Data collected during May–October, 2013 from four salt marshes in Waquoit Bay and adjacent estuary, Massachusetts, USA were utilized to evaluate the theoretically-derived dimensionless flux and various ecological driver numbers. Two meaningful dimensionless groups were discovered as the light use efficiency number  and the biogeochemical number . A semi-logarithmic plot of the dimensionless numbers indicated the emergence of a characteristic diagram represented by three distinct LUE regimes . The high regime corresponded to the most favorable  condition for CO2 uptake, whereas the low regime represented an unfavorable condition . The analysis identified two environmental thresholds , which dictated the regime transitions of CO2 uptake. The process diagram and critical thresholds provide important insights into the CO2 uptake potential of coastal wetlands in response to changes in key environmental drivers.We investigated the ecological parameter reductions (termed “similitudes”) and characteristic patterns of the net uptake fluxes of carbon dioxide (CO Understanding of the wetland carbon dioxide (CO2) exchange processes is pivotal to the ecological stability because of the vulnerability of wetland ecosystems to global environmental change4. The magnitude and variability of CO2 in coastal wetlands depend on their complex nonlinear interplay with the major environmental drivers 7. However, it remains unexplored whether the primary drivers of wetland CO2 fluxes can be grouped into a reduced set of emergent generalizable entities that represent the coastal wetland productivity potentials in response to a changing environment. Therefore, a fundamental scientific question is whether we can identify ecological parametric reductions, known as “similitudes” in the domain of fluid mechanics and hydraulic engineering, describing environmental regimes of salt marsh CO2 uptake. Do the salt marsh CO2 fluxes represent any characteristic process path  with the covarying environmental drivers? Are there any critical environmental thresholds, indicating shifts in the process regimes of CO2 fluxes? Answers to these scientific questions using similitude and dimensional analysis from fluid mechanics and hydraulic engineering may offer new insights into the underlying mechanistic principles and into the development of generalized predictive models of salt marsh CO2 fluxes in space and time.Coastal wetlands are among the most potent carbon sinks on earth, providing valuable ecosystem services such as global warming mitigation, storm protection, erosion control, and carbon sequestration2 during daytime due to photosynthesis and source during nighttime because of respiration8. There are multitudes of primary process drivers of salt marsh CO2 exchanges with the atmosphere. Light is the main driver of net CO2 uptake, whereas temperature mediates the photosynthesis process9. The temperature control on CO2 uptake is intertwined with the activity of the primary photosynthesis enzyme, RuBisCO—which has a positive linkage with ambient temperature10. In contrast, salinity significantly reduces the CO2 uptake rates and overall productivity in tidal wetlands by influencing the plants’ physiological responses to available light12. Usually, salt marsh plants are resilient to high salinity; however, previous studies reported a considerable negative impact of salinity on the productivity of the salt marshes14. A high saline condition favors the accumulation of toxic hydrogen sulfide in anaerobic wetland soils—which adversely influences the salt marsh productivity15. Furthermore, high salinity reduces the nutrient uptake  of the marshes, and thereby decreases the productivity16.Wetlands are, in general, net sink of CO2 uptake rates are higher during the high tides than the low tides, because salt accumulation during the low tides negatively impacts the productivity17. Abdul-Aziz et al.7 carried a comprehensive study to determine the major drivers of daytime CO2 uptake in Waquoit Bay and adjacent estuaries, MA, USA using a systematic data analytics and empirical modeling approach. The study reported a substantially higher control and linkage of photosynthetically active radiation, soil temperature, and porewater salinity on instantaneous CO2 uptake fluxes than that of pH, well water level, and soil moisture. Further, Abdul-Aziz et al.7 did not find any significant control of nitrogen loading gradient (5–126 kg/ha/year) on the CO2 uptake fluxes across the study marshes; however, the authors reported significantly higher CO2 uptake fluxes during high tides than the low tides.Typically, salt marsh CO18. As the Π numbers represent different factors pertinent to the process, the fundamental description of the system defined by the associated process variables remains unchanged. Dimensional analysis using Buckingham pi-theorem is a useful method to decrease dimensions in data, reduce the number of parameters, and achieve similitude. The method is particularly useful in systems where simple governing equations cannot be used to represent the primary processes19.A crucial advantage of identifying similitudes and dimensionless process groups is that information and underlying relationships and patterns can be generalized by utilizing data from different sites representing different gradients of response and predictor variables. In general, similitude represents the parametric reduction of a physical system by developing independent dimensionless groups or pi (Π) numbers, which functionally defines the mechanistic process of the system21. Here, the term “process diagram” refers to a graphical representation of different process states that a system follows in transition from one balanced condition to another. In “Moody Diagram”, seven original process variables of pipe flow were reduced to three process-based dimensionless groups using dimensional analysis, representing pipe friction and roughness across laminar, transitional, and turbulent flow regimes. Similarly, six process variables in rivers were used to formulate two meaningful dimensionless groups in “Shields diagram”, indicating different states of sediment transport across smooth, transitional, and rough flow regimes. Many studies in ecology, environmental sciences, and engineering utilized the concept of parametric reductions and dimensional analysis for process understanding and modeling30. West et al.22 employed parametric reductions and scaling to derive a single universal curve to describe the growth rate of diverse species. Warnaars et al.24 applied dimensional analysis for parametric reductions in stream biogeochemistry, and developed useful scaling relationships between dimensionless biotic and abiotic factors in various streams across North America.Classical examples of similitudes and dimensional analysis that led to the development of generalized process diagrams and scaling relationships include “Moody diagram” in fluid mechanics and “Shields diagram” in sediment transport2 uptake fluxes in coastal salt marshes follow emergent ecological parameter reductions  and distinct environmental regimes. The hypothesis was evaluated by formulating meaningful dimensionless numbers and defining different process regimes of salt marsh CO2 uptake fluxes, leading to a characteristics process diagram. The field data collected from four salt marshes on the southern shore of Cape Cod, MA, USA were used to examine the hypothesis.This study tests a fundamental hypothesis that the net CO32. On the basis of nitrogen loading influx, SL is in relatively pristine condition (~ 5 kg/ha/year), whereas HP (~ 29 kg/ha/year), EP (~ 63 kg/ha/year), and GP (~ 126 kg/ha/year) represent a medium to high nitrogen loading33. The vegetation community of the marshes is mostly dominated by Spartina alterniflora (a C4 plant) in the low marsh zone.Four salt marshes located in Waquoit Bay and adjacent estuaries at Cape Cod, MA, USA were used as the case study sites: 1) Sage Lot Pond (SL), (2) Eel Pond (EP), (3) Great Pond (GP), and (4) Hamblin Pond (HP)  of CO2 were made using a cavity ring-down spectrometer (CRDS) gas-analyzer  for different days during the extended growing season (May to October) in 2013 at the low marsh zones of the four salt marshes. The spectrometer analyzer was connected to the transparent, closed acrylic chamber (60 cm × 60 cm × 60 cm) through tubes. We calculated the molar concentrations of CO2 in the chamber using the ideal gas law. The instantaneous molar concentrations of CO2 were then linearly regressed with time (s). The regression slopes  were normalized by the chamber area  to compute the corresponding fluxes of CO2  between the wetland soil and the atmosphere inside the chamber for each sampling period 7. To avoid impacts of any experimental error, a coefficient of determination (R2) of 0.90 was set as the minimum threshold for the regression to qualify the computed CO2 fluxes as accurate and acceptable for analyses7.A comprehensive detail on the collections and processing of gas fluxes and environmental variables for the four salt marshes were presented in Abdul-Aziz et al.2 fluxes is a widely-used method in the carbon research domain37. The technique provides an effective way to measure surface-atmospheric gas fluxes. As demonstrated above, the method first involves the calculation of the gradient of molar concentrations of CO2 in time, which is then divided by the chamber area to compute the vertical CO2 fluxes. Since the measurement chamber is small  and enclosed, the vertical fluxes of CO2 between soil and atmosphere (and not the divergence of CO2 fluxes) drives the changes in CO2 concentrations with time inside the