 30 water-deficit (SD) and 34 salt stress (SS) candidate DNA motifs from relevant poplar transcriptomes. We selected three conserved water-deficit stress motifs (SD18, SD13 and SD9) found in 16 co-expressed gene promoters, and we discovered a well-conserved motif for salt response (SS16). We characterized several native poplar stress-inducible promoters to enable comparisons with our synthetic promoters. Fifteen synthetic promoters were designed using various SD and SS subdomains, in which heptameric repeats of five-to-eight subdomain bases were fused to a common core promoter downstream, which, in turn, drove a green fluorescent protein (GFP) gene for reporter assays. These 15 synthetic promoters were screened by transient expression assays in poplar leaf mesophyll protoplasts and agroinfiltrated Nicotiana benthamiana leaves under osmotic stress conditions. Twelve synthetic promoters were induced in transient expression assays with a GFP readout. Of these, five promoters (SD18-1, SD9-2, SS16-1, SS16-2 and SS16-3) endowed higher inducibility under osmotic stress conditions than native promoters. These five synthetic promoters were stably transformed into Arabidopsis thaliana to study inducibility in whole plants. Herein, SD18-1 and SD9-2 were induced by water-deficit stress, whereas SS16-1, SS16-2 and SS16-3 were induced by salt stress. The synthetic biology design pipeline resulted in five synthetic promoters that outperformed endogenous promoters in transgenic plants.

Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment.

Previous work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.

KEY MESSAGE: A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

Presentation and Investigation of Pediatric Bone and Joint Infections in the Pediatric Emergency Department.

OBJECTIVE: The objective of this study was to evaluate the presenting features of bone and joint infections with a view to identify distinguishing trends that will be useful for pediatric emergency departments. METHODS: We performed a retrospective review of patient records over a 12-year period in the pediatric emergency department of a large regional pediatric teaching center serving a diverse population. RESULTS: There were 88 cases of osteoarticular infections during the study period. Pain, fever, and impaired function were commonly reported, but overall, there was inconsistency in the presenting features. Inflammatory makers were sensitive tools, particularly in combination. When C-reactive protein, total white cell count, and erythrocyte sedimentation rate were all abnormal, 98% of bone and joint infections were identified.Causative organisms were identified in only 38% of cases, mostly from cultures of synovial fluid and bone. Streptococcal organisms were significantly more likely to be isolated in children under 5 years than in children over 5 years (P = <0.014). Staphylococcal organisms were significantly more likely to be isolated in children over 5 years than in children under 5 years (P = <0.001). Identification of virulent organisms such as Panton-Valentine leukocidin Staphylococcus aureus and methicillin-resistant S. aureus in our study should prompt review of diagnostic techniques and antibiotic choices. CONCLUSIONS: Overall, children under 5 years were significantly more likely to be diagnosed with septic arthritis than osteomyelitis (P = 0. 006). Children over 12 years were significantly more likely to be diagnosed with osteomyelitis than septic arthritis (P = 0. 019). These trends are useful to consider at presentation and in cases of diagnostic uncertainty.

An (E,E)-α-farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect-induced volatiles.

Plant terpene synthase genes (TPSs) have roles in diverse biological processes. Here, we report the functional characterization of one member of the soybean TPS gene family, which was designated GmAFS. Recombinant GmAFS produced in Escherichia coli catalysed the formation of a sesquiterpene (E,E)-α-farnesene. GmAFS is closely related to (E,E)-α-farnesene synthase gene from apple, both phylogenetically and structurally. GmAFS was further investigated for its biological role in defence against nematodes and insects. Soybean cyst nematode (SCN) is the most important pathogen of soybean. The expression of GmAFS in a SCN-resistant soybean was significantly induced by SCN infection compared with the control, whereas its expression in a SCN-susceptible soybean was not changed by SCN infection. Transgenic hairy roots overexpressing GmAFS under the control of the CaMV 35S promoter were generated in an SCN-susceptible soybean line. The transgenic lines showed significantly higher resistance to SCN, which indicates that GmAFS contributes to the resistance of soybean to SCN. In soybean leaves, the expression of GmAFS was found to be induced by Tetranychus urticae (two-spotted spider mites). Exogenous application of methyl jasmonate to soybean plants also induced the expression of GmAFS in leaves. Using headspace collection combined with gas chromatography-mass spectrometry analysis, soybean plants that were infested with T. urticae were shown to emit a mixture of volatiles with (E,E)-α-farnesene as one of the most abundant constituents. In summary, this study showed that GmAFS has defence roles in both below-ground and above-ground organs of soybean against nematodes and insects, respectively.

