The use of routine outcome monitoring (ROM) in the treatment of mental health has emerged as a method of improving psychotherapy treatment outcomes. Despite this, very few clinicians regularly use ROM in clinical practice. Online ROM has been suggested as a solution to increase adoption.The aim of this study is to identify the influence of moving ROM online on client completion rates of self-reported outcome measures and to identify implementation and utilization barriers to online ROM by assessing clinicians’ views on their experience using the online system over previous paper-based methods.Client completion rates of self-reported outcome measures were compared pre- and postimplementation of an online system of ROM. In addition, a survey questionnaire was administered to 324 mental health service providers regarding their perception of the benefits with an online system of ROM.Client completion rates of self-reported measures increased from 15.62% (427/2734) to 53.98% (1267/2347) after they were moved online. Furthermore, 57% (56/98) of service providers found the new system less time-consuming than the previous paper-based ROM, and 64% (63/98) found that it helped monitor clients. However, the perceived value of the system remains in doubt as only 23% (23/98) found it helped them identify clients at risk for treatment failure, and only 18% (18/98) found it strengthened the therapeutic alliance.Although the current study suggests mixed results regarding service providers’ views on their experience using an online system for ROM, it has identified barriers and challenges that are actionable for improvement. The prevalence of mental illness, accompanied by its social and economic burden on the individual and society, has gained global recognition ,2, creatThe use of ROM in clinical settings appears to have a positive effect on patient outcomes in several ways. One key benefit is that ROM can help clinicians identify clients at risk for treatment failure by limiting the effect of overestimating their own abilities (self-assessment bias) -12. BoswAnother positive effect of ROM in mental health treatment is quicker client improvement, which is tied to faster recovery resulting in fewer treatment sessions ,15. FeweThe use of ROM can also improve the therapeutic alliance between clinician and client. Clinicians who form stronger alliances with their clients can expect better outcomes ,15,18-21Despite these benefits of ROM and the fact that many countries are recommending the use of ROM in various mental health settings ,23-25, pPrevious studies evaluating the organizational benefits of ROM have shown that using online systems provides instantaneous feedback to clinicians and clients ,7,8,12. The objective of this paper is to identify if moving to an online system of ROM influences client completion rates of the outcome questionnaires. In addition, service providers’ views on their experience using an online system will be examined to identify implementation and utilization barriers and highlight actionable items where improvements can be made to ensure successful future implementations.The study was conducted at a large mental health and addiction facility in Canada, and 2 types of data were collected. First, retrospective data were gathered on completion of self-reported measures by clients. Second, a cross-sectional survey of mental health service providers was conducted to investigate their perception of the benefits of using an online ROM in their treatment of clients in the Depression Care and Trauma Care individual outpatient counseling programs. For the purposes of this study, clinicians are referred to as “service providers.” A research ethics board application was submitted, and ethics review was deemed to not be required.In this study, ROM refers to the repeated measurement of a client’s progress over the course of treatment according to standardized self-reported measurement questionnaires or assessments  by the clients as part of the ROM process were calculated as follows: each client that filled out at least 1 assessment battery at 1 time point during the course of treatment was calculated as 1 client with a completed assessment, both pre- and postimplementation. The total number of clients with a completed assessment was divided by the total number of clients. All clients in the Depression Care and Trauma Care programs were expected to complete assessments; all were included for the time period of 18 months prior to the implementation of the online ROM and again at 18 months postimplementation of the online ROM.A survey questionnaire was designed by the investigators for this study based on a literature review of research on utilization and attitudes toward ROM in the treatment of mental health and adaptation of the survey questions used in those studies ,30,32,33The questionnaire consisted of ten, 5-point Likert scale questions, asking service providers how they would best characterize their feelings toward the use of an online ROM system in the areas of time savings (Q1), client receptiveness (Q2), allowance for regular progress monitoring of clients (Q3), adequacy of training (Q4), strengthening of the therapeutic alliance (Q5), identification of clients at risk for failure (Q6), increased workload (Q7), help available as needed to assist in using the program (Q8), if confidence in usage increased over time (Q9), and if it positively impacted the care they provided (Q10). In addition, the questionnaire included 1 open-ended free-text question asking for further comments. The questionnaire was offered in both English and French to accommodate the bilingual nature of health care services in Canada . Of the 98 responses, 10 were completed in French.Client completion rates  of the self-reported measurement questionnaires using paper-based methods calculated over an 18-month period preimplementation of an online ROM were 15.62% (427/2734). Postimplementation of an online ROM, client completion rates calculated over an 18-month period were 53.98% (1267/2347).Survey responses provided the following results. Most service providers , responded that the online system was less time-consuming than were previous paper-based methods (Q1); however, only 31 out of 98 (31%) agreed that the system did not increase their workload (Q7).With regard to the online system of ROM allowing for regular progress monitoring of their clients (Q3), 63 out of 98 (64%) service providers responded that the online system did allow for regular progress monitoring of their clients. However, only 23 out of 98 (23%) responded that the online system helped identify earlier clients at risk for treatment failure (Q6), and only 18 out of 98 (18%) responded that the online system strengthened the therapeutic alliance with their clients (Q5). In addition, only 45 out of 98 (64%) service providers responded that their clients were receptive to using the online system to complete the self-assessment questionnaires (Q2), and only 38 out of 98 (38%) responded that the program positively impacted the care they provided (Q10).With regard to their ability to successfully use the system, only 36 out of 98 (37%) reported that help was available as needed to assist with using the program (Q8), 46 out of 98 (47%) responded that they received adequate training (Q4), and 50 out of 98 (51%) felt more confident in their ability to use the system since it was first introduced Q9; .“I like the paper savings, instant scoring and ability to visually monitor progress online”; and “Overall I think it is a great tool…”.Many of the 48 free-form responses were able to be split into several themes. The service providers who responded with positive feedback indicated their appreciation for the instant feedback on the progress of their clients: “I would say the best part is that it’s easier than paper and results are easy/instant”; Most service providers, however, stated difficulties navigating the software and understanding the process. Many of these respondents specifically stated that they received inadequate training, and many made a request for additional training. Due to their own difficulties using the software, some also described issues with assisting their clients in successfully using the software. In addition, some service providers stated they had to resort back to completing the measures for their clients or completing it with their clients on paper in session and faxing in the results. Reasons given by service providers for this were clients reporting technical difficulties, preferring paper and pen, reporting being too depressed already, or stating the process was frustrating and anxiety-provoking. Some service providers stated they found no benefit with using the online program over previous paper-based methods, some reported an increase in their workload, and some reported accessibility issues due to the platform not being entirely in French.The findings of this study suggest that an online system supports adoption of routine outcome monitoring in the treatment of mental health. Despite this study’s findings of low client receptiveness with service providers occasionally having to revert to paper-based completion of the measures, client completion rates increased significantly with the use of the online system of ROM. Previously with a solely paper-based system, only 16% of clients completed assessments; however, after implementation of the online system, the completion rates increased to 54%. A study evaluating the benefits, barriers, and disadvantages of electronic patient-reported outcome measures identified that electronic collection offers more advantages over paper-based methods. The study, conducted via a systematic review of articles that evaluated electronic patient-reported outcome measures identified advantages that included greater patient preference, lower costs, faster completion time, higher data quality, and higher response rates .Upon examination, the benefits of an online system over paper-based methods are clear; however, service providers’ perceptions are mixed. Contradictory results were found with regards to efficiency. Although 56 out of 98 (57%) service providers responded that the online system was less time-consuming than were previous paper-based methods, only 31 out of 98 (31%) responded that the system did not increase their workload. One of the main barriers identified in previous literature to the utilization of ROM has been the significant time burden involved, and it has been suggested that software systems may alleviate some of the burden ,35,36. TWith regard to the service providers’ views on their experience using an online system of ROM, 63 out of 98 (64%) agreed that the system of the online ROM did allow for regular progress monitoring of their clients. However, only a minimal number of service providers recognized benefits with the use of an online ROM for identifying clients at risk for treatment failure  or strengthening the therapeutic alliance . The online system of ROM that was implemented allowed service providers and clients to view progress in graphs in real time. The service provider was then able to determine if the client’s progress was going in a positive or negative direction. The online system also had a dashboard flag to allow the service provider to identify if the client was suicidal or displaying suicidal ideation. This flag is similar to other ROM systems such as the OQ-Analyst feedback system that provides a red alert signal to indicate that the client is responding poorly to treatment ,37. IncrOnly 45 out of 98 (46%) service providers reported that their clients were receptive to using the online system. This finding was underscored in the open-ended comments with some indicating that their clients refused to use the program, preferred paper and pen, or preferred to complete the questionnaires in session with the therapist. Clients also reported they were too depressed or lacked the motivation to use the program or felt the program provoked feelings of frustration, agitation, and anxiety. Similar findings regarding client receptiveness have been identified in the literature with some users expressing frustrations with a complicated or unintuitive interface, or technical issues and malfunctioning websites to the point of giving up . In a 20Finally, our study showed low results regarding service providers receiving adequate training or having help available when using the system was needed. Previous research concurs with these results in that it has been identified that lack of proper training and support is a barrier to successful implementation and utilization of a ROM program and that both these components are needed for successful adoption of an online system . TherefoA limitation in this study is the low survey response rate by service providers, with just 98 out of 324 30.2%) participants responding. Although this response rate and sample size are comparable to those of previous survey studies .2% parti, obtainiFor the purpose of parsimony, each question investigated a different construct. Future studies should use multiple items to enable a measurement of reliability and validity. Recommendations for future research also include an expansion of sociodemographic characteristics of service providers, which may help to explain the current findings. For example, service provider age, ethnicity, length of employment, years of experience, education details, and physical location they are servicing could be included as factors that may influence perceptions of the use of an online system for ROM. Previous research suggests that mental health therapists who have graduated more recently tend to value ROM and are more inclined to use it .A direct survey evaluation of client experience and perceptions using an online system for completing mental health questionnaires is also recommended for future research given the current low receptiveness of clients. This additional information would be beneficial in highlighting necessary improvements that should be made for future implementations. It also would be helpful to reevaluate service providers’ perceptions on the use of an online ROM after an enhancement is made in training and ongoing support to identify if this adjustment would glean improved results.Overall, the findings of this study suggest that online ROM has strong potential to lead to increased adoption of ROM, which has been associated with better outcomes for patients. This should encourage researchers and practitioners to identify and address the barriers and challenges, which, with limited intervention, could increase the chances of future implementation success, improved utilization, and completion of the measures.