chamber. Therefore, the chamber area-normalized rates of changes in CO2 molar concentrations represent the vertical CO2 gas fluxes between the soil and atmosphere inside the chamber.The employed enclosed chamber-based technique of measuring CO7. The corresponding observations of atmospheric pressure (Pa) were collected from the nearby NOAA National Estuarine Research Reserve System (NOAA-NERRS) monitoring station located at Carriage House, MA38. The filtered daytime net uptake fluxes of CO2  represented the measurements made between 8 a.m. and 4.30 p.m. , with the corresponding PAR higher than 1.5 µmole/m2/s. AT was used to calculate the fluxes of NEECO2,uptake using the ideal gas law7; AT was, therefore, excluded as an environmental driver from further analyses. Instead, soil temperature (ST) was considered to represent the impact of temperature on NEECO2,uptake. The dataset included 137 observational panels from the four study wetlands for 25 sampling days , air temperature (AT), soil temperature (ST), and porewater salinity (SS) were concurrently measured20. According to the pi theorem, a combination of CO2,uptake, PAR, ST, SS, Pa, and the time-scale of measurement or estimation (t) were used for the dimensional analysis. PAR, ST, SS were the most dominant drivers of NEECO2,uptake, as identified in the study of Abdul-Aziz et al.7. Furthermore, Pa negatively correlates with net photosynthesis as stomatal conductance increases with decreasing pressure39. The selected variables for the dimensional analysis included four fundamental dimensions  (Table p = 1.48 kJ/kg/K) was further incorporated in the dimensional analysis to normalize ST. Following the pi theorem, a functional relationship  and the potential predictors was expressed as follows:Dimensional analysis using Buckingham pi , whereas Therefore, dimensional analysis reduced the 7 original variables to 2–3 dimensionless numbers. Recalling the definition of similitude from the physical domain2 flux number) were expressed as a general function  among the environmental driver numbers in a multivariate space, identifying the relatively unbiased information on their individual linkages with the response41. To incorporate any non-linearity in the data matrix, observed  values of all pi numbers were log10-transformed, which were further standardized  as follows: 10-transformed pi number, The multivariate method of principal component analysis (PCA) was applied to the observational dataset from the salt marshes of Waquoit Bay to identify the important environmental driver number(s) that had dominant linkage(s) with the response pi number2 uptake relative to the available sunlight (PAR)44. Overall, the first two principal components (PCs) explained 90% of total variance in the dataset when PCA was performed using all unique dimensionless numbers  or low  regime . Overall, the developed curve indicated that CO2 uptake relative to light in coastal wetlands was characterized by the corresponding variability in temperature and salinity, and the interactions thereof. The shift from high to transitional regime was determined by the BGC value of 0.13 , LUE dropped sharply with the decrease of BGC values—forming the multivalue-based “loop” shape of the curve.Semi-logarithmic plot of the dimensionless LUE group as a function of the dimensionless BGC group showed a collapse of observed data from the four salt marshes into a unique dimensionless curve—indicating an emergence of similitude-based characteristic process diagram Fig. . Two disme Table . SimilarCO2,uptake and PAR varied within a wide range indicating their high variability, although the range of 2/s)46, the CO2 uptake , and low to high salinity 10–30 ppt) Table . The ass0 ppt Tab2 uptake followed the high regime when the temperature was approximately equal to or higher than 17 °C and salinity was lower or equal to 30 ppt (favorable condition for CO2 uptake). In contrast, the low LUE regime was subject to low temperature and high salinity (unfavorable condition for CO2 uptake)  relative to the absorbed PAR43. However, instead of NPP to PAR ratio, we defined LUE as the ratio of NEECO2,uptake to PAR in this paper. NEECO2,uptake includes gross primary productivity, and autotrophic as well as heterotrophic respirations during daytime hours. There is not much research available on the LUE of wetland plants, particularly for salt marshes. Overall, C4 plants such as Spartina sp. generally exhibit high photosynthetic efficiency relative to the C3 species47. For example, Jiang et al.48 reported a higher light utilization by the Spartina sp. plants than the C3 plants  in the Yangtze River estuary, China. The comparatively higher LUE of C4 plants stems from their ability to capture light for an extended growing period, higher uptake rates, and favorable physiology and biochemistry49. Previous studies indicated a substantial influence of climatic, physiological, and biogeochemical factors  on LUE for different species across ecosystems. Our study of the salt marsh CO2 fluxes and the corresponding LUE corroborates the finding of existing literature that LUE is characteristically linked with the dynamics of the BGC number . The impact of BGC group on the response LUE, which varied from approximately 0.000009 to 0.01, facilitated the identification of three underlying governing regimes  and emergence of a characteristics process diagram of the salt marsh CO10. Sage and Kubien10 also reported the role of photosynthesis enzyme, RuBisCo as a limiting factor on the CO2 fixation of C4 plants in a comparatively cool environment . The turnover rate of RuBisCo generally increases with temperature during daytime hours, and the rate corresponds to the release of RuBP  that facilitates the CO2 fixation50. Therefore, the low temperature in low LUE regime could represent the activation state of RuBisCo, where photosynthesis is extremely limited although the range of PAR in this regime was high enough to initiate a photosynthetic response  of 25.6 °C, which was obtained by developing a linear regression model of AT as a function of ST , the uptake efficiency of the marshes decreased in the transitional regime, although the range of temperature in the transitional regime was comparable to the high LUE regime  emissions56. Overall, the high LUE regime corresponds to the “most favorable” climatic-biogeochemical condition  of CO2 uptake, whereas the low LUE regime corresponds to the “least favorable” condition  of CO2 uptake. In contrast, the transitional regime represents relatively favorable climatic condition (relatively high temperature) but unfavorable biogeochemical condition  for CO2 uptake.The observed reduction of CO2 fixation efficiency in Spartina sp. marshes can also be low because of light saturation45. However, NEECO2,uptake was observed to have a linear increasing trend with PAR  fluxes . However, application of similitude and dimensional analysis requires a proper understanding of the underlying mechanisms and processes. Successful outcomes from the Buckingham pi theorem largely depend on an accurate selection of major processes and relevant variables. Involving too few or too many variables in the analysis would lead to misleading results. Further, the most meaningful outcomes  demand an exploration of all possible combinations of repeating and non-repeating variables in the analysis. However, once an appropriate and mechanistically meaningful set of dimensionless numbers such as LUE and BGC are identified through parametric reductions, these can indicate different environmental  regimes of the targeted GHG fluxes. For example, the process diagram of CO2 fluxes provided an overall understanding of the ecological health and carbon storage potential of the salt marshes in Cape Cod, MA. The information could be leveraged to spatially map coastal wetlands with a lower LUE  and distinct environmental regimes by using dimensional analysis. The research provided new insights into the first principles of wetland CO2 processes. Two meaningful and important dimensionless numbers  were identified based on an application of the Buckingham pi theorem. A semi-logarithmic plot of the dimensionless numbers demonstrated the emergence of a process diagram, which was characterized by three governing light use efficiency (LUE) regimes . Subject to the appropriate temperature and salinity conditions  suitable for photosynthesis, the high LUE regime favors the most efficient utilization of available sunlight by wetland plants. In contrast, the CO2 uptake rate relative to light was very low in the low LUE regime because of unfavorable ambient environmental conditions. Based on the characteristic process diagram, two critical environmental thresholds were identified  to indicate regime transitions and wetland productivity.The study successfully tested the hypothesis that the net uptake fluxes of CO2 uptake efficiency. The temperature and salinity thresholds render valuable knowledge of salt marsh productivity and resiliency in response to changes in key environmental drivers. The study also highlights the importance of developing environmental regime-specific predictive models of CO2 fluxes, compared to wetland-specific models, to minimize the site-specific model calibrations and uncertainty. The study can be considered a pioneering step in characterizing similitude-based emergent patterns and in identifying important process regimes of CO2 and other GHG fluxes in coastal salt marshes.The presented characteristic process diagram and environmental regimes would aid a targeted and efficient monitoring and management of tidal wetlands based on their COSupplementary Information.