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2-year comparative analysis of field-grown lines modified for target gene or genetic element expression.

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4-KD, miRNA156-OE, MYB4-OE, COMT-KD and FPGS-KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second- versus the first-year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second-year growth of transgenics targeted for wall modification, GAUT4-KD, MYB4-OE, COMT-KD and FPGS-KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next-generation bio-feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.

Multiple polyploidy events in the early radiation of nodulating and nonnodulating legumes.

Unresolved questions about evolution of the large and diverse legume family include the timing of polyploidy (whole-genome duplication; WGDs) relative to the origin of the major lineages within the Fabaceae and to the origin of symbiotic nitrogen fixation. Previous work has established that a WGD affects most lineages in the Papilionoideae and occurred sometime after the divergence of the papilionoid and mimosoid clades, but the exact timing has been unknown. The history of WGD has also not been established for legume lineages outside the Papilionoideae. We investigated the presence and timing of WGDs in the legumes by querying thousands of phylogenetic trees constructed from transcriptome and genome data from 20 diverse legumes and 17 outgroup species. The timing of duplications in the gene trees indicates that the papilionoid WGD occurred in the common ancestor of all papilionoids. The earliest diverging lineages of the Papilionoideae include both nodulating taxa, such as the genistoids (e.g., lupin), dalbergioids (e.g., peanut), phaseoloids (e.g., beans), and galegoids (=Hologalegina, e.g., clovers), and clades with nonnodulating taxa including Xanthocercis and Cladrastis (evaluated in this study). We also found evidence for several independent WGDs near the base of other major legume lineages, including the Mimosoideae-Cassiinae-Caesalpinieae (MCC), Detarieae, and Cercideae clades. Nodulation is found in the MCC and papilionoid clades, both of which experienced ancestral WGDs. However, there are numerous nonnodulating lineages in both clades, making it unclear whether the phylogenetic distribution of nodulation is due to independent gains or a single origin followed by multiple losses.

Engineered selective plant male sterility through pollen-specific expression of the EcoRI restriction endonuclease.

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen-specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible-to-no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand-crossed to both male-sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000-40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male-sterile tobacco, and 900-2100 seeds per male-sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI-driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.

Transgenic soybean overexpressing GmSAMT1 exhibits resistance to multiple-HG types of soybean cyst nematode Heterodera glycines.

Soybean (Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyses the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heterodera glycines Ichinohe. In this study, we produced transgenic soybean overexpressing GmSAMT1 and characterized their response to various SCN races. Transgenic plants conferred a significant reduction in the development of SCN HG type 1.2.5.7 (race 2), HG type 0 (race 3) and HG type 2.5.7 (race 5). Among transgenic lines, GmSAMT1 expression in roots was positively associated with SCN resistance. In some transgenic lines, there was a significant decrease in salicylic acid titer relative to control plants. No significant seed yield differences were observed between transgenics and control soybean plants grown in one greenhouse with 22 °C day/night temperature, whereas transgenic soybean had higher yield than controls grown a warmer greenhouse (27 °C day/23 °C night) temperature. In a 1-year field experiment in Knoxville, TN, there was no significant difference in seed yield between the transgenic and nontransgenic soybean under conditions with negligible SCN infection. We hypothesize that GmSAMT1 expression affects salicylic acid biosynthesis, which, in turn, attenuates SCN development, without negative consequences to soybean yield or other morphological traits. Thus, we conclude that GmSAMT1 overexpression confers broad resistance to multiple SCN races, which would be potentially applicable to commercial production.

Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis.

The tropane and granatane alkaloids belong to the larger pyrroline and piperidine classes of plant alkaloids, respectively. Their core structures share common moieties and their scattered distribution among angiosperms suggest that their biosynthesis may share common ancestry in some orders, while they may be independently derived in others. Tropane and granatane alkaloid diversity arises from the myriad modifications occurring to their core ring structures. Throughout much of human history, humans have cultivated tropane- and granatane-producing plants for their medicinal properties. This manuscript will discuss the diversity of their biological and ecological roles as well as what is known about the structural genes and enzymes responsible for their biosynthesis. In addition, modern approaches to producing some pharmaceutically important tropanes via metabolic engineering endeavors are discussed.

Manipulating microRNAs for improved biomass and biofuels from plant feedstocks.