TM M-450, M-270, and MyOneTM, respectively. The IL-6 and GFAP QLISAs were successfully multiplexed using a variable height channel that ranged in height from ~7.6 µm at the inlet to ~2.1 µm at the outlet. The IL-6, GFAP, and IL-8 QLISAs were also multiplexed using a channel that ranged in height from ~6.3 µm at the inlet to ~0.9 µm at the outlet. Our system can keep pace with TBI biomarker discovery and validation, as additional protein biomarkers can be multiplexed simply by adding in antibody-conjugated beads of different diameters.Traumatic brain injury (TBI) is a leading cause of global morbidity and mortality, partially due to the lack of sensitive diagnostic methods and efficacious therapies. Panels of protein biomarkers have been proposed as a way of diagnosing and monitoring TBI. To measure multiple TBI biomarkers simultaneously, we present a variable height microfluidic device consisting of a single channel that varies in height between the inlet and outlet and can passively multiplex bead-based immunoassays by trapping assay beads at the point where their diameter matches the channel height. We developed bead-based quantum dot-linked immunosorbent assays (QLISAs) for interleukin-6 (IL-6), glial fibrillary acidic protein (GFAP), and interleukin-8 (IL-8) using Dynabeads Traumatic brain injury (TBI) causes significant morbidity and mortality globally. In the United States alone, an estimated 2.87 million people sustain a TBI annually, and 50,000 of these individuals die of their injuries. Furthermore, 2% of the United States population is estimated to have a disability caused by TBI ,2. The hAssessment of TBI injuries is typically done through neurological examination and neuroimaging techniques. While these methods may identify direct tissue damage to the brain, they are more limited in their ability to monitor secondary damage stemming from the initial injury. The primary tissue damage sets off a cascade of secondary injuries, such as neuronal cell death, blood–brain barrier breakdown, edema, and upregulation of inflammatory markers ,5. Protein biomarkers have been proposed as a way of monitoring the progression of secondary TBI injury and of providing more sensitive diagnostic measures when used in conjunction with imaging and physical examination. Glial fibrillary acidic protein (GFAP) and ubiquitin c-terminal hydrolase L1 (UCH-L1) are currently FDA approved to aid in determining the need for evaluation with a head CT scan . HoweverMany microfluidic devices have been developed to facilitate multiplexed immunoassays. These devices typically employ surface patterning ,12,13, cTo overcome this limitation, we developed a microfluidic system capable of flexible and customizable multiplexing. In this study, we present bead-based quantum dot-linked immunosorbent assays (QLISAs) for GFAP, a marker of astrocyte injury ; interleEach bead-based QLISA was developed using the same set of reagent ratios. The volume of capture antibody-coated superparamagnetic beads was chosen such that there was a 5x molar excess of capture antibodies to the maximum protein  concentration of interest. The volume of detection antibody was chosen such that there was a 10x molar excess of detection antibodies to the maximum protein concentration of interest. Finally, the volume of quantum dots was chosen such that there was a 2x molar excess of quantum dot to detection antibody.TM M-270 Epoxy, InvitrogenTM, Waltham, MA, USA) using the DynabeadsTM Antibody Coupling Kit (InvitrogenTM). An amount of 1 mg of superparamagnetic beads suspended in dimethylformamide  was washed with 1 mL of the C1 reagent from the Antibody Coupling Kit. Next, 5 µg (5 µL) of Human GFAP Capture Antibody was added to the beads, followed by 45 µL of C1 reagent and 50 µL of C2 reagent. The mixture was incubated overnight (16–24 h) on a rocker at 37 °C. Following incubation, the antibody-coated beads were washed with 800 µL of HB reagent + 0.1% Tween 20 and 800 µL of LB reagent + 0.1% Tween 20. The antibody-coated beads were then washed three times with 800 µL of SB reagent followed by incubation with 800 µL of SB reagent on a rocker for 15 min at room temperature. The antibody-coated beads were then suspended in 100 µL of SB reagent and stored at 4 °C.Human GFAP Capture Antibodies from the Human GFAP Matched Antibody Pair Kit  were covalently linked to the surface of superparamagnetic beads . This stock solution was used to spike either normal human serum  or single donor human whole blood  such that the concentrations were 10,000 pg/mL, 5000 pg/mL, 1000 pg/mL, 500 pg/mL, 100 pg/mL, and 0 pg/mL GFAP. The serum and whole blood were rocked for 15 min before and after spiking with GFAP to ensure a homogeneous sample.Human GFAP Lyophilized Protein from the Human GFAP Matched Antibody Pair Kit  was reconstituted at 10 ng/µL in distilled water (Gibco®) along with 50 µL of 1X PBS + 0.1% Tween 20. The tube was then placed on a magnet , and the supernatant was removed. Next, 100 µL of either spiked human serum or whole blood was added to the beads, and the mixture was incubated at room temperature for 15 min on a rocker. The tube was placed on a magnet again, and the supernatant was removed. An amount of 0.3 µL of biotinylated Human GFAP Detector Antibody from the Human GFAP Matched Antibody Pair Kit, 1 µL of QdotTM 585 Streptavidin Conjugate (InvitrogenTM), and 50 µL of 1X PBS + 0.1% Tween 20 was then added to the beads, and the mixture was incubated at room temperature for 15 min on a rocker. The beads were then washed three times in 1X PBS + 0.1% bovine serum albumin (BSA) , washed once in 1X PBS, and resuspended in either 100 µL of 1X PBS or 100 µL of StartingBlockTM (PBS)  Blocking Buffer ).To run the assay, 0.75 µL of antibody-coated beads was placed in a 1.7 mL polypropylene microcentrifuge tube  using the DynabeadsTM Antibody Coupling Kit (InvitrogenTM). An amount of 200 µL of superparamagnetic beads was washed with 1 mL of the C1 reagent from the Antibody Coupling Kit. Next, 40 µg (40 µL) of Human IL-6 Capture Antibody was added to the beads, followed by 60 µL of C1 reagent and 100 µL of C2 reagent. The mixture was incubated overnight (16–24 h) on a rocker at 37 °C. Following incubation, the antibody-coated beads were washed with 800 µL of HB reagent + 0.1% Tween 20 and 800 µL of LB reagent + 0.1% Tween 20. The antibody-coated beads were then washed three times with 800 µL of SB reagent, followed by incubation with 800 µL of SB reagent on a rocker for 15 min at room temperature. The antibody-coated beads were then suspended in 200 µL of SB reagent and stored at 4 °C.Human IL-6 Capture Antibodies from the Human IL-6 Matched Antibody Pair Kit  were covalently linked to the surface of superparamagnetic beads (DynabeadsTM). This stock solution was used to spike Blocking Buffer (1X PBS + 0.1% BSA + 0.05% Tween 20) such that the concentrations were 25,000 pg/mL, 15,000 pg/mL, 10,000 pg/mL, 5000 pg/mL, 1000 pg/mL, and 0 pg/mL IL-6. All spiked Blocking Buffer samples were inverted to mix.Human IL-6 Lyophilized Protein from the Human IL-6 Matched Antibody Pair Kit  was reconstituted at 10 ng/µL in distilled water (Gibco®) along with 50 µL of 1X PBS + 0.1% Tween 20. The tube was then placed on a magnet , and the supernatant was removed. Next, 100 µL of spiked Blocking Buffer was added to the beads and the mixture was incubated at room temperature for 15 min on a rocker. The tube was placed on a magnet again, and the supernatant was removed. An amount of 0.72 µL of biotinylated Human IL-6 Detector Antibody from the Human IL-6 Matched Antibody Pair Kit, 2 µL of QdotTM 525 Streptavidin Conjugate (InvitrogenTM), and 50 µL of 1X PBS + 0.1% Tween 20 was then added to the beads, and the mixture was incubated at room temperature for 15 min on a rocker. The beads were then washed three times in 1X PBS + 0.1% BSA , washed once in 1X PBS, and resuspended in either 100 µL of 1X PBS or 100 µL of StartingBlockTM (PBS) Blocking Buffer (Thermo ScientificTM).To run the assay, 0.5 µL of antibody-coated beads was placed in a 1.7 mL polypropylene microcentrifuge tube . An amount of 10 µL of superparamagnetic beads was washed with 1 mL of 0.1 M sodium borate buffer (pH 9.5). Next, 94 µL of 0.1 M sodium borate buffer (pH 9.5), 40 µL of Human IL-8 Capture Antibody, and 66 µL of 3 M ammonium sulfate was added to the beads. The mixture was incubated overnight (16–24 h) on a rocker at 37 °C. Following incubation, the tube was placed on a magnet, and the supernatant was removed. An amount of 200 µL of 1X PBS + 0.5% BSA + 0.05% Tween 20 was then added to the beads, and the mixture was incubated overnight (16–24 h) again at 37 °C. Following the second incubation, the beads were washed three times with 1X PBS + 0.5% BSA + 0.05% Tween 20. The antibody-coated beads were then suspended in 100 µL of 1X PBS + 0.5% BSA + 0.05% Tween 20 and stored at 4 °C.Human IL-8 Capture Antibodies from the Human IL-8 Matched Antibody Pair Kit  were covalently linked to the surface of superparamagnetic beads (DynabeadsTM). This stock solution was used to spike Blocking Buffer (1X PBS + 0.1% BSA + 0.05% Tween 20) such that the concentrations were 1000 pg/mL, 800 pg/mL, 500 pg/mL, 200 pg/mL, 100 pg/mL, 10 pg/mL, and 0 pg/mL IL-8. All spiked Blocking Buffer samples were inverted to mix.Human IL-8 Lyophilized Protein from the Human IL-8 Matched Antibody Pair Kit  was reconstituted at 0.98 ng/µL in distilled water (Gibco®) along with 50 µL of 1X PBS + 0.1% Tween 20. The tube was then placed on a magnet , and the supernatant was removed. Next, 100 µL of spiked Blocking Buffer was added to the beads and the mixture was incubated at room temperature for 15 min on a rocker. The tube was placed on a magnet again, and the supernatant was removed. An amount of 0.72 µL of biotinylated Human IL-8 Detector Antibody from the Human IL-8 Matched