To describe ultrasonography as a diagnostic method of in vivo Descemet's membrane detachment (DMD) in horses. Animals studied: Seven horses , presenting with moderate‐to‐severe focal or diffuse corneal edema, in whom DMD was suspected on ultrasonographic examination and confirmed with histopathology, were studied.A retrospective analysis of case records of horses with suspected DMD was performed.Median age at presentation was 14 years (range 11‐24). Clinical signs in eyes with DMD were unilateral in all horses and included blepharospasm and epiphora (6/7), buphthalmos (5/7), moderate‐to‐severe focal or diffuse corneal edema (7/7), corneal epithelial bullae (4/7), corneal neovascularization (4/7), Haab's striae (2/7), corneal endothelial precipitates (1/7), fibrin in the anterior chamber (1/7), focal cataract (2/7), and pigment deposits on the anterior lens capsule (1/7). During transpalpebral ultrasonography, a distinct linear echogenic structure was noted in the anterior chamber, initially diverging from, and later running parallel to, the posterior lining of the cornea in all eyes studied. In all cases, the cornea was severely thickened and echogenic, consistent with edema, and DMD was suspected. In all horses, the clinical signs progressed and the affected eye was eventually enucleated. Histopathology revealed DMD (7/7), spindle cell proliferation (4/7), Descemet's membrane reformation (3/7), and inflammation of the anterior uvea (5/7). Overall incidence was 1.04%.Ultrasonography is an adequate tool in diagnosing DMD in horses. Descemet's membrane detachment should be included in the differential diagnosis in horses with dense focal or diffuse corneal edema. It is a homogenous acellular membrane containing various types of collagen but mainly Types III, IV, and VIII.Several diagnostic modalities for visualization of a DMD have been documented in human literature. In clear corneas, DMD can be observed with slit‐lamp biomicroscopy and gonioscopy.22.1Case records of horses that were presented to the Department of Equine Sciences of the Faculty of Veterinary Medicine, Utrecht University, The Netherlands, between January 2012 and August 2018, with suspected DMD on ultrasonography were reviewed. Only horses with confirmed DMD on histopathology were included in the present study. Breed, age at onset, clinical history, ophthalmic examination results, treatment, complications, and clinical and/or surgical outcome were recorded. A search of the database of the Pathology Department was performed using “Descemet's membrane” or “Descemet's membrane detachment” to possibly identify additional cases.2.2® Finland Oy) for all cases except for case 3 at the time of presentation for suspected DMD, fluorescein staining  and direct ophthalmoscopy . If necessary patients were sedated with detomidine hydrochloride , 0.01 mg/kg bodyweight iv ocular hypertension was defined as an increase in intraocular pressure (IOP) above 30 mm Hg.All horses were subjected to a complete ophthalmic examination of both eyes. Ophthalmic examinations were performed by an equine ECVS diplomate , and/or a board‐certified (ECVO) veterinary ophthalmologist , and a resident in veterinary ophthalmology , and included neuro‐ophthalmic testing, hand‐held slit‐lamp biomicroscopy , tonometry  and/or a board‐certified (ECVDI) veterinary radiologist (SV) and included imaging of the eye and retrobulbar area. Both eyes were examined. Standard transpalpebral ultrasonography of the equine globe was performed using a linear transducer in combination with a curvilinear transducer set between 8 and 12 MHz . The frequency applied was dependent on the depth of the ocular structure to be examined. Highest available frequency was applied for the cornea and anterior chamber. Mechanical and thermal indices were ranging between 0.7 and 0.9. If necessary, patients were sedated as described for “ophthalmic examination.”A small overlap exists between the range of reported ultrasonographic axial globe lengths for buphthalmic equine globes The corneal thickness was measured from the surface epithelium until the hyperechoic line representing the endothelial lining of the cornea in both eyes. At the region of the DMD, the corneal thickness was measured from the surface epithelium until the posterior margin of the corneal stroma . Descemet's membrane detachment was defined as the separation of the DM from the posterior corneal stroma in the presence of spindle cell formation and/or flattened endothelial cells and reformation of the DM.2.5The collected data are presented descriptively, with median and ranges included where appropriate. Incidence rates were calculated by dividing the number of new DMD cases per time period, with the total number of primary ophthalmic consultations at our facility in the respective time period.3Nine horses were identified with suspected DMD on ultrasonography, and two cases were excluded from this study as histopathology was not available . During the time of the study, 671 new ophthalmic cases were presented to the Equine Department, making the overall incidence 1.04% (7/671) for all confirmed DMD cases and 1.34% (9/671) for confirmed and suspected cases combined. Higher incidence rates for suspected and confirmed cases combined were seen in 2017  and 2018 .3.1The horses included in this study consisted of four geldings and three mares. The median age of the horses was 14 years (11‐24 years). Breeds included three Icelandic horses, two Dutch Warmblood horses, one Welsh pony, and one Appaloosa.3.2The clinical presentation, including the signalment, of the different cases is summarized in Table Four cases  subsequently developed epithelial bullae formation and became fluorescein positive. These cases had associated ocular discomfort . Case three was previously presented for recurring episodes of anterior uveitis, and case two revealed intraocular sequelae upon ophthalmic examination at our clinic, indicative of previous inflammatory episodes . Case four did not present nor develop signs of ocular discomfort. Corneal cytology was performed in case one and revealed normal epithelial cells and few inflammatory cells. Case seven presented with absent menace responses OU, negative direct pupillary light reflex (PLR) OS and negative consensual PLR from OD to OS , negative dazzle reflexes OU, bilateral anterior lens luxation, buphthalmos, and focal cataract. After consultation, the lens relocated posterior from the iris in the right eye. Only the right eye presented with extensive focal corneal edema, which progressed to dense diffuse corneal edema. There were otherwise no clinical signs of inflammation, and however, further diagnostic testing  was declined by the owner.Measurements of the IOP were available for six of the seven cases. One of the six horses undergoing tonometry presented with ocular hypertension. The IOP for case three was not available at time of presentation of suspected DMD. The horse was previously presented for equine recurrent uveitis, at which point the IOP was 27 mm Hg OU.Cases two and three also showed Haab's striae associated with stretching of the bulbus, and four cases showed perilimbal deep corneal neovascularization (cases 3‐6) suggestive of primary corneal inflammation, intraocular inflammation (uveitis), and/or glaucoma.In cases presented with extensive focal corneal edema  and dense diffuse corneal edema (cases 4 and 5) visualization, the posterior segment was not possible. In case 1, neuro‐ophthalmic examinations were normal and no fundus abnormalities were noted. Case 2 had a normal fundic examination and was visual at initial presentation, but as the edema progressed the pony lost its menace response and the dazzle and consensual pupillary light reflex. The remaining cases (cases 3‐7) had absent consensual PLRs, absent menace responses, and dazzle reflexes.According to the provided medical history, no (intra)ocular surgeries had been performed previously on any of the cases, except for case three. Case three had been treated with a suprachoroidal cyclosporine implant for equine recurrent uveitis a year prior to presentation for suspected DMD.Additional treatment options, such as modified Gundersen inlay flaps, or the use of topical hyperosmotics were discussed with the owners. One case (case 6) was additionally treated with sodium chloride hypertonicity ophthalmic ointment 5% q3h, but did not improve clinically. None of the owners elected surgical intervention. Ultimately, all owners decided to enucleate affected eye because of persistence or progression of the clinical signs despite medical therapy and poor prognosis regarding vision. Because of bilateral blinding disease, the owner of case 7 elected euthanasia. Corneal edema had existed for 6‐163 days prior to enucleation.3.3In all eyes examined, severe corneal thickening (1.9‐3.1 mm) with a diffusely increased echogenicity was noted, consistent with corneal edema. At the time of ultrasonographic examination, the corneal edema was focal in two cases (cases 1 and 2) and diffuses in five cases (cases 3‐7).All cases showed a thin, well‐defined hyperechoic line in the anterior chamber Figure A‐D. ThisAdditional findings in the ultrasonographic examination of the eyes included echogenic regions in the lens consistent with cataract , lens luxation , echogenic lines or particles in the vitreous , and thickened sclera/chorioretina .3.4Figure 4To the authors' knowledge, this is the first report describing ultrasonographic findings of DMD in horses, including confirmation of the diagnosis by histopathology. The findings of this study confirm the importance of an ultrasonographic examination, with special attention to the cornea and anterior chamber, as part of a standard ophthalmological work‐up in equine eyes with corneal edema.The clinical signs described in the present study are in agreement with previous studies concerning DMD in horses,Six cases presented with mild‐to‐moderate blepharospasm and epiphora. We associated these signs with ocular discomfort due to either an underlying primary ocular disease  or, subsequently, due to the bullous keratopathy and corneal ulcerations that resulted from the chronic corneal edema and/or glaucoma.The median age of the horses in this study was 14 years, which is considerably lower than the median age of 22 years in the study by Henriksen et al.The pathogenesis of DMD in horses is presently unknown. Based on previous reports and the findings of our study, there are multiple plausible causes and it is possibly multifactorial in some cases. Reports in human literature support this hypothesis as DMD in humans has not only been associated with various intraocular surgeries,In the present case series, there were two horses with signs of (pre‐existing) anterior uveitis noted upon initial ophthalmic examination. Histopathology confirmed ocular inflammation in these cases and revealed three additional cases with minor‐to‐moderate inflammation in the iris and/or ciliary body. In human literature, pre‐existing endothelial disease was found to be a significant preoperative risk factor associated with DMD after cataract surgery.Although the equine DM is likely to possess elastic properties,Descemet's membrane detachment on histopathologic examinations can be artifactual due to processing  fixation, paraffin embedding, and sectioning) of the enucleated globe. However, when spindle cell proliferation is present near the exposed corneal stroma, it helps in determining a clinical DMD . Spindle cell proliferation was a common finding in our study, however, in contrast to former studies,Limitations of the current study are the retrospective nature of the study design, lack of a control group, and the small sample size. Furthermore, a major limitation of the current study is the lack of detailed posterior segment evaluation. Especially as six of our seven cases lost PLRs and dazzle reflexes, which indicates a loss of retinal function and/or optic neuropathy. All owners from the horses in the present case study, except for the owners from case 1, declined histopathologic examination. However, we decided to send the samples of the remaining cases to our pathology department to see whether our ultrasonographic suspicion of DMD could be confirmed with histopathology. The eyes of horses were too large to encompass all parts of the globe within the paraffin blocks and were therefore hemidissected with only the anterior segments being processed and evaluated. Although ocular hypertension is a major contributing factor in glaucoma, vision can deteriorate with normal IOPs,In conclusion, ultrasonography is confirmed to be a useful and noninvasive diagnostic modality in evaluating the posterior part of the cornea for DMD in equine cases presenting with unilateral diffuse or focal corneal edema. Descemet's membrane detachment is increasingly being recognized and should be considered as a differential diagnosis in horses with dense focal and diffuse corneal edema, especially in otherwise salvageable globes as it has the potential to progress. Further studies are necessary to identify risk factors and treatment options.