Petroleum-based fuels are nonrenewable and unsustainable. Renewable sources of energy, such as lignocellulosic biofuels and plant metabolite-based drop-in fuels, can offset fossil fuel use and reverse environmental degradation through carbon sequestration. Despite these benefits, the lignocellulosic biofuels industry still faces many challenges, including the availability of economically viable crop plants. Cell wall recalcitrance is a major economic barrier for lignocellulosic biofuels production from biomass crops. Sustainability and biomass yield are two additional, yet interrelated, foci for biomass crop improvement. Many scientists are searching for solutions to these problems within biomass crop genomes. MicroRNAs (miRNAs) are involved in almost all biological and metabolic process in plants including plant development, cell wall biosynthesis and plant stress responses. Because of the broad functions of their targets (e.g. auxin response factors), the alteration of plant miRNA expression often results in pleiotropic effects. A specific miRNA usually regulates a biologically relevant bioenergy trait. For example, relatively low miR156 overexpression leads to a transgenic feedstock with enhanced biomass and decreased recalcitrance. miRNAs have been overexpressed in dedicated bioenergy feedstocks such as poplar and switchgrass yielding promising results for lignin reduction, increased plant biomass, the timing of flowering and response to harsh environments. In this review, we present the status of miRNA-related research in several major biofuel crops and relevant model plants. We critically assess published research and suggest next steps for miRNA manipulation in feedstocks for increased biomass and sustainability for biofuels and bioproducts.

Identification and overexpression of gibberellin 2-oxidase (GA2ox) in switchgrass (Panicum virgatum L.) for improved plant architecture and reduced biomass recalcitrance.

Gibberellin 2-oxidases (GA2oxs) are a group of 2-oxoglutarate-dependent dioxygenases that catalyse the deactivation of bioactive GA or its precursors through 2ß-hydroxylation reaction. In this study, putatively novel switchgrass C20 GA2ox genes were identified with the aim of genetically engineering switchgrass for improved architecture and reduced biomass recalcitrance for biofuel. Three C20 GA2ox genes showed differential regulation patterns among tissues including roots, seedlings and reproductive parts. Using a transgenic approach, we showed that overexpression of two C20 GA2ox genes, that is PvGA2ox5 and PvGA2ox9, resulted in characteristic GA-deficient phenotypes with dark-green leaves and modified plant architecture. The changes in plant morphology appeared to be associated with GA2ox transcript abundance. Exogenous application of GA rescued the GA-deficient phenotypes in transgenic lines. Transgenic semi-dwarf lines displayed increased tillering and reduced lignin content, and the syringyl/guaiacyl lignin monomer ratio accompanied by the reduced expression of lignin biosynthetic genes compared to nontransgenic plants. A moderate increase in the level of glucose release in these transgenic lines might be attributed to reduced biomass recalcitrance as a result of reduced lignin content and lignin composition. Our results suggest that overexpression of GA2ox genes in switchgrass is a feasible strategy to improve plant architecture and reduce biomass recalcitrance for biofuel.

Noninvasive in vivo optical detection of biofilm in the human middle ear.

Otitis media (OM), a middle-ear infection, is the most common childhood illness treated by pediatricians. If inadequately treated, OM can result in long-term chronic problems persisting into adulthood. Children with chronic OM or recurrent OM often have conductive hearing loss and communication difficulties and require surgical treatment. Tympanostomy tube insertion, the placement of a small drainage tube in the tympanic membrane (TM), is the most common surgical procedure performed in children under general anesthesia. Recent clinical studies have shown evidence of a direct correspondence between chronic OM and the presence of a bacterial biofilm within the middle ear. Biofilms are typically very thin and cannot be recognized using a regular otoscope. Here we report the use of optical coherent ranging techniques to noninvasively assess the middle ear to detect and quantify biofilm microstructure. This study involves adults with chronic OM, which is generally accepted as a biofilm-related disease. Based on more than 18,537 optical ranging scans and 742 images from 13 clinically infected patients and 7 normal controls using clinical findings as the gold standard, all middle ears with chronic OM showed evidence of biofilms, and all normal ears did not. Information on the presence of a biofilm, along with its structure and response to antibiotic treatment, will not only provide a better fundamental understanding of biofilm formation, growth, and eradication in the middle ear, but also may provide much-needed quantifiable data to enable early detection and quantitative longitudinal treatment monitoring of middle-ear biofilms responsible for chronic OM.