Antibody Pair Kit, 2 µL of QdotTM 655 Streptavidin Conjugate (InvitrogenTM), and 50 µL of 1X PBS + 0.1% Tween 20 was then added to the beads, and the mixture was incubated at room temperature for 15 min on a rocker. The beads were then washed three times in 1X PBS + 0.1% BSA , washed once in 1X PBS, and resuspended in either 100 µL of 1X PBS or 100 µL of StartingBlockTM (PBS) Blocking Buffer (Thermo ScientificTM).To run the assay, 0.5 µL of antibody-coated beads was placed in a 1.7 mL polypropylene microcentrifuge tube (VWRn = 10). The intra-assay CV was calculated as the SD divided by the mean of replicates (n = 3) of the same sample run on the same day. The inter-assay CV was calculated as the average of the CVs for the same sample from three different days.The limit of detection (LOD) and coefficient of variation (CV) for each QLISA were calculated as described in Krausz et al. . BrieflyTM220 photoresist was spin-coated using an ACS 200 Cluster Tool (Süss Microtec) and exposed for 15 s with 405 nm at 20 mJ/s using an MA/BA-6 Mask/Bond Aligner (Süss Microtec) and a low-cost photomask (Fineline Imaging). Exposed wafers were developed for 60 s in MF-319 using an ACS 200 Cluster Tool (Süss Microtec). Au and Cr layers were removed by etching for 72 s in TFA gold etchant (Transene) and 16 s in Chromium Etch 1020 .The variable height etching procedure presented here is a modified version of the original method described by Mena at al. . MDF polw/w HF diluted with deionized water) using a custom linear screw mechanism. After the channels were etched, the wafer was rinsed in deionized water for 5 min. The resulting etched channels were measured using a surface profilometer . The photoresist was then removed with acetone and isopropyl alcohol, followed by the removal of the Au/Cr layers with their respective etchants. The glass wafer was then dipped in buffered hydrofluoric acid (BHF) for 5 s to remove residual Cr.The masked glass wafers were gradually lowered into 15% hydrofluoric acid (HF) solution  heated to 70 °C. Drilling layouts were prepared using Autodesk Fusion 360 and exported as gcode to a CNC router (Tormach PCNC). A peck-drilling operation was used with a 750 µm diamond micro drill (Amplex S-Series 0.030”), a drill speed of 10,000 RPM, a peck height of 50 µm, and a feed rate of 5 mm/min. The drilled silicon wafer and etched glass wafer were anodically bonded on an SB-6E bonder (Süss Microtec) at 350 °C and an applied voltage of 100 V until the current reached 10% of its peak value.®). The microcentrifuge tube was adapted by punching a hole in the lid and a hole in the bottom using a scratch awl and rubber mallet. The pressure input was placed into the hole in the lid , and the tube  to deliver the sample to the device was placed in the hole in the bottom. The tubing and dispensing needle were secured with two-part epoxy (Gorilla). The tubing was connected to the variable height device channel inlet using a microfluidic probe with a compression sealing mechanism (CorSolutions microfluidic connectors BMP-LP-2X), and pneumatic pressure was applied to drive the sample into the channel that had been primed with either 5% BSA or StartingBlockTM (PBS) Blocking Buffer (Thermo ScientificTM). The sample flowed through the channel until a band of beads formed that was visible to the naked eye.The sample of processed and resuspended assay beads was placed into a modified 1.7 mL polypropylene microcentrifuge tube (VWRFluorescent images (1344 × 1100 pixels) of assay beads trapped in the variable height device were obtained using a Nikon Eclipse Ti inverted microscope and a Retiga R6 monochrome CCD camera (Teledyne Photometrics). A fluorescent image and an epi-illuminated image were obtained for each field of view. ImageJ macros were used to pseudo-flat field correct the epi-illuminated image and to background correct the fluorescent images. A Python script was used to perform the following operations: (1) threshold and binarize the pseudo-flat field-corrected epi-illuminated image to yield B’, (2) perform an elementwise multiplication of B’ and the corresponding background-corrected fluorescent image to yield F’, (3) calculate the sum of all values in B’ to yield b, (4) calculate the sum of all values in F’ to yield f, (5) determine the ratio of f to b to yield RFU/Bead Area. The ImageJ macros and Python script used for image analysis can be found on GitHub .TM 585 Streptavidin Conjugate as the fluorescent signal for both assays. Once again, a sample was used that contained both proteins. Third, the GFAP, IL-6, and IL-8 QLISAs were carried out as described in Multiplexed QLISAs were carried out in three different ways. First, the GFAP and IL-6 QLISAs were carried out as described in TM M-450 Epoxy, DynabeadsTM M-270 Epoxy, or DynabeadsTM MyOneTM Tosylactivated was placed on a glass slide under a coverslip with one drop (~50 µL) of SlowFadeTM Diamond Antifade Mountant (Molecular Probes #S36967). Beads were excited using a 532 nm laser and imaged from 554 nm to 697 nm with a Zeiss LSM 780 Confocal Microscope equipped with a 20X water immersion objective .An amount of 20 µL of either DynabeadsMaximum intensity Z-projections of each Z-stack were generated and thresholded (binarized) using ImageJ and the Default Local Threshold algorithm. The Analyze Particles plugin from ImageJ was used to identify and measure the area of each bead.TM can be found in The assay steps described in Images of the fluorescent bands were analyzed as described in The dynamic range of both the serum and whole blood curves was within the physiologically relevant range of GFAP concentrations post-TBI, 153–2089 pg/mL . The GFAThe LOD of the IL-6 assay in buffer C was 437We successfully developed three different QLISAs for three different TBI protein biomarkers using three different bead sizes and three different quantum dot colors. However, the system is not limited to the analytes, bead sizes, and quantum dots described above. If the chosen assay beads have surface epoxy or tosyl groups, the antibody pair is specific to the analyte of interest, and the appropriate microscope filters are available to analyze the chosen quantum dots, the design parameters discussed in ® assay [The GFAP standard curve A was use® assay ,31, one ®  assay is due to differences in the form of GFAP recovered by the antibodies. The Abcam antibodies (ab222279) used in the GFAP QLISA detect native GFAP (50 kDa). However, the Quanterix Simoa® assay uses antibodies from Banyan Biomarkers® that detect both native GFAP as well as GFAP breakdown products (GFAP-BDP) (50–38 kDa) [® assay to be higher than the GFAP QLISA, as seen in We hypothesize that the discrepancy between the concentrations from the GFAP QLISA and Quanterix Simoa–38 kDa) . GFAP is–38 kDa) ,34. TherAn outstanding question in TBI biomarker analysis is whether there are advantages to monitoring intact GFAP vs. GFAP-BDPs. It remains to be determined if intact GFAP or GFAP-BDPs better reflect the extent or kinetics of astrocyte injury following a TBI . Since tTM M-450 (~4.5 µm diameter) and DynabeadsTM M-270 (~2.8 µm) with completed sandwich complexes for IL-6 and GFAP, respectively, were introduced into the channel. The beads flowed through the channel and passively separated into two distinct bands where the diameter of the beads matched the height of the channel. Because the ~2.8 µm beads flowed through the band of ~4.5 µm beads as it was forming, it is possible that the ~2.8 µm assay beads could be trapped upstream of where the channel height matched their diameter.The design of the variable height device lends itself to multiplexing of bead-based immunoassays. Variable height channels with a height of ~7.6 µm at the inlet and ~2.1 µm at the outlet were used to multiplex the IL-6 QLISA and GFAP QLISA . A mixtuTM 525 as the fluorescent signal for the IL-6 QLISA and QdotTM 585 as the fluorescent signal for the GFAP QLISA. TM 525 and QdotTM 585 channels. Ideally, the IL-6 detection band should appear completely blue, and the GFAP detection band should appear completely yellow. TM 585 channel (yellow bar) was minimal in comparison with the intensity from the QdotTM 525 channel (blue bar) such that it would not influence the overall protein concentration determination. In the GFAP detection band , QdotTM 585 as the fluorescent signal for the GFAP QLISA (~2.8 µm assay beads), and QdotTM 655 as the fluorescent signal for the IL-8 QLISA (~1 µm diameter assay beads). A mixture of the different-sized assay beads with completed sandwich complexes for the proteins of interest was introduced into the channel and allowed to passively separate. TM 525, QdotTM 585, and QdotTM 655 channels. Ideally, the IL-6 detection band should appear completely blue, the GFAP detection band should appear completely yellow, and the IL-6 detection band should appear completely magenta. TM 585 channel (yellow bar) and the QdotTM 655 channel (magenta bar) was minimal in comparison with the intensity from the QdotTM 525 channel (blue bar). This was similarly the case in the GFAP detection band (center pair of bars in TM 585 channel (yellow bar) was the strongest, and in the IL-8 detection band (right pair of bars in TM 655 channel (magenta bar) was the strongest. As in the two-quantum dot color multiplex of IL-6 and GFAP, the signal from the QdotTM 525 channel (blue bar) in the GFAP detection band was due to strong autofluorescence of the assay beads at 525 nm.Following the successful multiplex of the IL-6 and GFAP QLISAs, all three QLISAs  were multiplexed using variable height channels with a height of ~6.3 µm at the inlet and ~0.9 µm at the outlet . We usedTM colors. Therefore, we performed a multiplex using QdotTM 585 as the fluorescent signal for both the IL-6 QLISA and GFAP QLISA by combining all assay reagents in the same microcentrifuge tube. The results of