Identification of the nucleotide sequences encoding antibiotic resistance elements and determination of their association with antibiotic resistance are critical to improve surveillance and monitor trends in antibiotic resistance. Current methods to study antibiotic resistance in various environments rely on extensive deep sequencing or laborious culturing of fastidious organisms, both of which are heavily time-consuming operations. Identification of the nucleotide sequences encoding antibiotic resistance elements and determination of their association with antibiotic resistance are critical to improve surveillance and monitor trends in antibiotic resistance. Current methods to study antibiotic resistance in various environments rely on extensive deep sequencing or laborious culturing of fastidious organisms, both of which are heavily time-consuming operations. An accurate and sensitive method to identify both rare and common resistance elements in complex metagenomic samples is needed. Referencing the sequences in the Comprehensive Antibiotic Resistance Database, we designed a set of 37,826 probes to specifically target over 2,000 nucleotide sequences associated with antibiotic resistance in clinically relevant bacteria. Testing of this probe set on DNA libraries generated from multidrug-resistant bacteria to selectively capture resistance genes reproducibly produced higher numbers of reads on target at a greater length of coverage than shotgun sequencing. We also identified additional resistance gene sequences from human gut microbiome samples that sequencing alone was not able to detect. Our method to capture the resistome enables a sensitive means of gene detection in diverse environments where genes encoding antibiotic resistance represent less than 0.1% of the metagenome. Antimicrobial resistance (AMR) is one of the most pressing challenges of the 21st century that poses a threat to modern medicine and food security 24. One of5–Profiling the resistomes of bacteria that are culturable is reasonably straightforward using whole-genome sequencing followed by analysis using algorithms, such as the Resistance Gene Identifier (RGI) in the Comprehensive Antibiotic Resistance Database (CARD) . In meta12–17–A more appropriate approach for the identification of resistomes in metagenomes is the use of a probe-and-capture strategy . Using t21–27–Here, we chronicle our targeted method for the analysis of antibiotic resistomes. We based our probe set design on stringently curated AMR gene (ARG) sequences from CARD , tiled across ARG sequences, combined with rigorous analysis to suppress off-target hybridization. This design enables a more cost-effective and sensitive method to sample the known resistance gene landscape . We testn = 37,826) span the protein homolog model of curated ARGs from CARD and represent nucleotide sequences  that are well characterized in the literature. Probes targeting genes for resistance conferred through single point mutations  in chromosomal metabolic genes (including DNA gyrase [gyrA] mutations associated with fluoroquinolone resistance and RNA polymerase subunit [rpoB] mutations associated with rifampin resistance) were purposefully not included in our design. Of the genes targeted by our probes, 78.03% mirrored the breakdown in CARD, dominated by genes encoding antibiotic inactivation mechanisms and by genes encoding the beta-lactamases  target a single gene, and the remainder target multiple genes ranging up to a maximum of 211 genes  due to sequence conservation within gene families . For example, a single probe initially designed to target 80 nucleotides of the beta-lactamase gene blaSHV-52 also targets an additional 208 genes, including other members of the SHV, LEN, and OKP-A/-B beta-lactamases, due to homology between these nucleotide sequences within AMR gene families. The combination of overlap in the utility of some 80-mer probes and partial hybridization can allow probes to target sequences that are divergent from their reference sequences and thus identify new alleles at the SNP level up to 15% divergence.A set of 80-mer nucleotide probes was custom designed and synthesized through the use of the myBaits platform . The probes  are among the genes with the highest probe coverage. The majority of genes  have greater than 80% length coverage by probes, 26 genes have less than 50% length coverage by probes , and only 1  has less than 5% length coverage by probes . Only 28 sequences from CARD have no probe coverage, due to filtering of candidate probes during the design. Overall, this probe set targets ∼1.77 megabases of antibiotic resistance-associated nucleotide sequences and greater than 83% of the nucleotide sequences curated in CARD. Additional metrics of the probe set are given in Fig. S1D to H.At the individual determinant level, the number of probes per gene  and the length coverage of a gene  vary . The majority of the targeted genes  are covered by at least 10 or more probes . Members of the beta-lactamase families . Genomic DNA from four different species was tested individually via enrichment on two different library preparations (the NEBNext Ultra II library preparation versus the modified Meyer and Kircher library preparation) of various insert sizes , referred to here as trial 1 and trial 2 (Table S2). Our enrichment approach is insensitive and tractable to different insert sizes, as there was a strong correlation between the read count on targeted regions for bacterial genomes enriched individually between the two trials  .mexW in Pseudomonas aeruginosa C0060 with a probe coverage of 2 had greater than 10 reads in both trials) and 80 bp . Other genes that had low probe length coverage included mdtA (22.4% coverage by 11 probes) in Escherichia coli C0002, which still retained over 100 reads in both trials, and a 140-bp region of aad(6) (16.8% coverage by 4 probes) in Staphylococcus aureus C0018 that was recovered with over 1,000 reads in both trials.This probe set is selective for regions associated with antibiotic resistance in these isolates, given that over 90% of the reads mapped to the respective draft bacterial genomes and the majority  of the reads mapped to the small proportion (<1%) of the genome associated with resistance . In 28/32 enrichments, 80% or more of the sequencing reads mapped to probe-targeted regions within the individual bacterial genome regardless of the pooling ratios . With these bacterial species tested, our probe set could successfully capture the resistome of these isolates, which represents less than 0.1% of the total DNA and even less at the individual gene level.At the isolate level, the percentage of the mock metagenome that was represented by probe-targeted regions in an individual isolate ranged from 0.0015 to 0.63% of the total DNA . In 21/32 enriched cases, over 90% of the probe-targeted regions were captured by 10 reads or more  by enrichment than by shotgun sequencing . A similE. coli C0002, which was previously used for enrichment with the mock metagenomes. In all cases, we identified the same genes, with a consistent number of reads mapping among these replicate enrichments , proving reproducibility regardless of the probe and library ratio . Within each set, we found an excellent correlation with the previous results seen with E. coli C0002  .In order to test the efficacy and the reproducibility of our enrichment in more complex samples, we performed enrichments on replicates from metagenomic libraries with DNA isolated from a healthy individual’s stool sample. We compared the results of the experiments with those of traditional shotgun sequencing, whereby selected libraries were sequenced to a depth of over 3.5 million paired reads (Table S2). We included a series of positive-control enrichments with genomic DNA from Across the enriched gut microbiome samples with the full number of reads and no filters, on average, 50.69% of reads mapped to sequences from CARD and 68 genes with at least 10 reads were identified, whereas 0.03% of reads mapped, on average, and 32 genes with at least 10 reads were identified in the shotgun libraries . We found significantly more genes with at least 1, 10, and 100 reads from each enriched sample than from the shotgun samples, and the average percent coverage of a gene by the number of reads in the enriched samples was 1.5-fold higher . When subsampled to the same depth as their enriched pairs , we identified, on average, 1  antibiotic resistance determinant with at least 10 reads after filtering in the shotgun samples, making comparisons at this level unrealistic (Table S8). Conversely, when subsampled to the depth of the sample with the lowest enriched read coverage , we identified, on average, 28 ARGs with at least 10 reads in the enriched libraries postfiltering (Table S8).n = 27), while we identified 16 genes of a total 32 (50.00%) that overlapped across all the shotgun libraries (n = 6) (Tables S7 and S9). When subsampled to the lowest enriched read coverage , there were no genes that overlapped across all 6 shotgun libraries, while 13/47 (27.66%) of the genes overlapped across all 27 enriched libraries (Table S10). Comparing the subsampled enriched libraries , the majority (31/34) of the genes missing in at least one sample were those with, on average, less than 20 reads across the 27 libraries . The order of genes with higher read counts  was consistent among the enriched and shotgun samples, and there was a more significant discrepancy between the two sets of samples for reads associated with lower-abundance genes . Thus, enrichment, in the same way as shotgun sequencing, does not in some way bias the prevalence of the rank order of AMR genes in these samples. Finally, both methods resulted in an excellent correlation among technical replicates individually .We combined the read counts for genes with at least 10 reads that passed the chosen filters within each set to compare the probe and library ratios in subsampled and full-read samples through both enrichment and shotgun sequencing. With the full number of reads, 24/70 (34.28%) of genes detected overlapped across all enriched libraries  were found in only a few shotgun samples with less than 30 reads and less than 20% read length coverage, on average . With the full number of reads in both methods (between 66- and 389-fold more in the shotgun samples than in the enriched samples), the average fold enrichment was >600-fold, and there were still 18 to 50 fewer genes in the shotgun samples than in the enriched samples ; Table 1n = 8 gene sequences) or had probes that were removed during design and filtering (n = 7 gene sequences). Of the four genes with predicted probes, cfxA6 was present in all enriched samples but was filtered out by mapping quality, vanSG was present in only 2/6 shotgun samples at less than 20% gene length coverage by reads, and cepA was found in enriched samples but at less than 10 reads; finally, we identified novA in all shotgun samples but in only a few enriched samples at less than 10 reads and less than 10% read length coverage. Despite the few (n = 4) genes that were missing from the enriched samples, even a 200-fold greater sequencing depth of our shotgun libraries could not provide results that match those shown by our enrichment data .We then compared the overlap between all 27 enriched samples and the 6 shotgun-sequenced samples and included genes found through shotgun sequencing without any probes mapping. We found a total of 89 genes with at least 10 reads between all libraries, of which 13 overlapped between methods, 57 were unique to the enriched libraries, and 19 were unique to the shotgun libraries . Only the blank samples from phase 1, trial 1, and phase 2, set 2, resulted in genes with at least 10 reads mapping .Increased interest in targeted capture approaches has resulted in the design of probe sets for the detection of viruses, bacteria, and, more recently, antibiotic resistance elements –29. Alth–30–gyrA, EF-Tu genes, etc.) that are likely to be found as homologs across many families of bacteria and that would likely have overwhelmed the probe set and sequencing effort with abundant, nonmutant antibiotic-susceptible alleles. Instead, we chose CARD’s protein homolog model  to focus our approach on genes that are likely to be acquired  and those that are unique to individual families of bacteria. Therefore, although we were unable to detect resistance conferred by SNPs in chromosomal metabolic genes, our probe set was capable of capturing the vast majority of resistance elements and those that were at a higher risk of being mobile. In future probe designs, the protein variant model of CARD  can be targeted using probes specific to the regions of a gene associated with a given set of SNPs, but they will need to be carefully tested in silico to ensure that they do not enrich unintended targets. Given that in certain populations  these variant sequences may be less abundant than their susceptible counterparts, careful and rigorous analysis will need to be implemented to identify the relevant variants .CARD was chosen as the reference database for our probe design (v1.0.1) and analysis (v3.0.0) due to its rigorous curation of antibiotic resistance determinants. We excluded some genes  for comparative analysis with our bait set designed in 2015. Since the design of our probes against v1.0.1 of CARD, the database has been updated and includes 264 additional genes. Despite these changes, our probe set targeted the majority  of known antibiotic resistance gene sequences from CARD (v3.0.0). In reality, the probes should target sequences with up to 15% nucleotide sequence divergence from a reference sequence, suggesting a wider applicability and target capacity toward newly characterized members of AMR gene families, which often differ from other members by only a few nucleotides. Of the 264 genes added to CARD (v3.0.0), our existing probes capture 75 of these genes that are sufficiently similar to other targeted members of the same AMR gene family . Of the remaining genes, the sequences of 60 have been newly identified since 2015, and the other genes, although mentioned in the literature prior to 2015, were added due to increased efforts of curation of CARD.To address our probe set’s compatibility with a frequently updated database, we chose a more recent version of CARD  significantly smaller than the amount recommended by the manufacturer (up to 2 μg of DNA for metagenomic samples), setting our method apart from other methods for the targeted capture of AMR genes , 28. OthfosA2 (ARO:3002804) in K. pneumoniae C0050, retained good probe coverage, despite a low percent identity (71.32%) to the CARD reference sequence. The poor performance in enrichment may suggest the limit of sequence similarity (>30%) that can be captured by probes designed against a single reference sequence. In addition, the high GC content of certain genes in the K. pneumoniae isolates and of many regions of the P. aeruginosa isolates  likely reduced the capture efficiency in the more complex pooled samples, resulting in less than 10 reads for targeted genes. The conditions of hybridization may need to be further optimized for targets with higher GC contents. Regardless of this limitation, the enriched data provide significantly more read coverage for antibiotic resistance genes at a lower depth of sequencing than shotgun sequencing of these mock metagenomes.The sensitivity of our probe set was tested using individual bacterial genomes and mock metagenomes, wherein the percentage of total DNA represented by probe-targeted antibiotic resistance genes ranged from 0.0015% to 0.97%. A successful enrichment in our trials was considered when greater than or equal to 90% of the probe-targeted regions with 10 or more reads were captured. When tested individually, enrichment was able to successfully capture all probe-targeted ARGs (100% with more than 10 reads) in the four bacterial species tested, with >85% of sequenced reads mapping to the targeted regions (<1%) of the genome. With the mock metagenomes, the probe-targeted regions of each isolate represented a smaller proportion, and there were 11 cases in which enrichment was not successful under the above-described criterion. In 7 instances, the given isolate represented less than 10% of the total pool, and many of the probe-targeted regions that were missing were short (<200 bp), and less than 5 probes for these regions were designed . One particular predicted resistance gene that was not captured in 2 cases, E. coli C0002), processed alongside the other samples, ensured that our methodology and probes were performing optimally at the time of hybridization. We also introduced negative controls to measure the extent of exogenous DNA contamination, which is ubiquitous in all laboratory settings and reagents (Escherichia coli) and encode efflux systems or other intrinsic resistance determinants . The two genes that were unique to the results for the blanks  are associated with mobile genetic elements in Enterobacteriaceae, and the latter has previously been associated with laboratory reagent contamination (Standardization (including reproducibility) in enrichment studies remains sorely lacking. In this study, we attempted to reduce bias and assess enrichment by using the same DNA extract, library preparations, and enrichment in triplicate. Even among replicate libraries and shotgun sequencing runs, the differences in the number of genes identified at various sequencing depths highlight the inherent variability in metagenomics . The positive control  compared to that obtained by other published capture-based methods, 30.26%  , highlignovA, which has a 70.51% GC content, there was a gap in the tiling of probes across the gene, and the hybridization conditions were perhaps not sufficient to capture this gene by our method. Additional probes or denser tiling along high-GC-content (>65%) sequences may facilitate successful capture. Another gene that we could not identify, the variant of the vanS  sensor from vancomycin resistance gene clusters, was covered by less than 20 reads in the shotgun samples, suggesting a very low abundance in the metagenome. Finally, the beta-lactamase genes cepA and cfxA6 had been excluded from the enriched results after filtering due to low mapping quality or less than 10 reads. The low mapping quality suggests that reads are mapping to other beta-lactamase genes in the reference database.Although shotgun sequencing can provide additional information on other functions and genes of interest, our targeted capture provides a more robust and a more reproducible profile of antibiotic resistance genes from metagenomes at a fraction of the sequencing cost. Only a few genes were absent in the enriched libraries but present in the shotgun libraries. In the case of All current methods to detect antibiotic resistance genes have limitations. Culturing, although time-consuming, remains the standard for diagnosing infections through the identification of both the pathogen and its susceptibility to a panel of antibiotics. Other biochemical techniques have been developed but are often organism specific and require additional assays for confirming ARGs . When stCompared to the other probe sets designed for AMR –29, ours–In conclusion, we have rigorously measured the performance of our probe design and methods to satisfy many of the parameters in targeted capture routinely discussed . The senn = 2,129) from CARD   was compared against the human reference genome and GenBank’s nonredundant nucleotide database through BLAST (blastn) analysis . Probes with HSPs of less than 50/80 nucleotides to bacterial sequences were additionally discarded, resulting in a set of 32,066 probes. The candidate list was further filtered to omit probes that had bacterial HSPs that had <95% identity, resulting in a candidate list of 21,911 probes.Our reference for probe design was the protein homolog model of antibiotic resistance determinants (er 2015) . Using Per 2015) . To prevanalysis , 45. ProProbe sequences, along with 1 to 100 nucleotides upstream and downstream of the probe location on the target gene, were sent to Arbor Biosciences  for probe design. These sequences are contained within the open reading frame of the target gene and allow probe sequences to be modified, if needed , ensuring that the desired probe coverage of the target genes is attained. An additional 80 nucleotide probes were created across the candidate probe and flanking sequences at a tiling density of four times, resulting in 226,440 probes. Sequences with 99% identity over 87.5% of their length were collapsed using the USEARCH program , resulting in a set of 37,826 final probes . Filterihttps://gist.github.com/wdecoster/8204dba7e504725e5bb249ca77bb2788. The melting temperature (mT) was determined using the OligoArray function melt.pl    alignmenPseudomonas aeruginosa C0060 was additionally extracted using a varied genomic lysis/binding buffer . We obtained a human stool sample from a healthy volunteer for the purpose of culturing the microbiome with consent from the Hamilton Integrated Research Ethics Board . DNA was extracted on the same day following a modified protocol described elsewhere . Genomic DNA was isolated from a cell pellet using the PureLink genomic DNA mini kit . If DNA was not isolated on the same day, we stored the cell pellets at −80°C. While genomic DNA from all other isolates was extracted only once, DNA from a cell pellet of http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and de novo assembled using SPAdes (v3.9.0) software  using the Nextera XT DNA library preparation kit . Libraries were sequenced using an Illumina HiSeq 1500 or Illumina MiSeq v3 platform and v2 (2 × 250-bp) chemistry. Paired sequencing reads were processed through a Trimmomatic (v0.39) trimmer to remove adapters, checked for quality using the FASTQC program  (Tables S1 and S3). In addition, various nanogram amounts  of each genome were combined prior to library preparation to create mock metagenomes, referred to as pool 1 , pool 2 , and pool 3 . The amounts of the genome of each isolate in each pool varied between trials (Table S4). Phase 2 consisted of 3 replicates, referred to as set 1, set 2, and set 3, wherein a DNA extract from one individual human stool sample was split evenly into each set. From these aliquots, we generated 9 individually indexed sequencing libraries and performed capture with various library and probe ratios (Table S3). In all trials and sets, a blank DNA extract was