this single microcentrifuge tube multiplex are presented in There was minimal crossover between the detection bands when the two-plex and three-plex were run using different Qdott-test (p = 0.42), indicating that the co-trapped beads would not influence the protein concentration determination. Additionally, the bright spots in the GFAP detection band in To investigate the potential of the bead crossover between detection bands to generate false positive signals, we statistically compared the fluorescent intensity of the IL-6 detection bands in TM colors as well as a two-plex using the same QdotTM color for both assays. During the two-plex assays, the mixture of assay beads separated into distinct assay bands after 4 min, but during the three-plex assay, the mixture of beads took 40 min to separate. To shorten the time it takes to form detection bands, care should be used when selecting the bead size associated with each QLISA. The bead diameter determines the height profile of the channel, and the height profile in turn determines the flow rate according to Equation S15 in the We successfully performed a two-plex and three-plex using different QdotHere was presented a variable height microfluidic device that is capable of passively multiplexing bead-based QLISAs for IL-6, GFAP, and IL-8, three TBI protein biomarkers. The assay design principles presented can be applied to any analyte of interest for which a sandwich or competitive immunoassay is feasible, making the QLISA method widely applicable to diagnosing and monitoring conditions that impact human health. Our platform decouples the QLISAs from the variable height channel, allowing for highly flexible multiplexing. Any mixture of antibody-conjugated beads of different diameters can be introduced into the channel, and the beads will passively separate into distinct bands as they flow through the channel, resulting in a custom barcode of biomarker concentrations. There is no need to actively pattern antibodies in the channel during fabrication, as the differences in bead size coupled with the channel height gradient lead to passive patterning. We used the variable height device to successfully multiplex QLISAs for IL-6 (~4.5 µm diameter assay beads) and GFAP (~2.8 µm diameter assay beads) as well as QLISAs for IL-6, GFAP, and IL-8 (~1 µm diameter assay beads). However, additional biomarkers could be multiplexed simply by adding in antibody-conjugated beads of a different diameter.Ideally, the variable height device can be used to multiplex up to 20 different biomarkers. Each band of trapped beads is approximately 500 µm wide, and a channel height profile slope should be chosen that results in at least a 500 µm separation between the detection bands. Therefore, a 20 mm long channel could accommodate 20 different bands of beads, effectively multiplexing 20 different biomarkers. It is imperative to select assay beads that are homogeneous in size to facilitate the multiplex. Bead sizes should also be selected such that the channel outlet height is 1.5 µm or greater to ensure that the flow rate in the channel is sufficient to separate the beads within 15 min and prevent bead aggregation and adsorption. As more bead sizes are added to the channel, bead crossover between detection bands will increase. Each band of beads acts as a leaky filter as it forms, trapping some smaller diameter assay beads while allowing the majority to pass through. Detection band crossover can be mitigated by optimizing the number of beads of each size introduced into the channel such that fewer larger diameter beads are introduced, delaying the formation of upstream detection bands while downstream bands form. Of course, multiplexing of biomarkers in the variable height device is limited by antibody specificity and affinity for an analyte of interest. If an antibody is cross-reactive, multiplexing will be limited.Since the assay beads spatially separate into distinct bands due to the ramp-like height profile of the channel, the same quantum dot can be used to detect each biomarker, simplifying the development of a point-of-care optics module. In this work, a fluorescence microscope was used to analyze the fluorescence intensity of the bands of beads, but a portable optics module would allow our device to be used wherever it is needed. A portable platform could be used by military personnel for rapid triage in the field, by coaches to assess injured players on the sidelines, by paramedics to assess a patient’s condition before arrival at a hospital, by clinicians to monitor a patient’s response to neuroprotective treatments, and by clinical research teams to accurately enroll and group patients for clinical trials. Ultimately, our platform has the potential to not only transform the field of TBI diagnosis but to expand along with it.


The emergence of SARS-CoV-2 variants complicates efforts to control the COVID-19 pandemic. Increasing genomic surveillance of SARS-CoV-2 is imperative for early detection of emerging variants, to trace the movement of variants, and to monitor effectiveness of countermeasures. Additionally, determining the amount of viable virus present in clinical samples is helpful to better understand the impact these variants have on viral shedding. In this study, we analyzed nasal swab samples collected between March 2020 and early November 2021 from a cohort of United States (U.S.) military personnel and healthcare system beneficiaries stationed worldwide as a part of the Defense Health Agency's (DHA) Global Emerging Infections Surveillance (GEIS) program. SARS-CoV-2 quantitative real time reverse-transcription PCR (qRT-PCR) positive samples were characterized by next-generation sequencing and a subset was analyzed for isolation and quantification of viable virus. Not surprisingly, we found that the Delta variant is the predominant strain circulating among U.S. military personnel beginning in July 2021 and primarily represents cases of vaccine breakthrough infections (VBIs). Among VBIs, we found a 50-fold increase in viable virus in nasal swab samples from Delta variant cases when compared to cases involving other variants. Notably, we found a 40-fold increase in viable virus in nasal swab samples from VBIs involving Delta as compared to unvaccinated personnel infected with other variants prior to the availability of approved vaccines. This study provides important insight about the genomic and virological characterization of SARS-CoV-2 isolates from a unique study population with a global presence. The COVID-19 pandemic is currently a leading cause of death globally and new SARS-CoV-2 variants continue to emerge. Genomic surveillance of variants is necessary to characterize mutations that could affect transmissibility and spread, antigenicity or virulence. Furthermore, wet lab studies are necessary to evaluate how genetic differences may affect viral fitness and transmissibility. The Delta variant is currently the predominant strain globally and determining factors that drive its ability to spread rapidly is important. In this study, we characterized SARS-CoV-2 positive samples from U.S. military personnel and their beneficiaries by next-generation sequencing and isolation of viable virus. Consistent with other studies, we found higher levels of infectious virus from Delta samples when compared to non-Delta infections. Strikingly, we found the difference in titer between Delta and other strains to be so profound as to be unaffected by vaccination status, suggesting that increased transmissibility of the Delta variant is in part due to higher amounts of virus shedding. This helps explain the rapid spread of the Delta variant and provides the impetus to increase control measures such as vaccination, boosters, masking and distancing requirements. It will be necessary to continue genomic and virological characterization of new variants, such as Omicron.The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has led to a historic global pandemic. Multiple SARS-CoV-2 genetic variants are circulating globally, some of which have mutations of concern or mutations of interest that could impact transmissibility and spread, antigenicity or virulence . The DelQuantitative real time reverse-transcription PCR (qRT-PCR) is the benchmark method for COVID-19 diagnosis, but an important limitation of this approach is the assay only detects RNA and does not detect infectious virus. This can be problematic since several studies have described the persistence of SARS-CoV-2 RNA within different body sites –10 and vin vitro viral culture on susceptible cell lines. However, the ability of viral culture methods to provide insight on infectious shedding is hampered by the fact that this method is labor-intensive and requires biosafety level 3 (BSL-3) facilities for SARS-CoV-2. There are also assay variables such as the cell line and methods used to quantify the amount of virus. Vero cells and their subclones or derivatives are commonly used to culture SARS-CoV-2 and Vero E6 cells expressing human transmembrane serine protease 2 (TMPRSS2) have demonstrated enhanced susceptibility and isolation of SARS-CoV-2 (50) assays are commonly used to quantify viable virus, but new methods have been described like the S-Fuse assay that uses a cell reporter system and is based on the detection of GFP positive syncytia formation between infected and neighboring cells   was used to collate SARS-CoV-2 consensus genomes . Brieflyangolin) .2 flasks. For the plaque assay, duplicate wells were infected with 0.2 mL aliquots from serial 10-fold dilutions in Minimum Essential Medium (MEM) followed by a 1 h incubation at 37°C, 5% CO2 to allow virus adsorption to occur. After incubation, cells were overlaid with MEM containing 0.5% agar supplemented with 5% heat-inactivated FBS, 1% Penicillin/Streptomycin and incubated for 48 h