carried throughout library preparation and enrichment, while an additional negative reagent control was introduced during enrichment.We performed two phases of experiments. The first was with genomic DNA from cultured multidrug-resistant bacteria (phase 1), and the second was with metagenomic DNA from a human stool sample (phase 2). The two trials in phase 1 differed in their library preparation methods, as described below (the major difference being the library fragment size obtained by sonication). In both trials, we tested genomic DNA from isolates individually . Based on absorbance and fluorometer values , we sonicated approximately 1 μg of individual bacterial genomic DNA or pools of genomic DNA to 600 bp and prepared dual-index libraries with a size selection for 500- to 600-bp inserts. Postlibrary quality and quantity verification was performed using a high-sensitivity DNA kit for the Agilent 2100 bioanalyzer  and quantitative PCR using a Kapa SYBR Fast qPCR master mix for Bio-Rad machines , primers for the distal ends of Illumina adapters, and the following cycling conditions: (i) 95°C for 3 min, (ii) 95°C for 10 s, (iii) 60°C for 30 s, (iv) a repeat of steps (ii) and (iii) for 30 cycles total, (v) 60°C for 5 min, and (vi) hold at 8°C. We used Illumina’s PhiX control library  as a standard for quantification.P. aeruginosa C0060, which was reisolated, was used for library construction through a modified protocol   . BrieflyEscherichia coli C0002 genomic DNA (40 ng of sonicated DNA) and a negative control with a blank DNA extract.We divided one DNA extract from a donor stool sample into three 50-μl aliquots of approximately 3,150 ng each (based on QuantiFluor fluorometer results). DNA was sonicated to 600 bp and split into 9 individual library reaction mixtures (350 ng in 5.55 μl). We prepared dual-index libraries  with a size selection for 700- to 800-bp library fragments and 6 (set 1), 7 (set 2), or 8 (set 3) cycles of amplification. The McMaster Genomics Facility performed library quality control . We generated positive-control libraries using We performed enrichments in a PCR clean hood, with a water bath, thermal cyclers, and heat blocks being located nearby. The probe set was provided by Arbor Biosciences  and diluted with deionized water. For enrichment of bacterial genomes in trial 1, we used 100 ng of probes and 100 ng of each library, following the instructions in the myBaits manual , at a hybridization temperature of 65°C for 16 h . After hybridization and capture with Dynabeads MyOne streptavidin C1 beads , the resulting enriched library was amplified through 30 cycles of PCR  using Kapa HiFi HotStart polymerase with library-nonspecific primers . A 2-μl aliquot of this library was amplified in an additional PCR for 3 cycles (under the same conditions described above) and then purified. We performed the capture in trial 2 in the same manner described above for trial 1 but applied 17 cycles of amplification postcapture . The McMaster Genomics Facility performed library quality control as described above. The libraries were pooled in equimolar amounts and sequenced to an average of 94,117 clusters by use of an Illumina MiSeq sequencer . Preenrichment libraries for the mock metagenomes were sequenced in a separate Illumina MiSeq  run from the enriched libraries to an average of 93,195 clusters each. From both trial 1 and trial 2, negative controls consisting of blank extractions carried through library preparation and enrichment were sequenced on separate individual Illumina MiSeq (2 × 250-bp) runs. After demultiplexing of the blank, all possible index combinations were retrieved to identify potential cross-contamination of libraries as well as exogenous bacterial contamination.2O to replace the volume normally required for library input. We performed enrichment following the instructions in the myBaits manual  at a hybridization temperature of 65°C for 24 h. After hybridization and capture with Dynabeads , the resulting enriched library was amplified through 14 cycles of PCR using Kapa HiFi HotStart ReadyMix polymerase with library-nonspecific primers and the same conditions described above . The resulting products were purified using Kapa Pure beads  at a 1× volume ratio and eluted in 10 mM Tris, pH 8.0. Purified libraries were quantified through qPCR using 10× SYBR Select master mix  for Bio-Rad Cfx machines, Illumina specific primers , and Illumina’s PhiX control library  as a standard. Cycling conditions were as follows: (i) 50°C for 2 min, (ii) 95°C for 2 min, (iii) 95°C for 15 s, (iv) 60°C for 30 s, and (v) a repeat of steps (iii) and (iv) for 40 cycles total. We pooled the enriched libraries in equimolar amounts based on qPCR values, and the McMaster Metagenomic Sequencing facility performed library quality control as described above. Finally, we sequenced the enriched libraries  and the preenrichment libraries  with an Illumina MiSeq sequencer . The negative controls consisting of blank extractions carried through library preparation and enrichment were sequenced on separate individual Illumina MiSeq (2 × 250-bp) runs. After demultiplexing, all possible index combinations were retrieved.Based on qPCR values and the average fragment sizes of each library generated from the human stool DNA extract, we combined various nanogram amounts of library  and probes  for enrichment (Table S3). Along with the negative-control (blank) library, we introduced additional negative controls during enrichment, using distilled Hhttps://github.com/BioInfoTools/BBMap)  program . We useds/BBMap) , 56. Alis/BBMap) , 49, 57.s/BBMap) . The perhttps://github.com/lh3/seqtk). We used the bwt feature in RGI  to map trimmed reads, using the Bowtie2 (v2.3.4.1) program, to the sequences in CARD (v3.0.0), generating alignments and results without any filters (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) programs, log transformation, and clustering arrays with an uncentered correlation (Pearson) and average linkage. For rarefaction analysis, we first aligned trimmed reads against the sequences in CARD (v3.0.0) using the Bowtie2 program, followed by filtering for a mapping quality of ≥11 (http://megares.meglab.org/amrplusplus)  program  , with suPRJNA532924. All metagenomic sequencing results, enriched or shotgun, were deposited in NCBI under BioProject accession number PRJNA540073. The probe set sequences and annotations are available at https://card.mcmaster.ca/download.Raw sequencing reads (FASTQ) for the IIDR clinical isolate collection bacterial isolate genome assembly were deposited in NCBI under BioProject accession number


The 1H-NMR profiles highlighted a similar go and return pattern in the metabolism of the BRAF, NRAS, and cKIT mutated cell lines. Indeed, melanoma cells exposed to mutation-specific inhibitors underwent metabolic disruptions following acute exposure but partially recovered their basal metabolism in long-term exposure, most likely acquiring resistance skills. The protein microarrays inquired about the potential cellular mechanisms used by the resistant cells to escape drug treatment, by showing decreased levels of proteins linked to the drug efficacy, especially in the downstream part of the MAPK signaling pathway. Integrating metabonomic and proteomic findings revealed some metabolic pathways  and key proteins  as potential targets to discard drug resistance.Treatments of metastatic melanoma underwent an impressive development over the past few years, with the emergence of small molecule inhibitors targeting mutated proteins, such as BRAF, NRAS, or cKIT. However, since a significant proportion of patients acquire resistance to these therapies, new strategies are currently being considered to overcome this issue. For this purpose, melanoma cell lines with mutant BRAF, NRAS, or cKIT and with acquired resistances to BRAF, MEK, or cKIT inhibitors, respectively, were investigated using both  Melanoma is the deadliest form of skin cancer . Its incAltered metabolism is a hallmark of cancer . This caTo date, studies on melanoma cell metabolism under targeted therapies or after resistance acquisition mainly focused on cells treated with BRAF inhibitors. These studies indicated that BRAF inhibitors used in BRAF-mutated melanoma cells led to a decreased expression of glycolytic enzymes associated with lower glucose consumption. These studies indicated that the resistance to BRAF inhibitors was linked to an increased oxidative metabolism associated with an increased mitochondrial dependency ,22,23. TMAPK and PI3K-AKT, the two major oncogenic signaling pathways in melanoma, have been extensively studied. It turns out that resistance to BRAF and MEK inhibitors mainly occurs through new modifications in these signaling pathways ,26,27,28In the present work, we evaluated which metabolic pathways are affected in melanoma cells that are exposed to targeted therapies, in cells with acquired resistance to these drugs and their parental counterpart. The aim was to understand the metabolic changes occurring among melanoma lines, either sensitive or resistant to targeted therapy, with the perspective of identifying new targets for combinatorial studies in order to overcome or at least delay the development of acquired resistance. As the current literature concerning metabolic changes in resistant melanoma cells mainly focuses on BRAF mutated cells, we decided to expand our studies to other important mutational profiles. We used three different cell lines with frequent gene mutations in melanoma. The two mutually exclusive BRAF and NRAS mutations account for about 85% of the mutations affecting the MAPK pathway ,32. The D820YcKIT), MM074 (V600EBRAF), and MM161 (Q61RNRAS). 