at 37°C, 5% CO2. Cells were fixed in 10% formalin prior to staining with crystal violet. For the CPE assay, cells were seeded in T25 cm2 flasks and each flask was infected with 0.5 mL aliquots from a 1:10 dilution in Dulbecco's Modified Eagle Medium (DMEM) followed by a 1 h incubation at 37°C, 5% CO2. After incubation, 5 mL of DMEM supplemented with 5% heat-inactivated FBS, 1% Penicillin/Streptomycin was incubated for 4 days at 37°C, 5% CO2. CPE was monitored daily. After 4 days, supernatant was passed onto fresh cells to allow additional time to amplify. The presence of SARS-CoV-2 was confirmed by lateral flow immunoassay using the Quidel Sofia 2 analyzer and/or qRT-PCR.Patient samples were cultured for SARS-CoV-2 using a standard plaque assay on Vero E6 cells expressing TMPRSS2 [4] in six-well plates and a cytopathic effect (CPE) assay on Vero E6/TMPRSS2 cells in T25 cmStatistical analysis was performed using GraphPad Prism. Tukey's multiple comparisons test was used to compare the amount of viable virus between the unvaccinated and vaccinated groups.We have sequenced over 2,300 nasal swab samples collected from March 2020 until early November 2021 and of those, we produced 1,304 coding complete genomes for which we then determined the Pango lineages . Smaller10 PFU/mL, which is similar to the amount of virus detected from VBIs associated with all variants except for Delta (3.1 log10 PFU/mL). Significantly more virus was detected in VBIs associated with the Delta variant when compared to unvaccinated personnel. The mean amount of virus detected in VBIs associated with the Delta variant was 4.6 log10 PFU/mL, which was statistically significant by Tukey's multiple comparisons test (p = 0.0001) when compared to the amount of virus detected in unvaccinated personnel prior to the emergence of VOCs. In contrast to the unvaccinated samples, almost all the VBI samples associated with the Delta variant had detectable virus that was above the level of detection for the plaque assay and one sample had a titer as high as 7 log10 PFU/mL. Samples from unvaccinated personnel infected with the Delta variant were not available for this study since the majority of military personnel were fully vaccinated when the Delta variant became the predominant strain. The amount of virus detected in VBIs associated with the Delta variant compared to VBIs with other variants was also statistically significant by Tukey's multiple comparisons test (p = 0.0132) although with less power because of the low number of VBI samples associated with other variants. The results are further broken down by vaccine manufacturer (p = 0.0106) and Johnson & Johnson (p = 0.0427) vaccines. Interestingly, VBI associated with the Delta variant that received the Moderna vaccine was not statistically significant when compared to the unvaccinated group. This may be a reflection of the limited number of samples in our analyses.A subset of samples were selected to determine if viable virus was present where we compared viral titers in samples collected from unvaccinated individuals and cases of VBI . A summafacturer  where weThe Delta variant displaced the Alpha variant and was identified as the predominant strain in military personnel starting in July 2021. Currently, multiple sublineages of B.1.617.2 continue to be identified. A greater proportion of the VBI cases in our dataset have been associated with the Delta variant, commensurate with its current preponderance in the population at this point in time. We quantified the amount of viable virus in a subset of samples, and our results suggest that the increased transmissibility of the Delta variant is in part due to higher amounts of virus shedding. We detected 50-fold more infectious virus in VBIs involving Delta compared to VBIs with other variants. We also detected 40-fold more infectious virus in VBIs involving Delta compared to unvaccinated personnel infected with non-Delta variants.Our results are similar to what has been reported for the rest of the population in the U.S. where the Delta variant is currently the predominant strain and is more than twice as infectious as previous strains. The improved viral fitness of the Delta variant has been associated with mutations in the furin cleavage site that allow the virus to enter cells more efficiently , 28. MosNumerous studies measure viral load by the amount of viral RNA in a clinical sample, but do not determine the amount of viable virus. This is likely due to the cumbersome nature of the assays used to quantify levels of infectious virus coupled with the requirement to perform these assays in a BSL-3 laboratory. However, a recent study found a significant amount of variability in the relationship between viral titers for infectious virus and viral RNA levels . Their rOther studies have also found that viable virus is more likely to be isolated from clinical samples from Delta compared to Alpha . The autOur results highlight the importance of characterizing the SARS-CoV-2 genome and infectious viral titers of emerging variants isolated from unvaccinated and vaccinated personnel. This work contributes to the growing body of evidence that the increased fitness of Delta is primarily due to viral genetic determinants that increase infectivity or replication. This helps to explain the rapid spread of the Delta variant and provides the impetus to increase control measures such as vaccination, boosters, masking and distancing requirements to prevent ongoing transmission. Furthermore, this study demonstrates the value of pairing genomic and virological characterization when assessing novel SARS-CoV-2 variants. These parallel approaches should be applied to newly emerging variants, such as Omicron, and other variants that may arise in the future.https://www.ncbi.nlm.nih.gov/genbank/) and GISAID (https://www.gisaid.org/). The names of the repository/repositories and accession number(s) can be found in the The datasets presented in this study can be found in online repositories: GenBank , Global Emerging Infections Surveillance (GEIS) Branch, ProMIS IDs P0013_AH_01.01, P0166_20_NM, P0093_21_NM, and P0199_21_NM to KB-L as well as Navy Work Unit Number (WUN) A1417.The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of Defense, the Navy, or the U.S. Government. Several of the authors are U.S. Government employees. This work was prepared as part of their official duties. Title 17 U.S.C. § 105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.' Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.CS was employed by company Parsons. ML, LV, KL, GR, AL, HM, LG, AB, RM, and FM were employed by company Leidos. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


FMR1 gene. These disorders are named for FRAXA, the folate-sensitive fragile site that localizes with the CGG-repeat in individuals with FXS. Two pathological FMR1 allele size classes are distinguished. Premutation (PM) alleles have 54–200 repeats and confer the risk of fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI). PM alleles are prone to both somatic and germline expansion, with female PM carriers being at risk of having a child with >200+ repeats. Inheritance of such full mutation (FM) alleles causes FXS. Contractions of PM and FM alleles can also occur. As a result, many carriers are mosaic for different sized alleles, with the clinical presentation depending on the proportions of these alleles in affected tissues. Furthermore, it has become apparent that the chromosomal fragility of FXS individuals reflects an underlying problem that can lead to chromosomal numerical and structural abnormalities. Thus, large numbers of CGG-repeats in the FMR1 gene predisposes individuals to multiple forms of genome instability. This review will discuss our current understanding of these processes.The Fragile X-related disorders (FXDs), which include the intellectual disability fragile X syndrome (FXS), are disorders caused by expansion of a CGG-repeat tract in the 5′ UTR of the X-linked  FMR1 gene (MIM* 309550) [FMR1 encodes FMRP, an RNA-binding protein important for learning and memory. The repeat tract, which is coincident with the fragile site at Xq27.3, explains the unusual inheritance pattern that had become known as the Sherman paradox; the repeat is expansion prone, tending to gain repeats with each successive generation, with symptoms of FXS only becoming apparent when the repeat number exceeds 200 [FMR1 transcript  for the fragile site that once served as a diagnostic feature of FXS [In the late 1940s, J. Purdon Martin and Julia Bell described an extended family with 13 cases of intellectual disability and an unusual pattern of X-linked inheritance in which the traits could be transmitted by males who displayed no clinical symptoms . Origina MIM* 30950 [FMR1 eeds 200 . FM alleeeds 200 . Both maeeds 200 . PM pathe of FXS .FMR1 gene or critical regions of exon 1. This can result in a deficit of FMRP, resulting in a phenocopy of the symptoms seen in FM carriers [In addition to the expansions that cause these disorders, contractions of the repeat and deletions associated with the loss of flanking sequences are also associated with PM and FM alleles. In some cases, this generates a normal sized allele ,9,10,11.carriers ,16. Contcarriers , but concarriers ,18,19,20carriers . This mocarriers ,23,24. Icarriers . A high carriers . Thus, eFMR1 locus. These diseases belong to a much larger group of clinical conditions known as the repeat expansion diseases that all result from expansion of a disease-specific short tandem repeat tract [FMR1 locus in PM and FM carriers is still an open question. This review will discuss evidence from PM and FM carriers and patients with other repeat expansion diseases that allows us to narrow our focus to a subset of possible mechanisms that account for these events.There are several other diseases resulting from unstable CGG-repeat tracts and their associated folate-sensitive fragile sites . The mecat tract . Whetherat tract ,30,31 foFMR1 region [By metaphase, chromosomes are normally fully condensed in preparation for anaphase and cytokinesis. However, at this stage, fragile sites have a microscopic appearance consistent with chromatin that has not yet been condensed. In the case of FRAXA, expression of the fragile site is limited to FM alleles that are transcriptionally silenced . This do1 region ,34.FMR1 locus [In contrast to the many fragile sites induced by aphidicolin, a DNA polymerase inhibitor, FRAXA and other fragile sites associated with long CGG-repeat tracts are induced by treatments that negatively impact thymidylate synthase, and thus the size and composition of the nucleotide pools available for replication . CGG-repR1 locus . These uR1 locus . ExtendeR1 locus .FM alleles undergo mitotic DNA synthesis (MiDAS) in response to folate stress . MiDAS iFMR1 locus is known to be directly related to the length of the repeat tract and inversely related to the number of AGG interruptions often present at the 5′ end of the repeat tract [The risk of CGG-repeat expansion at the at tract ,49,50. Iat tract . Increasat tract . While Fat tract  and smalOnce alleles expand into the FM range and become methylated, the repeat tract ceases to be expansion-prone ,54. It iWhile the hypothesis that contractions and expansions result from the same underlying process is reasonable, there is evidence to suggest that this is not the case. For example, slowing replication with FdU in lymphoblasts from human PM carriers results in contractions, but not expansions . While iFMR1 locus. However, expansions in the FXDs share many similarities with other repeat expansion diseases. This includes the small number of repeats gained with each expansion event, their frequency, their dependence on transcription, and the fact that expansion occurs in non-dividing cells. In fact, expansions in non-dividing striatal neurons in Huntington disease can result in the gain of many hundreds of repeats during an individual’s lifetime [To date, no genome-wide association studies of PM carriers has been done to identify trans-acting genetic factors involved in the CGG-repeat expansion process at the lifetime . Work frlifetime ,61. Speclifetime .These same proteins play equivalent roles in promoting and protecting against both somatic and germline expansions in a knock-in mouse model of the FXDs ,64,65,66MSH2, the binding partner of MSH3 in the MutSβ complex, is also required for expansion in the FXD mouse model , while MFMR1 locus [Why transcription is required for repeat expansion is unknown. One early idea in the field was that it could be related to problems associated with long CGG-repeat tracts that were resolved by transcription coupled repair (TCR), the major DNA repair pathway limited to transcribed regions of the genome . HoweverR1 locus ,78,79,80R1 locus , expansiR1 locus ,82. R-loR1 locus . A role R1 locus ,84.FMR1 locus likely forms an intra-strand structure in patient cells [As illustrated on the right-hand side of nt cells ,80. Resont cells . Cleavagnt cells . Thus, MA CGG-reporter construct integrated into a murine erythroid leukemia cell line shows both expansions and contractions occurring at approximately equal frequencies. These events depend upon the Polδ subunit POLD3 and the recombination proteins RAD51 and RAD52 . It is pWhile the loss of MMR proteins such as MLH3 eliminates most, if not all, expansions in mice, it could be argued that, in humans, some expansions arise via an MMR-independent mechanism. In particular, it has been suggested that the large increases in repeat number associated with FM alleles inherited from female PM carriers might arise from a mechanism that generated a large expansion in a single event. BIR has been proposed as just such a mechanism . HoweverWhen cells from PM carriers are grown in the presence of FdU, contractions rather than expansions are observed . This miSilenced FM alleles do contract ,58, thusFMR1 locus that reduces the sequence space, it is possible that such MHs reflect the mechanism involved in the generation of these deletions. Such MHs are the hallmarks of a process known as microhomology mediated end joining (MMEJ). As illustrated in Notably, most of the breakpoints of larger deletions seen in the children of PM and FM carriers that have been characterized at the sequence level are associated with microhomologies (MHs) of 2–9 nucleotides ,100,101.EagI site associated with a contraction of the repeat have been reported in humans [EagI recognition site is CGGCCG, the new site could arise either from a base substitution or from the insertion of a CCG triplet. We have observed the de novo generation of a SmaI site (CCCGGG) within the repeat in our FXD mouse model that might have arisen the same way . Thus, MMEJ and Polθ-mediated end-joining, in particular, may be an appealing candidate for the process that generates contractions or deletions in PM and FM carriers.Breaks with minimal resection could result in deletions within the repeat (contractions), while more extensive resection would result in deletions that extend into the flanking regions. MMEJ is often accompanied by nucleotide insertions at break sites. The insertions are often derived from sequences close to the breaks or are added de novo because of non-templated extension of the 3′-termini of breaks. These insertions are characteristic of Polθ, the major polymerase implicated in MMEJ . A 7 bp n humans ,104,105.FMR1 locus represent a triple threat to the genome. As illustrated in In summary, expanded repeats at the FMR1 locus, but, by extension, are likely to be relevant to the many other CGG-repeat tracts that are seen in human genomes. While the current data indicate that the sources of genetic instability at the FMR1 locus, expansion, contractions/deletions, and chromosome fragility result from different problems caused by the expanded CGG repeat tract, more work is needed to better understand the mechanisms responsible and what, if any, overlap there is in the pathways involved. While, at this point, there is no evidence for a role of MMR in chromosome fragility, the bulk of the evidence points to an important role for MMR proteins in many, if not all, expansions, both in the FXDs and in other repeat expansion diseases. This is relevant as there are some encouraging signs that it may be possible to reduce MMR-dependent expansions by targeting components of the MMR machinery like PMS1 that are not associated with significantly increased cancer risk [These threats are not limited to the cer risk .


Tobamovirus genus and TSWV prevailed under greenhouse conditions (28.9% and 5.2%) when compared to open field (11.2% and 1.1%), respectively. Single (28%) and multiple infections (8.9%) were found. All PMMoV isolates were classified as pathotype P1.2. Survey results indicated that tobamovirus and PVY resistance genes would be the most appropriate to be included in breeding programs with these sensitive pepper landraces.Viral diseases have become one of the main phytosanitary problems for pepper growers in the Basque Country (northern Spain). In 2014, a survey was carried out to determine the prevalence of the most common viruses found in Gernika pepper and Ibarra chili pepper landraces. A total of 97 plots were surveyed and classified according to the crop system. Within these plots, 1107 plants were sampled and tested for tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), tobacco mild green mosaic virus (TMGMV), pepper mild mottle virus (PMMoV), paprika mild mottle virus (PaMMV), potato virus Y (PVY) and tomato spotted wilt virus (TSWV) applying a DAS-ELISA test. PaMMV was verified by the non-radioactive molecular hybridization technique and it was found to be negative. All viruses were detected, but the most prevalent viruses were PVY and TMGMV . Differences among cultivation systems were found for most of the tested viruses. PVY had a higher level of infection under open field conditions (27.3%) than under greenhouse conditions (12.3%). Inversely, the viruses belonging to the  Pepper is one of the main vegetable crops in the Basque Country (northern Spain), with a cultivated area of 250 ha in 2018. Pepper crops of this region are mainly concentrated in the Atlantic humid temperate climate zones [Capsicum spp.) are relatively susceptible to viruses that can cause important production losses in this crop worldwide [Potyvirus, Tobamovirus, Tospovirus, Cucumovirus, Polerovirus and Begomovirus [Tobamovirus), tobacco mosaic virus , tomato mosaic virus , potato virus Y , tomato spotted wilt virus , broad bean wilt virus , tomato yellow leaf curl virus , tobacco mild green mottle virus , pepper vein yellows virus  and parietaria mottle virus  [Cucumovirus) and TSWV. Less frequently, alfalfa mosaic virus , BBWV and ToMV have also been found in open field conditions [Cultivated peppers  . Howevernditions . FurtherTobamovirus resistance in pepper is considered to be provided by four allelic genes  [Tobamovirus—P0, P1, P1,2 or P1,2,3-, which are determined by their ability to overcome L gene-mediated resistance [Frankliniella occidentalis in a persistent propagative manner [The viruses that cause the highest crop losses in the region are the group of tobamoviruses, PVY and TSWV. Firstly, tobamoviruses represent the most important phytosanitary problem for protected pepper crop viability in the study region. These viruses are seed-borne and can be mechanically transmitted very efficiently . Tobamov and L4) ,14. Thersistance ,16. Secosistance ,18 by vasistance  and it hsistance . Severalsistance . Finallye manner . A single manner . Howevere manner .Research on the occurrence, incidence and distribution of viral diseases is essential