1H-NMR spectra of the cellular extracts of each cell line were generated. For each cell line, three main experimental conditions were conducted. The first one consisted of sensitive cells free of any drugs . The second one corresponded to sensitive cells exposed to their corresponding targeted therapy for a short time (24 and/or 72 h) for results purposes . The latter included the resistant counterpart for each cell line considered. Those resistant cells were produced in-lab using a continuous exposure of melanoma cells to increasing concentrations of their corresponding targeted therapy for at least 12 weeks .We performed our analyses on three different cell lines: HBL  was next applied to the data to highlight the most reliable discriminating metabolites in group separation, resulting in a scores plot a and its1H-NMR intracellular (IC) spectra were further investigated using the MestRenova software. Indeed, some metabolites can be hidden due to spectral overlapping or low intensity. Using the MestRenova Peak Picking tool, numerical data were successfully collected and separated from the two metabolic groups identified in the loadings plot. Thus, the resonances of PCho and GPC, as well as Cr and PCr, were successfully separated from each other. Furthermore, five extra metabolites with a low intensity were detected thanks to additional spectral investigations, being acetate, aspartate, glutathione (GSH), succinate, and formate. The global signature was next processed through statistical analyses  returned the same “roundtrip” pattern as the one already described in the IC signature  and their parental drug-sensitive counterparts . We used the log2 transformed data , enablinThe first plot a shows tConsidering the HBL-resistant cell condition b, higherFinally, a bench of nine proteins with a modified abundance were highlighted in the MM161-resistant case thanks to the fold change analysis c. Thus, Using the Human Reference Protein Interactome Mapping Project, protein–protein interaction (PPI) analysis was next applied on the 18 most abundant proteins identified in the resistant cases to get some understanding of the resistance mechanisms. The first PPI network d returnep-values were calculated and used for classification. Focusing on the pathways that come out from the MSEA analysis, from a cancer point of view, special attention is paid to: ‘Glutamate metabolism’, ‘glutathione metabolism’, ‘Warburg effect’, ‘aspartate metabolism’, ‘alanine metabolism’, ‘malate-aspartate shuttle’, ‘alanine metabolism’, and ‘glycine and serine metabolism’.All 15 discriminant metabolites were selected for a metabolic set enrichment analysis (MSEA) using the MetaboAnalyst 4.0 online software. MSEA can connect the metabolites set presenting relative concentration variations to biological pathway(s), thus improving data interpretation. The resulting bar chart a highligA joint pathway analysis was also performed using both discriminant metabolic and protein levels. The analysis returned a metabolome view b, where The aim of this work was to combine both metabonomic and proteomic approaches in order to better understand the resistance adaptations occurring in metastatic melanoma exposed to targeted therapies and to identify new targets paving the way for novel combinatorial drug strategies. We used three different cell lines with frequent gene mutations in melanoma .The metabonomic analysis of HBL cells indicates that the treatment of these HBL cells with Dasatinib leads to a disruption of their metabolism in first place, rapidly followed by a metabolic adaptation to survive under Dasatinib chronic exposure. The same dynamic evolution of metabolism is observed in both IC and EC compartments. To understand how these cells can survive under chronic treatment and become resistant, we were particularly puzzled by the behaviors of IC lactate, aspartate, and glutamine, as well as EC lactate and glutamine. Both lactate and glutamine (IC and EC) levels decreased after a short exposure to Dasatinib and stabilized after resistance acquisition. The aspartate level decreased slightly after acute exposure but more intensively after chronic treatment. Lactate is the end-product of glycolysis and is overproduced in many cancer cells under the Warburg effect . Its decThe metabolomic analysis of the BRAF mutated cell line MM074 exposed to Dabrafenib (1µM) for 24 and 72h indicated a metabolic switch in the IC score plot, followed by a return to a previous state when becoming resistant. The same “roundtrip” pattern was also observed in the EC compartment. Once again, we focused on those metabolites that did not follow the “roundtrip” evolution after resistance development, namely aspartate and alanine. They both increased after short exposure to 1µM Dabrafenib and kept relatively high levels after resistance acquisition. As already mentioned, an increase in the aspartate level can be indicative of a higher production of mitochondrial ATP . It has Finally, metabolomic analysis of the NRAS mutated cell line MM161 under either short or chronic exposure to the MEK inhibitor (1 µM Pimasertib) indicates the same switch and return pattern. In this case, the metabolites of interest are mainly found in the IC with an increase in the glutamine level after a short exposure that lasted after resistance development, pointing to a higher activity of glutaminase enzymes. Indeed, a major role of glutaminases as oncogenic drivers has been highlighted in many cancer types ,47,48. TBased on a better understanding of the mechanisms of resistance, the aim of our work was to find new biomarkers linked to drug resistance development and/or new targets, allowing such resistance to be delayed or overcome. We studied three melanoma cell lines with representative mutations in order to find a common pattern of resistance to different targeted therapies. Regarding the metabolism, the common point between our different cell lines is that while acquiring resistance, cells tend to switch to a metabolism relying more on glutamine and mitochondria for ATP production. This opens some interesting perspectives to disrupt this resistant metabolome by using mainly two kinds of metabolic inhibitors. First, glutaminolysis  could beD820YcKIT), MM074 (V600EBRAF), and MM161 (Q61RNRAS). These lines are sensitive to targeted therapies, but we also worked with their resistant counterparts. To develop these resistances, cells have been chronically exposed to increasing doses of targeted therapies for 12 weeks (0.01–1 µM) by M. Krayem and A. Najem. This model mimics what happened in patients developing resistances to targeted therapies. Acute drug exposure effects were generated by exposing sensitive cells to 1 µM of their corresponding drug during 72 h. Due to Dabrafenib’s impact on the MM074-sensitive cell density, a shortened time-point of 24 h of exposure was also considered.Metastatic melanoma cell lines were derived from tumors by the Laboratory of Oncology and Experimental Surgery at Institut J. Bordet, Université libre de Bruxelles . For the work, we selected 3 cell lines harboring different mutations: HBL  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin  at 37 °C in a humidified 95% air and 5% COThe BRAF inhibitor Dabrafenib, used for BRAF mutated cells, and the MEK inhibitor Pimasertib, used for NRAS mutated cells, were from Selleck Chemicals . The tyrosine kinase inhibitor Dasatinib, used for cKIT mutated cells, was from Bristol-Myers .3 flasks of each cell line were cultivated to confluency. Concerning the culture media collection, they were replaced 48h before sample collection. They were then collected and stored at −20 °C until analysis. Concerning the cell collection, we washed them twice with cold DPBS (4 °C)  and used 3 mL of cold methanol to quench their metabolism. Finally, cells were collected using a scraper. Prior to −80 °C storage, cell pellet was de novo quenched using a liquid nitrogen immersion for a few seconds.Before sample collection, 675-mm1H-NMR samples’ preparation, an additional methanol:water:chloroform 1:0.9:1 extraction was required for cell pellet preparation, to separate the polar metabolites from macromolecules. The resulting polar phase containing the metabolites held a mix of methanol and water, which interacted with the NMR signal. The solvents were therefore dried using a SpeedVacuum.Before 1H-NMR sample preparation, either the cellular extracts or culture media were mixed with phosphate buffer , previously prepared in a mixture of H2O/D2O (80:20). For the intracellular samples, 700 µL of buffer were added to the dried cell pellets, whereas 250 µL of buffer were mixed with 500 µL of the culture media. To remove residual macromolecules, samples were centrifuged for 10 min at 13,000 g. Next, 650 µL of each supernatant were mixed with 50 µL of a solution of Trimethylsilylpropanoic acid (TSP) solubilized in D2O, 7mM for cell pellets, and 14 mM for culture media. For 1H-NMR spectra acquisition, the previous described samples were loaded into 5-mm NMR tubes. Spectra acquisition was processed on a Bruker 600-MHz Advance spectrometer, using the NOESYPRESAT-1D pulse sequence and 256 scans. For Both the phases and baseline of the obtained spectra were corrected using the MestreLab Research 10.0.2 software . Next, spectra were merged into one page using the Stack tool, and the water peak region ranging from 4.20 to 5.20 ppm excluded. After ensuring good spectra alignment, spectra were binned into small subregions of 0.04 ppm width, and the area under the curve (AUC) of each descriptor calculated. Each 0.04-ppm width AUC was normalized to the total spectrum AUC.2 and Q2 values, based on the Umetrics recommendations. The supervised PLS-DA technique enabled variable importance in projection (VIP) calculation, with ones greater than the 1 value retained for further analyses.Binned spectra were investigated using the SIMCA-P+ 12.0 software . To ensure the quality of the spectra processing, principal component analysis (PCA) was always carried out on the dataset prior to partial least square discriminant analysis (PLS-DA). The quality of the PLS-DA models was ensured by the two parameters RThe lack of sensitivity of the multivariate data analysis, as well as metabolites overlapping within the 0.04 ppm width, can hide some valuable information. Therefore, a semi-quantification analysis of the spectra within the MestRenova software was essential and returned additional spectral areas of interest. Next, the selected spectral areas were identified as metabolites using the Chenomx NMR suite software (version 8.1.1) and the human metabolome database (HMDB) .p-values were Holm-corrected. Identified metabolites were then submitted to statistical analysis. First, the precise AUC of each metabolite was calculated within MestRenova using the Peak Picking tool. Then, the obtained numerical data were submitted to a Kruskal–Wallis test followed by a Dunn test. The considered The relative intensity of each discriminant metabolite was illustrated as a heatmap using the Morpheus online software.Protein microarray was performed by RPPA analysis at the Functional Proteomics Core Facility at The University of Texas MD Anderson Cancer Center .Protein–protein interaction studies were conducted according to the Human Reference Protein Interactome Mapping Project .Connection of the discriminant metabolites to metabolic pathways was performed using the Metabolic Set Enrichment Analysis (MSEA) tool from the online available software Metaboanalyst 4.0 , using tData integration of both metabolite and protein sets was performed using the Joint Pathway Analysis of MetaboAnalyst, with the Homo Sapiens Pathway Library.As metastatic melanoma is still one of the deadliest forms of cancer with a 5-year survival below 15% and as there is a lack of therapies effective in the long term due to the hypermutable genome of melanoma cells inducing the development of resistance to targeted therapies, it is crucial to better understand how these mechanisms take place. It is now clear that metabolic reprogramming plays a key role in resistance development. Our study indicated that common patterns took place in cells with different mutations exposed to different molecules of targeted therapy. As we showed that these common patterns indicate a particular role of glutaminolysis, glycolysis, and oxidative phosphorylation, we successfully targeted these processes and showed the disruption of the metabolome associated with the resistance to various targeted drugs.Our integrative metabolomic and proteomic approach also pointed out the importance of cross-talk between metabolic enzymes and oncogenic drivers in the MAPK and PI3K-AKT pathways. This once again highlights the importance of integrative studies involving metabolic approaches in order to better understand the resistance mechanisms occurring in cancer cells.


Little is known about the trajectory of tinnitus over time. This study addressed (1) how often tinnitus remitted in patients with chronic tinnitus; (2) how subjective reported tinnitus characteristics, such as loudness, laterality, and type and measures of burden, such as tinnitus distress, depression, and quality of life, changes over time; (3) how often tinnitus-specific treatments were undertaken and the prevalence of comorbidities, (4) if the number of treatments and comorbidities were associated to changes in tinnitus distress over time. Data from 388 patients with chronic tinnitus who visited a tertiary tinnitus clinic between 2012 and 2017 were interrogated via a mail survey in 2018. Tinnitus characteristics were measured with the Tinnitus Sample Case History Questionnaire and numeric rating scales; tinnitus distress with Tinnitus Handicap Inventory (THI) and the Tinnitus Questionnaire (TQ), depression with the Major Depression Inventory and Quality of life with the World Health Organisation Quality of Life BREF at both time points and the clinical global impression scale. Comorbidities experienced and undertaken treatments were assessed with an in-house survey. Three participants (0.8% of the sample) reported tinnitus remission between both assessments. A decrease in the THI and TQ, and numeric ratings for tinnitus severity, annoyance, unpleasantness, and discomfort was observed, but no differences in tinnitus characteristics, depression, quality of life or overall health status. 64% presented at least one comorbidity, and 88% sought at least on tinnitus-specific treatment. Common comorbidities were psychological and sleeping problems, and the most common interventions were going to the dentist, taking medications, and wearing hearing aids. Our results suggest that full remission of tinnitus is a rare condition, that tinnitus distress on average decreases over time, and that tinnitus characteristics, quality of life, and depression tend to remain unaltered. The high number of interventions and comorbidities displayed minimal association to the changes in tinnitus distress, highlighting the substantial and durable burden of tinnitus sufferers. Although a considerable number of patients report little to no impact in their daily lives due to this condition, tinnitus can also be exasperating3. Previous studies from 2005 and 2013 estimated that, at the time, 13 million tinnitus patients were actively seeking support in Europe and in the United States5. Furthermore, the costs associated with tinnitus were estimated at 750 million pounds per year in the United Kingdom6. Overall, tinnitus can be described as a burdensome condition both at the individual and societal level.Tinnitus can be described as the perception of sounds, usually inthe form of ringing or hissing, without an external source7. Based on clinical experience it is generally assumed that tinnitus becomes less burdensome over time, but systematic data supporting this assumption are scarce. One mechanism of amelioration of tinnitus over time may be related to tinnitus habituation, first described by Hallam8. It is also a key component of the so-called neurophysiological tinnitus model, and it offers a potential explanation why the condition is so burdensome for a subset of patients9. According to this model, distress originates from aberrant activation in areas of the brain other than the auditory pathway, such as the limbic and autonomic systems, which prevents that patients habituate to tinnitus by repeated emotional and stressful ratings of the condition9. This concept is supported by recent imaging data demonstrating the involvement of non-auditory brain areas such as the frontal cortex, the amygdala and the parahippocampal cortex in tinnitus pathophysiology11.The reasons why only a subset of patients are burdened by the condition are unclear. Moreover, evidence suggests tinnitus can change over time even when presented chronically13. However, very little is known about the course of tinnitus over several years and about the facets of tinnitus that might vary over time, even if this knowledge would be of exceptional interest for clinicians, researchers and patients15.Recent studies using ecological momentary assessment indicate that tinnitus distress fluctuates over time and that these fluctuations are related to emotional factors16 conducted a 15-year longitudinal study on noise-exposed workers in which the authors identified tinnitus as an early indicator of hearing loss. In a 7-year longitudinal study, Andersson et al.17 found that tinnitus severity shows signs of improvement over time after clustering patients in three groups based on different degrees of distress. However, the authors did not use any distress-specific, numeric questionnaire to compare baseline and follow-up values. Olderog and colleagues18 conducted a study with 44 patients suffering from tinnitus for less than four weeks with a 6-month follow-up survey. A stepwise regression with the predictors “sleep disturbance”, “anxiousness”, and “life satisfaction” collected at baseline could explain 56% of the variance (R2) of tinnitus distress measured with the Tinnitus Questionnaire (TQ) in the 6-month follow up. It remains unclear, however, whether these effects would persist in a larger, chronic tinnitus sample. Erlandsson and Persson19 found that the Beck Depression’s Inventory (BDI) and the trait and state anxiety (quantified by Spielberger’s State and Trait Anxiety Inventory) decreased over a period of 18 months only among patients without a personality disorder. Lastly, Folmer20 measured sleep quality, depression (measured by the BDI), and tinnitus distress  in 190 patients. The author reported a decrease in the TSI, especially among patients whose sleep patterns improved and whose BDI decreased by at least 3 points between the two assessments.In the following, some of the longitudinal studies, alongside their main findings and limitations are briefly discussed. Gr iest and BishopWe investigated whether tinnitus changes over time in a large cohort of patients with chronic tinnitus from a tertiary clinic in a retrospective mail survey to further understand the development of tinnitus over time. Our research questions were (1) how often tinnitus remits in patients with chronic tinnitus; (2) how tinnitus characteristics and burden caused by tinnitus  change over time; (3) how often patients enrolled in a tinnitus-related treatment protocol and which treatments were most often sought; (4) if number of treatments or comorbidities were associated with changes in tinnitus distress over time.Three patients (0.8%) reported losing their tinnitus at T2. Table Table p = 0.12) and p = 0.23) respectively, whereas Fig. p = 0.64) and p = 0.49) respectively.Most patients (64%) reported having at least one comorbidity, with psychological and sleeping problems being the two most often reported Fig. a,b. Visimentclass2pt{minimmentclass2pt{minimmentclass2pt{minim21. Three patients (0.8% of our sample) reported full remission of tinnitus at T2. No common factors or characteristics were identified distinguishing those patients from the whole sample  and the variability of treatments and comorbidities included in this analysis. Only a subset of patients reported to our mail survey; therefore, the possibility of a selection bias must be considered. Additionally, the observed improvement might reflect the tendency to the mean, that is, patients visit the clinic when they are severely impaired, and a later improvement may reflect spontaneous fluctuations. Future studies should assess both subjective and objective  tinnitus characteristics at all time points.Our analysis was limited to factors such as demographics, tinnitus distress and tinnitus characteristics, quality of life and depression. However, other confounding variables such as socio-demographics, coping strategies, life history, and personalityWe investigated the longitudinal trajectory of tinnitus characteristics, tinnitus distress, depression and quality of life among chronic patients from a tertiary tinnitus clinic. Tinnitus disappeared from three chronic patients, suggesting that, albeit rare, remission is possible even after years of having tinnitus. Future research should further investigate which factors are associated with tinnitus remission. Our findings should also encourage future research to focus not only on tinnitus management but also on interventions to suppress it, as tinnitus remission may be possible also in the chronic manifestation of the condition.Our sample consisted of patients from the Tinnitus Center of the University of Regensburg. Participants were invited to this study via a letter containing questionnaires and a consent form. The letters were sent to 1213 out-patients who visited the clinic between 2012 and 2017. 388 letters were sent back, resulting in a 32% response rate. The study was approved by the ethical committee at the faculty of medicine of the University of Regensburg (study number 18-1041-101). All the protocols and guidelines of the institution were followed, and only patients from whom we obtained signed informed consent were included in the final analysis.30, WHOQOL-Bref), health status according to Clinical Global Impression and the Tinnitus Sample Case History Questionnaire(31 TSCHQ) were collected at T1 and T2, and an informal in-house questionnaire asking patients which treatments they tried, and which comorbidities they experienced since T1 was obtained at T2 .Henceforward, the patients’ first visit to the clinic between 2012 and 2017 will be described as T1, and the assessment through mail conducted in 2018 will be described as T2. The Tinnitus Handicap Inventory (THI), Tinnitus Questionnaire (TQ), Tinnitus Numeric Ratings Scales for severity, loudness etc., Major Depression Inventory (MDI), World Health Organization Quality of Life(32), alongside the ”tidyverse” package33. After checking whether variables presented normal distributions and homogeneous variance, t-tests were used to compare mean differences in numeric items between T1 and T2; categorical items were tested with Pearson’s Chi-Squared test. Two previous studies defined an improvement in tinnitus distress, measured by the THI, to be clinically significant if above 20 and 7 points24. Differences in the THI between T1 and T2 were used to calculate the percentage of patients who clinically improved under these definitions. Missing observations in pairwise comparisons were excluded from the analysis. Multiple comparisons were corrected with the Holm-Bonferroni method34. Following convention, p values below the threshold 0.05 were considered statistically significant and p values below 0.1 were considered trend significant. Effect sizes were calculated with the Cohen’s d using the ”effsize” package36. The package Igraph was used to represent the association between comorbidities and treatments tried as a network37. In this context, the node’s size represents the number of people who experienced a comorbidity or attempted a treatment protocol, and the link’s thickness between two nodes represents the number of people who experienced a given pair of comorbidities or two attempted treatment protocols between T1 and T2.Differences between T1 and T2 (T2–T1) were calculated and reported with a Supplementary Information 1.Supplementary Information 2.