for developing diagnostic tools and appropriate control measures  such as 22,1107 = 0.88; p = 0.6443) or the type of cultivation system   tested positive for at least one of the viruses analyzed . Under o 0.2796) .22,97 = 1.08; p= 0.5807) and the cultivation system   .22,593 = 12.12; p = 0.0023) when it was produced under greenhouse conditions (56.1% in soil greenhouses and 62.5% in hydroponic greenhouses) than when it was produced under open field conditions (34.5%)  .22,290 = 8.20; p = 0.0165) and hydroponic greenhouse  conditions. Under the soil greenhouse conditions, the viral infection was 56.1% in chili pepper, 31.5% in Gernika pepper and 45.8% in roasting pepper. In the hydroponic greenhouses, it was 62.5% in chili pepper, 39.1% in Gernika pepper and there was no viral infection in roasting pepper.Significant differences were also found in viral infection among pepper types produced under soil greenhouse  were plants with symptoms and 354 (32%) were asymptomatic plants. Among symptomatic plants, 343 (45.6%) were positive for any of the viruses analyzed. In contrast, among asymptomatic plants, 64 (18.1%) were positive. The most important viruses detected among the symptomatic plants were PVY (24.5%) and TMGMV (12.1%), as well as in the asymptomatic plants .With respect to the cultivation system, among the plants sampled in open field conditions, 509 (91.9%) were symptomatic and 45 (8.1%) were asymptomatic. In this cultivation system, the number of positive plants to viruses was 185 (36.3%) in symptomatic and 2 (4.4%) in asymptomatic plants. In protected crops, 309 (55.9%) were symptomatic plants and 244 (44.1%) were asymptomatic. In this cultivation system, the number of positive plants to viruses was 158 (64.7%) in symptomatic and 62 (20.1%) in asymptomatic plants.22,1107 = 1.40; p = 0.4975)  and TMV . Plots contaminated by PVY were 77.8% under open field conditions, 40.5% in soil greenhouses and 28.6% in hydroponic greenhouses. Plots infected by TMV were 40.7%, 9.5% and 21.4%, respectively. PVY had a greater presence in the plants taken under open field conditions (27.3%) than in the greenhouse plants (14.8% in soil greenhouses and 9.5% in hydroponic greenhouses) . Nevertheless, TSWV and all the tobamoviruses, except TMV, showed higher sample positivity in greenhouse conditions (p < 0.05)  . However < 0.05) .Tobamovirus predominated over the rest of the viruses analyzed. These viruses were found in 20.1% of the sampled plants  were observed in 91.4% of double infections, and in 100% of triple, quadruple and quintuple infections .All the isolates of PMMoV that were inoculated caused disease in all the plants of the sensitive Negral cultivar, showing typical symptoms of PMMoV (mosaic in upper non-inoculated leaves) . These sThe main pepper landraces grown in the Basque Country were infected by some of the viruses that are usually the most important in pepper crops, in accordance with other research on pepper crops worldwide ,8. In thIn our survey, plants with virus-like symptoms showed negative results for the viruses analyzed by DAS-ELISA. These symptoms could have been caused by the presence of already known pepper viruses not evaluated in this study  or even Capsicum spp. plants using RNA-seq analysis [Tobamovirus that may be in soil-substrate organic matter or in infected seed [Multiple virus infections were found and plants with up to quintuple co-infections were identified. Synergistic viral interactions that result in devastating diseases often occur when two or more unrelated plant viruses infect a plant simultaneously . These manalysis ,33,34. TAccording to other previous studies, the most frequent virus was PVY, which had a greater presence in open field conditions than in greenhouse conditions ,35,36,37Conversely, the incidence of tobamoviruses was greater under greenhouse conditions than under open field conditions. In intensive greenhouse farming systems, the high number of harvests can favor the spread of tobamoviruses in the crops through its mechanical transmission by workers , as it oTobamoviruses may be spread during transplanting and at harvest after primary infections via infected seeds . The relIn this survey, the TMGMV incidence was higher in hydroponic greenhouses than in soil greenhouses. These results appear to be well substantiated by a higher transmission of tobamoviruses through the nutrient solution in the hydroponic cultivation system . It was The use of cultivar resistance against tobamoviruses might be an effective method to control these viral diseases  in the aAs occurred with the tobamoviruses, TSWV incidence was greater under protected crops. The highest incidence of TSWV in this cultivation system could be explained by the lack of insect screens in the greenhouses, the transmission mode of this virus, and the faster life cycle of the insect vector . The milRegarding the cultivation system and virus control strategies, under soil greenhouses, the solution to inactivate tobamoviruses would be to perform biodisinfestation with organic amendments or to compost crop residues for the control of other soil pathogens ,58,59. UThe current status of virus infection in the surveyed area, and the use of landraces or cultivars without virus resistance genes, could promote virus outbreaks in pepper crops. Therefore, backcross breeding programs have been started in order to introduce virus resistance genes to tobamoviruses , PVY and2 (61.8%), 6 samples were surveyed; 10 samples were sampled in plots between 1000–2000 m2 (18.8%); 16 samples were surveyed in plots between 2000–4000 m2 (12.3%) and 40 samples in plots larger than 4000 m2 (7.1%). Each crop plot was completely surveyed and leaves showing virus-like symptoms were given preference when collecting samples with the aim of knowing which viruses are the ones that create the greatest damage in pepper crops in the study area. In those plots with not enough samples with symptoms, asymptomatic plants were randomly sampled.In October 2014, at the end of the crop cycle, a survey of viruses was carried out in 44 counties of the Basque Country . In thisA total of 97 plots were surveyed, which represented an area of 24.3 ha. The surveyed surface corresponded to the open field cultivation system (78%), soil greenhouses (7%), and hydroponic greenhouses (15%). Out of 1107 plants analyzed, 50% were taken under open field conditions and 50% under greenhouse conditions. Under protected conditions, 53% of the samples were taken in soil greenhouses and 47% of the samples were taken in hydroponic greenhouses. Out of the 1107 samples, 53% were samples of chili pepper type, 42% were Gernika pepper type and 5% were roasting pepper type .The samples were tested by the double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA)  techniqu2) tests for equality of distributions. The statistical significance level was fixed at p < 0.05.The significance of differences between the percentages of infected plants and infected plots among pepper type and crop system was tested using Chi-square (χPMMoV pathotypes resistance genes confer resistance to the rest of viruses belonging to the group of tobamoviruses  which arTobamovirus), “Padua RZ F1”  and “Giulio RZ F1” . Furthermore, in each of the three pepper cultivars, three control treatments were included in the inoculation test: Ctr-: Negative control of non-inoculated plants; Ctr + P1.2: Positive control of an isolate belonging to PMMoV pathotype P1.2; Ctr + P1.2.3: Positive control of an isolate belonging to PMMoV pathotype P1.2.3.The leaf samples in which PMMoV infection was detected (36 samples) were frozen at −80 °C. These samples were inoculated in three different pepper cultivars: “Negral”  50 mM at pH 7.5 and 1 g of frozen fresh leaf tissue which had been previously crushed in a mortar to achieve homogenization. Carborundum powder was added for an abrasive effect. Tested plants were inoculated by rubbing the two true basal leaves with the virus inoculum suspension. For the negative control of non-inoculated plants, a suspension was prepared with the tissue of healthy pepper leaves, phosphate buffer, and carborundum. After inoculation, plants were kept in a growth chamber maintained at 25 °C during the day and 20 °C at night, with a 14 h photoperiod. Symptoms were recorded in each of the inoculated plants at 15 and 30 days after inoculation (DAI). When plants were sensitive to the virus, mosaic (M) symptoms were observed on the non-inoculated upper leaves. The plants were considered resistant when necrotic spots (NS), followed by leaf abscission (LA), were observed in the inoculated leaves . These she plant . The DASThe tobamovirus group was predominant in protected crops of the Basque Country, while PVY was predominant in open field conditions. Within the tobamoviruses, TMGMV was the most important. TSWV was also more important in protected cultivation. The seven analyzed viruses  tested positive by DAS-ELISA in all the evaluated cropping systems. However, when PaMMV was analyzed by the non-radioactive molecular hybridization technique, it was found to be negative. It may be possible that it was cross-positive with other tobamoviruses. Various combinations of multiple infections were found, with coinfections of up to five viruses.All PMMoV isolates studied in the bioassay belong to the pathotype P1.2 which could be controlled with the L3 resistance gene in the sensitive pepper landraces grown in the region.The application of anti-insect mesh could be effective to reduce the risk of infection by PVY or TSWV in open field and greenhouse cultivation systems, respectively. Crop infection by tobamoviruses in the greenhouse cultivation system could be reduced by the use of resistance genes combined with the use of certified virus-free disinfested seed and the inactivation of virus inoculum in the crop residues.


The four serotypes of the mature dengue virus can display different morphologies, including the compact spherical, the bumpy spherical and the non-spherical clubshape morphologies. In addition, the maturation process of dengue virus is inefficient and therefore some partially immature dengue virus particles have been observed and they are infectious. All these viral particles have different antigenicity profiles and thus may affect the type of the elicited antibodies during an immune response. Understanding the molecular determinants and environmental conditions  in inducing morphological changes in the virus and how potent antibodies interact with these particles is important for designing effective therapeutics or vaccines. Several techniques, including cryoEM, site-directed mutagenesis, hydrogen-deuterium exchange mass spectrometry, time-resolve fluorescence resonance energy transfer, and molecular dynamic simulation, have been performed to investigate the structural changes. This review describes all known morphological variants of DENV discovered thus far, their surface protein dynamics and the key residues or interactions that play important roles in the structural changes. Dengue virus (DENV) is one of the most important viruses transmitted to humans by the bite of the infected Aedes mosquitoes . DENV isDENV consists of four different serotypes (DENV1-4) ,8. The fThe inside of the mature DENV particle contains a positive-sense single stranded 10.7 kb genomic RNA complexed with capsid (C) proteins . This isThe infection cycle of mature DENV into cells  starts wImmDENV is assembled in the neutral pH environment of the ER. The C, prM and E proteins are the first proteins being translated by ribosomes on the cytoplasmic side of the ER membrane ,33. The The newly formed immDENV contains 180 copies of prM-E proteins assembled into 60 copies of inverted tripod structures, thus making the viral surface look spiky a 14,30],3014,30]We often observed a mixture of mature, partially mature and highly immature virus particles in a mature virus preparation ,42. ThisAs even the highly immDENV has been shown to be infectious to DC-SIGN-bearing cells  and alsoIn the immDENV spiky neutral pH structure, the trimeric prM:E spikes are criss-crossing on the virus surface. Parts of the lipid bilayer are exposed, particularly on the 5- and 3-fold vertices a. The prIt is speculated that the spiky immDENV, after gaining entry into the cell, will undergo massive structural surface rearrangement in the endosome, stimulated by the low pH environment—this motion will likely be similar to that of the newly synthesized immDENV in the cell when it undergoes maturation in the acidic transgolgi-network. The endosome also contains furin protease, and thus will allow cleavage of the prM. However, in the last step of the normal immature virus maturation process, an increase in pH is required  for the pr to detach from the E protein fusion loop, but this is not possible when the virus is in the endosomes, as the environment will continue to become more acidic with time. The question is then how the immDENV when complexed with anti-prM antibody could help to overcome this in the endosome. Wirawan et al.  showed tSome DENV serotypes and strains generate high percentage of mature compact DENV particles, e.g., DENV2 strain PVP94/07  and DENVOn the surface of the mature compact structure, there are 180 copies of E proteins arranged as 90 head-to-tail homodimers a, and thDENV can change from smooth to bumpy-surfaced particles when incubated at 37 or 40 °C, as observed in DENV1 strain WestPac74 and DENV2 strains NGC a 46], S, S46], SThe gaps between the E protein layer and the lipid bilayer of Class II and III particles are likely caused by the E protein ectodomains loosening their interactions with neighboring E proteins and also the E and M protein TM helices on the viral lipid membrane. The Class III cryoEM map b 46] sh sh46] shThe DENV2 strain NGC, which changed from a smooth compact surface to a bumpy surface morphology when the temperature is switched from 28 to 37 °C, has a long passage history in mosquito C6/36 cell lines grown at 28 °C. Meanwhile, the surface morphology of the same NGC strain that was passaged in mammalian BHK21 cells at 37 °C  58] rem rem58] rExamination of four naturally circulated DENV2 strains  showed that at 37 °C, the surface of three of these DENV2 strains  remained smooth, whereas strain PVP103/07 turned into the bumpy morphology . At 40 °Most lab-adapted viruses, with a long passage history in C6/36 mosquito cell lines at 28 °C, have a bumpy surface morphology, while the clinical DENV isolates which circulate between human and mosquito hosts are largely smooth surface particles ,58. The The low resolution cryoEM maps of bumpy surfaced DENV2 strain NGC at 37 °C indicate that the E proteins on virus surface are moving, since the E protein densities are weak and “smeared”. This does not allow us to observe the dynamic motions of structural proteins of the virus. Hence, the amide hydrogen-deuterium exchange mass-spectrometry (HDXMS) method was used to identify important regions in E, M or C proteins at peptide resolution that could play important roles in the structural changes . This teThe DENV surface E proteins are arranged in an icosahedral symmetry with 60 asymmetric units ,14. EachIn contrast with the DENV2 strain NGC, there was no differences observed in the deuterium exchange across peptides of E and M proteins in DENV1 strain PVP159 at 37 °C compared to 28 °C . This suThe movement of the whole E protein shell away from the viral lipid membrane on DENV2 strain NGC and DENV1 strain PVP159 can also be measured by using time-resolved fluorescence resonance energy transfer (TR-FRET) experiments . In the Combining the results from cryoEM, HDXMS and TR-FRET experiments shows that different virus serotypes and strains undergo conformational changes at different temperatures, and also these viruses moved in a different way involving different regions of the E protein on the virus surface. The intradimer E-to-E interaction at the icosahedral 2-fold vertices is shown to be the most mobile part of the DENV2 strain NGC structure, whereas for DENV1 strain PVP159, it is the interactions between E proteins around the 3-fold and/or 5-fold. This is consistent with the previous cryoEM structural analysis comparing the contacts between E-proteins in the DENV1, DENV2 and DENV4 structures . It was A new morphological flavivirus variant adopting a temperature-induced clubshape (ClubSP) morphology has been discovered . It usuaThe clubSP contains a head and a tail. The sizes of the head and the length of the tail vary between particles . HoweverThe helical structure of the Fab ZIKV-Fab C10 catSP shows the E protein dimers arranged parallel to each other in an identical way to that observed between the E protein dimers within a raft of the ZIKV-Fab C10 spherical virus complex structure. In the 4 Å resolution spherical ZIKV-Fab C10 complex structure, Fab C10 not only binds across E protein protomers at the intradimer interface, but it can also lock different E protein dimers together by engaging residues across the inter-dimer interface . Thus, tAmong all DENV serotypes, DENV4 strain 06K2270DK1 has been reported to be the most stable virus structure so far  51]. Si. Si51]. The ability to change into different morphologies and thus their antigenicity properties suggest the virus could use this as a way to escape from the immune system . Below iPreviously, it has been shown that serotype-specific highly neutralizing human monoclonal antibodies (HMAb), such as 1F4, 5J7 or 14c10, recognize the quaternary-dependent epitope presented only on the smooth compact DENV surface ,62,63. TThe ClubSP particles have a head and a tail, and hence the E proteins might have different antigenic characteristic on different parts of the particle. Several antibodies were tested on DENV3 strain CH53489 to determine their ability to bind and neutralize. A mouse monoclonal antibody 8A1 , which rAntibody 4G2, which binds to the DII fusion loop, when mixed with the clubSP particles, showed that the Fab fragment only binds to the head of the clubSP . This isA group of antibodies binding across E protomers within a dimer are named E-protein dimer epitope (EDE) antibodies; some of these antibodies include HMAb 2D22, C8 and C10. Their epitopes in general involve part of DIII on one E protein protomer and also the fusion loop of DII on the opposite protomer within a dimer. While HMAb 2D22 is DENV2 specific ,65, HMAbThere are antibodies that recognize hidden or partially hidden epitopes on the compact smooth surfaced DENV. Thus, whether the antibodies could neutralize them may partly depend on the mobility/transient exposure of these epitopes. As shown by a combination of cryoEM and HDXMS, different virus strains could have different level of E protein mobility at 37 °C — some become bumpy surfaced particles with increased solvent accessibility, while others, that may appear smooth-surfaced under cryoEM, could have local mobility/dynamics of the E protein. The regions with increased dynamics on the virus surface also vary among different DENV serotypes or strains. The increased incubation temperature  and high fever temperature (40 °C)) also caused increased dynamics. Indeed, at 40 °C, most DENV strains, even those exhibiting smooth compact-surfaced morphology at 37 °C, can become bumpy-surfaced, suggesting antibodies that bind well to bumpy surface particles may be a better therapeutic agent. However, for prophylactic treatment, where the antibody is injected prior to infection and hence the antibodies will likely encounter virus at 37 °C, a mixture of antibodies that bind well to both smooth compact-surfaced and bumpy-surfaced particles may work better.Below are examples of antibodies that bind better when temperature is increased to cause viruses to either become bumpy-surfaced or to increase the E protein dynamics on smooth-surfaced virus particles. A study on West Nile virus (WNV), another flavivirus, showed that a fusion loop binding antibody, E53, has a time- and temperature-dependent neutralization profile . This fiAnother study also found that in addition to increased dynamics of E proteins on virus surface, high affinity of antibodies could help them to bind to cryptic epitopes. HMAb 1C19 binds to a partially hidden epitope (hi- and ij-loops) on DII . HMAb 1CMature DENV particle can adopt several different morphologies. At 28 °C, most DENV particles have a spherical smooth surface morphology. However, at higher temperatures, they can either remain as spherical smooth surface particles, or turn into spherical bumpy particle or clubSP. The ability of E proteins to undergo some movements depends on the residues that are involved in the intra-dimer, inter-dimer and inter-raft interactions, and also the residues that are involved in the interactions between E protein ectodomain and stem helices of the E and M proteins.Some highly neutralizing HMAb have been shown to recognize quaternary structure-dependent epitopes on the spherical smooth-surfaced particle; however, these antibodies may not be able to neutralize the bumpy-surfaced particles or clubSP. Conversely, antibodies that recognize partial or completely hidden epitope, such as fusion loop antibodies or DIII-binding antibodies, could bind well to bumpy-surfaced virus particles due to their looser E protein arrangement and the higher solvent accessibility of the epitopes. Bumpy morphology virus could thus likely elicit more cross-reactive fusion loop or DIII antibodies. ClubSP has a heterogenous way of displaying E protein epitopes—different parts of the clubSP have different levels of exposure of different epitopes. Altogether, this suggests that the morphological changes on the DENV particle may facilitate the virus to escape from the immune system. Therefore, it is important to understand all possible morphological structures of flavivirus in order to design effective therapeutics or vaccines.